Aldose Reductase Inhibitors

Pharmac. Ther.Vol. 54, pp. 151-194,1992 0163-7258/92$15.
00
Printed in Great Britain. All rights reserved 1992PergamonPress Ltd
Specialist Subject Editor: B. L. FURMAN
ALDOSE REDUCTASE INHIBITORS A N D

DIABETIC COMPLICATIONS
DAVID R. TOMLINSON, GARY B. WILLARS a n d ANNE U CARRINGTON

Department of Pharmacology, Medical Sciences, Queen Mary and Westfield College,
Mile End Road, London E1 4NS, U.K.
Abstract--Aidose reductase inhibitors impede flux of glucose through the sorbitol

pathway in diabetes mellitus. They therefore reduce the accumulation of the pathway
metabolites, sorbitol and fructose, reduce the impact of the flux on the cofactors
used by the pathway and reduce other derived phenomena, such as osmotic stress and
myo-inositol depletion. As drugs, their targets are the chronic complications of diabetes--
neuropathy, retinopathy, nephropathy and vasculopathy. In experimental models there is
proof of activity against biochemical, functional and structural defects in all of the involved
tissues, but we await full clinical verification of this potential.
CONTENTS
1. Introduction 151
2. Cataracts 153
3. Keratopathy 155
4. Blood 156
4, I. Erythrocytes 156
4,2.White blood cells and platelets 157
5. Vascular Function 158
5,1. Localization of aldose reductase 158
5,2.Vascular and cardiac function 159
6. Microangiopathy 160
6,1. Capillary basement membrane thickening 160
6.2. Endothelial cell tight junctions 161
6.3. Vascular permeability 161
7. Retinopathy 162
8. Kidney 166
8.1. Distribution of aldose reductase 166
8.2. The physiological role of aldose reductase in renal osmoregulation 167
8.3. Diabetic nephropathy 167
8.4. Functional effects of sorbitol pathway activity and aldose reductase
inhibitors 169
9. Neuropathy 169
9.1. Localization of aldose reductase in peripheral nerve 169
9.2. Defects of nerve function in diabetes and diabetic neuropathy 170
9.3. Experimental studies with aldose reductase inhibitors 171
9.3.1. Functional abnormalities 171
9.3.2. Structural abnormalities 174
9.4. Clinical studies with aldose reductase inhibitors 175
10. Potential Role for Aldose Reductase in Other Diabetes-related Disorders 177
I 1. Synopsis and Conclusions 177
Acknowledgements 178
References 178
1. I N T R O D U C T I O N
The sorbitol (or polyol) p a t h w a y comprises conversion o f glucose to the polyhydric alcohol
(polyol), sorbitol, by the enzyme aldose reductase ( a l d i t o l : N A D P oxidoreductase; E.C. 1.1.1.21),
151
152 D.R. TOMLINSONet al.
followed by conversion of the sorbitol to fructose by sorbitol dehydrogenase (L-iditol:NAD

oxidoreductase; E.C. 1.1.1.14). These reactions are illustrated in Fig. 1. Sorbitol is also produced
by related enzymes known as aldehyde reductases. Aldose reductase belongs to a family of
NADPH-dependent oxidoreductases, which are collectively known as aldehyde reductases. The
nomenclature of these enzymes is confusing and they vary in cellular and species distribution as
well as in their affinities for various substrates (Dvornik, 1987).
In tissues which take up glucose independently of insulin and contain aldose reductase, the
flux through the pathway under normoglycemic conditions is limited by the relatively low
cellular glucose concentration and the low affinity of aldose reductase for glucose. Under
these conditions glucose is metabolized predominantly by hexokinase. In hyperglycemia, glucose
levels are elevated within these tissues, hexokinase is saturated and the fraction of glucose
metabolized by aldose reductase increases. Presumably, accumulation is progressive until clear-
ance gradients cause disappearance to match production and something approaching a new
steady state is reached. Certainly, various tissues in diabetic models and in patients contain
persistent and--where repeat measurements have been made--relatively consistent accumulations
of sorbitol and fructose (Gabbay et al., 1966; Stewart et al., 1967; Dyck et al., 1980; Mayhew
et al., 1983; Sherman and Dyck, 1983). The reader is referred elsewhere for further details
of the biochemistry of this pathway (Crabbe et al., 1981; Kador, 1988; Kinoshita, 1990; Dvornik,
1987).
The increased longevity given to diabetics, by the capacity to manage acute hyperglycemia
and ketoacidosis, has revealed the development of chronic complications of the disease. Thus,
morbidity and mortality are related to vasculopathy, neuropathy, retinopathy and nephropathy
plus a range of less major complications. Specific syndromes associated with ulceration of the
lower extremities, giving chronic infective lesions (so-called 'diabetic foot') arise from a combi-
nation of neuropathy and vasculopathy, together with a generalized impairment in combating
infection and wound healing (see Boulton, 1988 for a recent review). Attempts to develop
management or prophylactic strategies for these complications have focused on the notion that
biochemical consequences, deriving from hypoinsulinemia and/or hyperglycemia are ultimately
responsible for the generation of the functional and structural defects which present as the
complications of the disease.
Given that many secondary complications of diabetes occur in tissues which take up glucose
independently of insulin, the sorbitol pathway has been a logical and feasible drug target. Thus,
inhibitors of aldose reductase have been developed in the hope that reduction of the flux through
the pathway might attenuate the development or progression of diabetic complications. The main
inhibitors which have been used for research and, in some cases, developed for clinical use are
cited in Table I.
Evidence linking direct consequences of exaggerated flux through the sorbitol pathway
to the chronic complications of human diabetes mellitus is scarce. Of course, such evidence
is hard to get and the best instruments to obtain it are aldose reductase inhibitors. This
generates a vicious circle, because clearance for clinical work demands indications of clinical
efficacy. Normal recourse to animal experiments proves less than satisfactory because the
fidelity of rodent models to human long-term diabetic complications is questionable. None-
theless, research has developed via a series of hypotheses relating the sorbitol pathway to
secondary biochemical defects. This offers sequences of events which can be modelled
accurately and used to test drugs. The relevance of the relationships arrived at can then be
tested in man. A classical example of this approach is given by the established relation-
ship between exaggerated sorbitol pathway flux and depletion of nerve myo-inositol in diabetic
rats (Mayer and Tomlinson, 1983; Finegold et al., 1983; Gillon and Hawthorne, 1983). Thus it
has been argued that sorbitol accumulation, via reduced levels of free myo-inositol and a
sequence of events linking reductions in phosphatidylinositol-4,5-bisphosphate, diacylglycerol,
protein kinase C activity and Na /K -ATPase activity, results in nerve conduction disturbances
(Mayer and Tomlinson, 1983; Finegold et al., 1983; Gillon and Hawthorne, 1983; Greene and
Lattimer, 1983, 1984). The relationship between impaired Na /K -ATPase activity and conduc-
tion disturbances became contentious, has not been proven in man and appears to be a
phenomenon restricted to diabetic rats (Whiteley and Tomlinson, 1985; Calcutt et al., 1988;
Aldose reductase inhibitors 153
TABLE1. The Main Aldose Reductase Inhibitors Referred to in the Review
Inhibitor Company of origin Major dedicated publication(s)
Sorbinil Pfizer (Peterson et al., 1979a,b)
Tolrestat Wyeth-Ayerst (Sestanj et al., 1984)
Ponalrestat ICI (Stribling et al., 1985)
Epalrestat ONO (Kikkawa et al., 1983;
Ishida et aL, 1989)
See Dvornik (1987) for chemical structures of all these compounds.
Tomlinson, 1989) and only then under particular experimental conditions (Sredy et al., 1991).
Nonetheless, the ' s o r b i t o l - m y o - i n o s i t o l hypothesis' (Simmons et al., 1982; Winegrad et al., 1983;
Finegold et al., 1983) has stimulated an enormous amount of valuable work and has given a logical
framework that has argued cogently for clinical trials culminating in the introduction, in several
countries, of aldose reductase inhibitors to the market for diabetic complications. Thus, even if this
hypothesis is abandoned completely, it has been instrumental in supplementing crude pragmatism
and enabling development of a class of drugs which may alter profoundly the natural history of
diabetes mellitus.
This review attempts to evaluate the potential of these drugs as agents to reduce the incidence
or slow the progression of the major complications of diabetes mellitus. There is little information
about the impact of these drugs on the real end points which mark the critical stages of the
complications in humans. We are, therefore, concerned with their effect on related--and arguably
progenitive--biochemical aberrations and pathology. Our account is not exhaustive; another
source gives a compendious review of work up to the mid 1980s (Dvornik, 1987). We have,
therefore, attended more to recent information and have concentrated upon studies we consider
to be particularly revealing. Since this review appears in a source aimed at pharmacologists, we
have also given some background pathology to put the drug effects into context.
2. CATARACTS
Exaggerated formation of sorbitol in diabetes was first reported in the lens of diabetic rats, thus
indicating aldose reductase activity, by van Heyningen (1959). Previous work had suggested an
osmotic mechanism contributing toward the formation of cataracts in both diabetic and galacto-
semic rats. This information with subsequent experimentation, particularly by Kinoshita, led to the
osmotic or polyol hypothesis of cataract formation (for review see Kinoshita, 1986). Briefly, glucose
enters the lens independently of insulin and thus in hyperglycemia, levels within the lens are
elevated. This results in an increased flux through the polyol pathway with the resultant formation
of both sorbitol and fructose. Aldose reductase is located in epithelium and cortical fibers (Akagi
et al., 1984b) and the generally poor ability of polyols to penetrate membranes and the limited
removal of fructose by subsequent metabolic steps results in their accumulation within these cells.
The resultant hypertonicity is not offset and water is drawn in, causing the lens fibers to swell. This
increases membrane permeability and/or disturbs ion pumps and exchanges, such that materials
O=C--H
I ?H=OH ? I-I=OH
H--C--OH
I A/don H--C--OH
I Sorbltol C=O
I
HO--C-- H reductm#e HO-- C-- H dehydrogenne HO--C-- H
I i I P I
._c_o.. ._c_o., ._c_o.,
H--C--OH NA H--C--OH N H--C--OH
I I I
CH=OH NADP+ CI'I=OH NADH CH=OH
D-GLUCOSE SORBITOL D-FRUCTOSE
FIG. 1. The sorbitol (polyol) pathway showing the processing of glucose as a substrate.
are able to diffuse down their concentration gradients and substances such as K +, amino acids and
myo-inositol leak out, while Na and CI- leak in, further contributing toward lens fiber swelling.
These changes result in the formation of vacuoles, but ultimately cortical and nuclear opacity
occurs with reductions in protein synthesis, proteolysis, protein leakage and a complete loss of
cellular integrity.
The development and testing of this hypothesis has evolved not only through the use of
experimentally diabetic animals but also through the use of other in vivo models of sugar cataract
and the in vitro exposure of lenses to media with an elevated sugar content. In particular, in vitro
and in vivo exposure to high levels of galactose has been employed as cataracts develop more rapidly
than in diabetic models (Kinoshita et al., 1962). Galactose and other aldoses are also substrates
for aldose reductase. Indeed the enzyme has a higher affinity for galactose than glucose and this
enzymatic reduction of galactose produces dulcitol (galactitol) (Kinoshita, 1965). Dulcitol is not,
however, a substrate for sorbitol dehydrogenase, the next enzyme of the polyol pathway. Thus,
for equivalent loads of galactose and glucose, dulcitol will accumulate more rapidly and to higher
levels than those of sorbitol and any complications related to excessive aldose reductase activity
should occur more rapidly in galactosemia than diabetes (Kador and Kinoshita, 1985a,b).
Although this may account for the more rapid development of opacification in vivo under a
galactose load than a glucose load (Kinoshita et al., 1962) this may also be the result of a lack
of a balancing osmotic effect from the vitreous which occurs to some extent in diabetes but not
galactosemia (Xiong and Cheng, 1989). In addition there are other differences between the diabetic
and galactosemic lenses (Cheng et al., 1990) although their importance is not clear. Despite these
differences, the efficacy of many aldose reductase inhibitors in reducing polyol accumulation and
delaying or preventing sugar cataract formation, both in vitro and particularly in diabetic or
galactosemic rats has been demonstrated (see, for example, Kinoshita, 1974; Kinoshita et al., 1968;
Dvornik et al., 1973; Kador and Kinoshita, 1984; Peterson et al., 1979a; Fukushi et al., 1980a;
Poulsom et al., 1983b; Simard-Duquesne et al., 1985; Varma and Kinoshita, 1976; Varma et al.,
1977). This inhibition of cataractogenesis is associated with the attenuation or prevention of the
biochemical and structural alterations linked, in the osmotic hypothesis, to cataract formation by
exaggerated aldose reductase activity.
Despite overwhelming evidence that aldose reductase inhibitors are able to prevent or attenuate
the development of sugar cataracts, there is some evidence that mechanisms other than the
accumulation of polyols with subsequent osmotic stress may also play a role in cataractogenesis.
Thus, loss of energy producing capacity, increased oxidative stress (Cheng and Gonzalez, 1986) and
increased non-enzymatic glycosylation particularly of lens crystallins (Stevens et al., 1978; Monnier
et al., 1979) have all been implicated.
In addition to the alterations in polyol pathway metabolites and the changes in lens constituents
through altered membrane permeability, there are other metabolic disturbances in lenses exposed
in vitro or in vivo to high levels of glucose (Cheng and Gonzalez, 1986; Gonzalez et al., 1983, 1986).
These include increases in ~-glycerophosphate and glycerol-3-phosphate with decreases in ATP,
reduced glutathione, phosphorylcholine, glycerophosphorylcholine and glycerophosphorylethanol-
amine suggesting alterations in respiration, oxidative resistance and membrane metabolism (Cheng
and Gonzalez, 1986). Flux through the polyol pathway consumes both NADPH and NAD and
the ability of aldose reductase inhibition to prevent the above changes, particularly in glycerol-3-
phosphate, ATP and glutathione, may be related to the maintenance of a normal redox potential
(Cheng and Gonzalez, 1986). The maintenance of reduced glutathione levels by aldose reductase
inhibition may have other consequences related to its ability to prevent cataract formation. Thus,
there is evidence that oxidative damage may play a significant role in the development of
experimental sugar cataracts (Srivastava and Ansari, 1988; Creighton and Trevithick, 1979;
Trevithick et al., 1981; Ross et al., 1982; Linklater et al., 1986; Creighton et al., 1985) and the
inhibition of aldose reductase may reduce the competition between aldose reductase and gluta-
thione reductase for NADPH thus allowing maintenance of reduced glutathione. Interestingly,
glutathione may also inhibit non-enzymatic glycosylation of lens crystallins (Huby and Harding,
1988), thus providing another link by which aldose reductase may be related to a potential
pathogenic mechanism of cataract formation. Indeed, the increase in fluorophores in lens crystallins
of galactosemic rats, possibly the result of nonenzymatic glycosylation and browning (Maillard
reaction), is attenuated by sorbinil (Nagaraj and Monnier, 1990). This occurs despite the apparent
inability of sorbinil to interfere with the binding of galactose to crystallins (Chiou et al., 1980). Such
alternative hypotheses of cataract formation also stress the importance of a consideration of the
flux through the polyol pathway as opposed to the level of metabolite accumulation per se.
Thus, although the etiology of sugar cataract formation may be multifactorial, aldose reductase
inhibitors would appear to have at least the theoretical ability to interfere with the major suggested
pathogenic mechanisms. This may be a particularly important consideration given that the precise
mechanisms of the approximate fivefold increase in senile cataract in diabetic patients compared
to the nondiabetic population are unclear. The lenses of diabetic patients are rarely exposed to
the levels of sugars employed in the models, although this could still be of importance if a very
localized accumulation of polyol pathway products occurs. On the other hand, if cataractogenesis
is of a multifactorial origin the relative contributions of the various pathogenic mechanisms may
differ between the models and diabetic patients. This may be of little consequence if aldose
reductase inhibitors can influence all of the mechanisms, but the ability of aldose reductase
inhibitors to arrest or prevent cataract formation in diabetic patients has yet to be confirmed. Such
studies are difficult given the lack of methodology for the prediction, detection or quantification
of early cataracts and the unrealistic target of reversal of established cataracts (Stribling, 1990).
3. KERATOPATHY
The term diabetic keratopathy applies to changes occurring to the cornea of diabetic patients
which range from punctate epithelial erosions to neurotrophic (noninfectious) corneal ulceration
(Cobo, 1984). Although trivial changes are common in diabetic patients, sight-threatening
ulceration is rare. However, the clinical importance of diabetic keratopathy became apparent
following the introduction of vitreous surgery (Perry et al., 1978; Brightbill et al., 1978; Foulks
et al., 1979). Frequently during intraocular surgery the corneal epithelium has to be removed to
allow sight of the posterior segment of the eye (Blankenship and Machemer, 1978; Faulborn et al.,
1978; Pfister et al., 1971). Although re-epithelialization occurs rapidly in nondiabetic subjects,
healing is slow and incomplete in diabetic patients (Perry et al., 1978), often requiring special
post-operative care (Foulks et al., 1979). Other stresses such as contact lenses (Eichenbaum et al.,
1982) and manipulation during retinal photocoagulation (Pfister et al., 1971; Kanski, 1975) are not
well tolerated by the cornea of diabetic patients (Cobo, 1984).
A delay in corneal re-epithelialization also occurs in diabetic and galactosemic rats after scraping
of the epithelium and the newly formed cornea is often edematous and cloudy (Kinoshita et al.,
1979; Fukushi et al., 1980b; Datiles et al., 1983). This delay in re-epithelialization was prevented
both in diabetic and galactosemic rats by either the topical (Kinoshita et al., 1979; Awata et al.,
1988) or systemic (Fukushi et al., 1980b; Datiles et al., 1983) administration of an aldose reductase
inhibitor. A role for exaggerated polyol pathway flux in the development of this defect in
experimental and clinical situations is consistent with the accumulation of dulcitol in the corneal
epithelium of galactosemic rats (Awata et al., 1988) and the localization of aldose reductase in the
epithelium of the human cornea (Akagi et al., 1984b). It has been suggested, however, that it may
be the flux through aldose reductase, with the subsequent reduction in NADPH availability, that
is responsible for the corneal complications rather than the accumulation of metabolites per se (Hay
et al., 1986). A topical aldose reductase inhibitor has been applied to the eyes of diabetic patients
suffering from corneal lesions resulting in a dramatic improvement in their condition (Cobo, 1984;
Ohashi et al., 1988). However, the benefit of widespread clinical use of aldose reductase inhibitors
in spontaneous or trauma-induced keratopathy remains to be established.
Aside from the keratopathic lesions, diabetic patients also have a reduced corneal sensation
(Schultz et al., 1981) and a greater corneal thickness (Foulks et al., 1979; Busted et al., 1981)
particularly in patients with proliferative retinopathy (Busted et al., 1981) and persistent stromal
edema (Foulks et al., 1979). These alterations suggest cornea endothelial abnormalities. Indeed an
increased coefficient of variation of cell size and a reduction in the proportion of hexagonal cells
has been reported in the corneal endothelium of type I and type II diabetic patients while patients
with type I diabetes also had a reduced endothelial cell density (Schultz et al., 1984). Such changes,
particularly those found in type II diabetic patients, are also found in diabetic rats (Matsuda et al.,
1987; Meyer et al., 1988) and dogs (Yee et al., 1985). Aldose reductase is localized in human corneal
endothelium (Akagi et al., 1984b) and a role for its involvement in endothelial abnormalities is
supported by the occurrence of the changes in galactosemic dogs (Datiles et al., 1990) and their
prevention, attenuation or reversal by a number of aldose reductase inhibitors in experimental
diabetes (Akagi et al., 1987b; Matsuda et al., 1987; Terubayashi et al., 1988; Meyer et al., 1988)
and galactosemia (Datiles et al., 1990). A delay in the regeneration of damaged corneal endothelium
also occurs in galactosemic rats which is prevented by aldose reductase inhibition (Akagi et al.,
1988).
There are, therefore, several abnormalities of the corneal epithelium and endothelium in diabetic
patients which appear to be the result of an exaggerated polyol pathway flux and as such may be
alleviated by aldose reductase inhibition.
4. BLOOD
4.1. ERYTHROCYTES
Aldose reductase activity is present in erythrocytes as evidenced by the sorbitol accumulation
which occurs in human (Malone et al., 1980) and rat (Ao et al., 1991b) erythrocytes incubated
in vitro in high glucose. Elevated levels of sorbitol are also found in erythrocytes from diabetic
humans (Robison et al., 1989b; Malone et al., 1980) and rats (Kikkawa et al., 1983; Yue et al.,
1984). The in vitro accumulation of sorbitol under conditions of elevated glucose has been
attenuated or prevented by a number of aldose reductase inhibitors including tetramethylene
glutaric acid (Malone et al., 1980), tolrestat (Raskin et al., 1985), ponalrestat (Rillaerts et al., 1988)
and FR74366 (FK366) (Ao et al., 1991b). Many studies have also demonstrated the ability of aldose
reductase inhibitors to reduce erythrocyte sorbitol content in diabetic patients (Martyn et al., 1987;
Jaspan et al., 1985; Price et al., 1988b; Raskin et al., 1985; Peterson et al., 1986) and diabetic rats
(Yue et al., 1984; Kikkawa et al., 1983; Ao et al., 1991b; Peterson et al., 1986). However, whether
or not the reduction or prevention of sorbitol accumulation is beneficial in terms of erythrocyte
function requires clarification.
Altered blood rheology may play a role in the development of diabetic microangiopathy
(McMillan, 1983). This complication is discussed in more detail below. One important factor in
the altered blood rheology is the reduction in erythrocyte deformability (Schmid-Sch6nbein and
Volger, 1976; McMillan et al., 1978; Drouin et al., 1981; Juhan et al., 1981, 1982) and this along
with altered rheological properties of white cells (Vermes et al., 1987), elevated plasma viscosity
and increased erythrocyte aggregation may underlie the increase in whole blood viscosity in
diabetes (Schmid-Sch6nbein and Volger, 1976). Improved diabetic control can reverse reduced
erythrocyte deformability (Schmid-Sch6nbein and Volger, 1976; Drouin et al., 1981; Juhan et al.,
1982) and although insulin itself may increase deformability (Juhan et al., 1981, 1982; Bryszewska
and Leyko, 1983) and membrane fluidity (Bryszewska and Leyko, 1983; Dutta-Roy et al., 1985)
little is known of the mechanisms, including the role of exaggerated polyol pathway flux, in altered
erythrocyte rheology.
Deformability relates to the ability of erythrocytes to pass through capillaries whose luminal
diameters, at physiological perfusion pressures, may be smaller than their own and any reductions
in such deformability are likely to impede flow. Measurement of the viscosity of erythrocyte
suspensions may be used as an index of erythrocyte deformability (Chien, 1977; Bareford et al.,
1985). Under these conditions viscosity at high shear rates is mainly influenced by intracellular
viscosity but at low shear rates the influence is more by membrane properties and cell geometry
(Chien, 1977). A number of studies have reported normal high-shear but increased low shear
viscosity in erythrocyte suspensions from diabetic patients (McMillan and Utterback, 1981;
Rillaerts et al., 1986). Others have also reported an increased low-shear viscosity in patients with
insulin-dependent diabetes mellitus but there was no correlation to the levels of erythrocyte sorbitol
(Rillaerts et al., 1988). In addition, although incubation of erythrocytes from control and
insulin-dependent diabetes mellitus subjects in elevated glucose resulted in sorbitol accumulation,
it did not affect viscosity (Rillaerts et al., 1988). Despite this, aldose reductase inhibition did
improve viscosity, particularly at low shear rates. Thus, although sorbitol accumulation may not
directly influence erythrocyte viscosity, the aldose reductase inhibitor may have improved viscosity
independently of its ability to inhibit aldose reductase. This was also suggested by a study which
determined erythrocyte deformability by their filterability (Robey et al., 1987). This study indicated
a partial prevention, by sorbinil, of the reduced deformability of erythrocytes from streptozotocin-
diabetic rats. However, the authors argued that the level of sorbitol was insufficient to produce
osmotic stress and therefore, the impact of sorbinil on erythrocyte deformability may be, at least
in part, via mechanisms unrelated to aldose reductase inhibition.
Alterations in low-shear viscosity of erythrocytes from diabetic patients (Rillaerts et al., 1988;
McMillan and Utterback, 1981; Rillaerts et al., 1986) suggest an effect of diabetes on membrane
properties and cell geometry. The potential of an aldose reductase inhibitor to influence erythrocyte
low-shear viscosity independently of aldose reductase inhibition (Rillaerts et al., 1988) suggests a
direct effect on these perturbations. It is of interest, therefore, that aldose reductase inhibitors bind
to membranes of renal glomeruli (Cohen and Klepser, 1985), bovine renal Na+/K-ATPase
(Garner and Spector, 1987) and human erythrocyte Na /K -ATPase (Umeda et al., 1989). Indeed
in some instances binding to the Na /K -ATPase was accompanied by pump activation but the
implications of this require further investigation.
It should be noted that, despite the reports of altered erythrocyte properties in diabetic patients,
there is conflicting evidence concerning the effects of diabetes on erythrocyte rheology (Juhan et al.,
1979; Stuart et al., 1983; Hanss et al., 1983; Ritchie, 1985; Sewchand et al., 1981; Marigo et al.,
1981; Bidet et al., 1979). This lack of clarity may arise from methodological differences (Bareford
et al., 1986).
One such study which found no differences in physical properties of erythrocytes from diabetic
patients, including filterability through straight or tortuous channel pores and elongation over a
range of extracellular osmolalities, did find that incubation of erythrocytes, from controls or
diabetics, in 50 mM glucose reduced filterability and that this was prevented by sorbinil (Bareford
et al., 1986). However, as the reductions in filterability were small (12-13%) despite erythrocyte
sorbitol levels twice that in poorly controlled diabetics, these authors argued that sorbitol
accumulation does not play an important role in the rheological abnormalities. This has been
supported to some extent by the observation that sorbinil, despite preventing sorbitol accumu-
lation, failed to prevent reduced deformability of erythrocytes incubated in elevated glucose (Robey
et al., 1986) and that despite very high dulcitol levels in erythrocytes of galactosemic dogs, whole
blood filterability was unchanged (Kern et al., 1986). Thus, the role of exaggerated polyol pathway
flux in diabetes-associated alterations in erythrocyte rheology remains to be established.
4.2. WHITE BLOOD CELLS AND PLATELETS
Diabetic patients have an increased prevalence of bacterial and fungal infections (Rayfield et al.,
1982) suggesting an alteration in white cell, particularly neutrophil, function. Indeed the movement
(Molenaar et al., 1976), phagocytosis (Bagdade et al., 1974), killing (Nolan et al., 1978) and
oxidative metabolism (Nath et al., 1984) of neutrophils from diabetic patients are adversely
affected. Sorbitol accumulation has been identified in neutrophils of diabetic patients and in
neutrophils from diabetics, but not from controls, incubated in elevated glucose (Wilson et al.,
1987). In addition, dulcitol accumulated in neutrophils of both control and diabetic subjects
incubated in galactose (Wilson et al., 1987). Aldose reductase activity is, therefore, present in
neutrophils and, moreover, was reported to be greater in diabetic patients with complications than
those without (Dent et al., 1991). Although others have reported no differences in the phagocytosis
of neutrophils from controls or diabetics incubated in elevated glucose, the ability of cells from
diabetics to kill was reduced (Tebbs et al., 1991). This deficit was prevented by the inclusion of
an aldose reductase inhibitor during the in vitro incubation thus implicating exaggerated polyol
pathway flux in the reduced oxidative killing.
The platelets of patients and animals are also adversely affected by diabetes demonstrating an
increased sensitivity to aggregating agents (Bern, 1978; Butleus et al., 1980; Colwell et al., 1976;
Eldor et al., 1978; Johnson et al., 1979a) and increased production of thromboxane (Lagarole et al.,
JPT 54/2--C
1980; Ziboh et aL, 1979; Subbiah and Deitemeyer, 1980; Halushka et al., 1981). The administration
of the aldose reductase inhibitor, sorbinil, to diabetic patients reduced the in vitro platelet
responsiveness to collagen and ADP compared to an increased responsiveness in a placebo treated
group (Jennings et al., 1990). Others have demonstrated that while the elevation of glucose levels
in citrated whole blood increased the spontaneous platelet aggregation, this was unaffected by
aldose reductase inhibition with tetramethylene glutaric acid (May et al., 1990).
Thus, while exaggerated polyol pathway flux may play a role in some aspects of altered
neutrophil and platelet function in diabetes, these are not precisely defined. In addition, the ability
of aldose reductase inhibitors to influence in vivo function and the clinical manifestations of altered
function remain to be established.
5. VASCULAR FUNCTION
5.1. LOCALIZATIONOF ALDOSEREDUCTASE
Determination of the cellular localization of aldose reductase has centered essentially on the
assessment of aldose reductase product accumulation in cells or tissues cultured with high
concentrations of glucose and on the immunolocalization of aldose reductase.
Elevated glucose levels were shown to increase sorbitol content in a number of cell preparations
in culture including human umbilical vein endothelial cells (Hawthorne et al., 1989; Lorenzi et al.,
1987), porcine, canine and bovine retinal capillary endothelial cells (Kennedy et al., 1983; Lee et al.,
1989), bovine aortic endothelial cells (Yorek and Dunlap, 1989) and bovine cerebral microvascular
pericytes and endothelial cells (Sussman et al., 1988). Aldose reductase activity has also been
demonstrated by this method in bovine retinal microvessels, cerebral cortex microvessels, retinal
pericytes (Kennedy et al., 1983) and mouse cerebral microvessels (Yorek et al., 1991). In addition,
canine retinal microvessels have been reported to contain a hexitol producing activity (Kern and
Engerman, 1985) and isolated cerebral microvessels from streptozotocin-diabetic rats and alloxan-
diabetic rabbits show slight elevations in sorbitol (Sussman et al., 1988). Aldose reductase inhibitors
prevented the increased levels of sorbitol in cultured endothelial cells from human umbilical vein
(Lorenzi et al., 1987), bovine aorta (Yorek and Dunlap, 1989) and bovine retinal capillaries (Lee
et aL, 1989) cultured in high glucose. Although the observed increases in the sorbitol content of
cells cultured in high glucose are small compared to levels in diabetic animal tissues which take
up glucose independently of insulin, it must be remembered that the level of sorbitol accumulation
does not necessarily reflect the magnitude of aldose reductase activity. It is of interest, therefore,
that mouse cerebral microvessel endothelial cells show only a small increase in sorbitol when in
long-term culture with an elevated glucose concentration but much greater increases in dulcitol
content when cultured in the presence of high levels of galactose (Yorek et al., 1991). This would
suggest that, like mouse nerve (Calcutt et al., 1988), endothelial cells in culture have an aldose
reductase-like activity but may not accumulate excessive levels of sorbitol due to the capacity of
subsequent metabolic or removal processes. Taken together, the above studies indicate the presence
of aldose reductase activity in cultured endothelial cells but direct extrapolation to the in vivo
situation may be unwise.
Aldose reductase has been immunolocalized in endothelial cells of the aorta in dogs (Kern and
Engerman, 1982), the cornea, aorta and muscular arteries of the rat (Ludvigson and Sorenson,
1980b) and the human cornea (Akagi et al., 1984b). Immunoreactivity was not detected in the
endothelium of retinal or glomeruli capillaries of the rat (Ludvigson and Sorenson, 1980a), in
capillaries of the cerebrum, kidney, eye (iris, retina, choroid), abdominal muscle, heart, lung, liver,
peripheral nerve and testes in the non-diabetic dog (Kern and Engerman, 1982) or in endoneurial
vessel endothelia and pericytes in rat peripheral nerve (Powell et al., 1991). Other studies have,
however, demonstrated aldose reductase immunoreactivity in retinal capillary endothelial cells and
pericytes of non-diabetic Bio-Breeding (BB) rats (Chakrabarti et al., 1987) while in the dog and
human retinal microcirculation such immunoreactivity was located in pericytes but not endothelial
cells (Akagi et al., 1983, 1984b, 1986). The failure to demonstrate aldose reductase immuno-
reactivity must be set against methodological difficulties and the fact that, in general, tissues have
been taken from non-diabetic, non-galactosemic sources. There is evidence that elevation of
substrate levels or osmotic stress may activate and induce aldose reductase. Thus, elevation of
substrate in vitro activates aldose reductase of many human tissues (Das and Srivastava, 1985;
Srivastava et al., 1986) and, compared to control rats, increased levels of aldose reductase have
been demonstrated immunohistochemically in lenses of galactosemic and diabetic rats (Akagi et al.,
1984a, 1987a) and in kidneys of diabetic rats (Ghahary et al., 1989). In addition, increased aldose
reductase activity has been reported in kidney of streptozotocin-diabetic rats (Ghahary et al., 1989)
and erythrocytes of diabetic patients (Hamada et al., 1991) while compared to controls, increased
aldose reductase mRNA has been reported in kidney of diabetic rats (Ghahary et al., 1989), lens
epithelial cells of galactosemic rats (Lightman et al., 1990a) and human diabetic retinal pigment
epithelial cells (Vinores et al., 1988). In genetically diabetic rats, aldose reductase activity was
increased in lens, kidney, sciatic nerve, skeletal muscle, retina and spinal cord (but not in aorta,
seminal vesicle, lung, brain, testes, adrenal gland or heart) compared to non-diabetic controls
(Ghahary et al., 1991). In most tissues showing increased aldose reductase activity, aldose reductase
immunoreactivity and mRNA were also increased. These increases were attenuated by insulin
treatment but, while the aldose reductase inhibitor ponalrestat reduced aldose reductase activity,
it had no effect on its immunoreactivity or mRNA levels. Similarly, aldose reductase inhibition
failed to prevent increased aldose reductase mRNA in dog lens epithelial cells cultured under
hypertonic conditions (Carper et al., 1990).
5.2. VASCULAR AND CARDIAC FUNCTION
The precise distribution of aldose reductase in the intact vascular system of both man and
animals is still uncertain and its role in vascular complications of diabetes is perhaps even less clear.
The possible involvement of vascularly-located aldose reductase in the retinopathic, nephropathic
and neuropathic complications of diabetes is discussed elsewhere. However, it is important to note
that the micro- and indeed macroangiopathic lesions of diabetes extend beyond these tissues.
Although altered hemeorheology may play a part in reduced vascular perfusion, particularly in
small exchange vessels, a role for exaggerated flux through the polyol pathway, particularly in the
vascular wall, cannot be discounted in altered vascular function. There is evidence to suggest that,
with the possible exception of the kidney where early increases in prostaglandin production may
mediate early increases in renal blood flow (Craven and De Rubertis, 1989; Craven et al., 1987;
Kreisberg and Patel, 1983; Schambelan et al., 1985), the production of the vasodilator and
anti-platelet agent, prostacyclin is reduced in experimental and clinical diabetes (Johnson et al.,
1979b; Subbiah and Deitemeyer, 1980; Silberbauer et al., 1979, 1980; Rosenblum and Hirsh, 1984;
Harrison et al., 1978; Gerrard et al., 1980; Carreras et al., 1980; Lubawy and Valentovic, 1982;
Pace-Asciak et al., 1988). Conversely, the production of the vasoconstrictor and platelet aggregat-
ing agent thromboxane A2 is increased (Lagarole et al., 1980; Ziboh et al., 1979; Hui et al., 1989;
Pace-Asciak et al., 1988; Rosenblum and Hirsh, 1984; Subbiah and Deitemeyer, 1980). The
production of the potent vasoconstrictor endothelin may also be increased in diabetic animals
(Takeda et aL, 1991) and patients (Takahashi et al., 1990) while endothelium dependent relaxation
is reduced in diabetic animals (Durante et al., 1988; Oyama et al., 1986; Pieper and Garrett, 1988;
Hattori et al., 199 l; Abiru et al., 1990). In addition, vascular smooth muscle from diabetic animals
appears to be more sensitive to vasoconstrictors and less sensitive to vasodilators (Legan, 1989;
MacLeod and McNeill, 1985; Abebe and MacLeod, 1991; Le Blanc and Poduslo, 1990; Owen and
Carrier, 1979; Gebremedhin et al., 1988; Mayhan et al., 1991). Although not all studies have
confirmed these findings (Rosen et al., 1983; Rosen and Schror, 1980; Davis et al., 1979; Hui et al.,
1989; Fulton et al., 1991; Takahashi et al., 1991; Predel et al., 1990), the presence of one or more
of the above alterations, unless compensated, is likely to cause inadequate perfusion. The etiology
of these changes is, however, unclear. Few studies have examined the role of aldose reductase. One
study found a small effect of aldose reductase inhibition, enhancing prostacyclin release from the
aortae of diabetic rats (Wakasugi et al., 1991) while others have failed to find an effect on the
increased pressor response to angiotensin II in isolated perfused lung of diabetic rats (Stevens et al.,
1992). Sorbinil has, however, been demonstrated to prevent the increased glomerular production
of prostacyclin and prostaglandin E 2 in acute diabetes in rats (Craven and De Rubertis, 1989).
160 D, R. TOMLINSONet al.
Although disturbances of the autonomic nervous system influence aspects of cardiovascular

function in diabetic patients (Hosking et al., 1978; Clarke et al., 1979), changes in the contractile
properties, particularly of the ventricular muscle could account for the shortened left ventricular
ejection time, a longer pre-ejection period and prolonged isovolumetric relaxation (Cameron et al.,
1989b). These changes may, therefore, be a reflection of a true cardiomyopathy (Cameron et al.,
1989b). Similar changes occur in experimental models of diabetes and a role for exaggerated polyol
pathway flux in their development is suggested by the attenuation of both ventricular sorbitol
accumulation and deficits in the speed of contraction and relaxation by aldose reductase inhibition
in diabetic rats (Cameron et al., 1989b). In the rat, aldose reductase inhibition has also been
demonstrated to attenuate diabetes-associated increases in ventricular noradrenaline content and
turnover (Lucas and Qirbi, 1989), atrial muscarinic receptor numbers (Kofo-Abayomi and Lucas,
1987) and ventricular fl-adrenoceptor responsiveness and receptor number (Austin and Chess-
Williams, 1991). Although there is evidence that aldose reductase inhibition may influence several
aspects of cardiac function in diabetic rats, it is possible that these occur in conjunction with effects
on the autonomic nervous system and independence from these effects cannot be assumed. Thus,
whilst aldose reductase inhibition influences some responses of atria from diabetic rats to
transmural sympathetic nerve stimulation, it has no influence on the decreased positive
chronotropic and inotropic responses to exogenous noradrenaline (Hashimoto et al., 1990).
Clinically the ability of aldose reductase inhibitors to influence cardiac function may well be related
to their effects on the autonomic nervous system as discussed later.
6. MICROANGIOPATHY
6.1. CAPILLARY BASEMENT MEMBRANE THICKENING
Capillary basement membrane thickening is considered one of the classical ultrastructural
findings in diabetic microangiopathy (Rohrbach and Martin, 1982) and occurs in most tissues of
diabetic patients and animals including retinal, skeletal muscle and glomerular capillaries
(Williamson and Kilo, 1977, 1983; Efimov et al., 1980; Chandler et al., 1984). While the basement
membrane may impose a size-selective barrier for molecular filtration, the charge distribution,
particularly of the anionic sites of the glycosaminoglycans, may provide a charge-selective filtration
barrier. Thus, the basement membrane plays a role in the determination of capillary permeability
and in the formation of other filtrates such as the urinary filtrate of the kidney (Kanwar and
Farquhar, 1979; Brenner et al., 1978). Increased capillary basement membrane thickness per se has,
therefore, the potential to interfere with normal capillary function by altering the permeability
characteristics and influencing the mechanical properties of the vessels. However, there is evidence
of increased capillary permeability in both clinical and experimental diabetes (Vinores et al., 1990;
Lightman et al., 1990b; Jakobsen et al., 1987; Williamson et al., 1985; Cunha-Vaz et al., 1985a;
Seneviratne, 1972; Ohi et al., 1985). Increased thickness is unlikely, therefore, to account for such
permeability changes. Although altered perfusion characteristics and endothelial barrier function
may play a role in the altered capillary permeability of diabetes, altered chemical composition of
the basement membrane has been implicated. Thus, various studies, primarily in animal models
of diabetes have indicated increased type IV collagen and laminin production in certain tissues
(Grant et al., 1976; Cohen and Khalifa, 1977; Brownlee and Spiro, 1979; Cohen et al., 1981;
Hasslacher et al., 1982) but a reduction in the basement membrane heparan sulphate proteoglycan
content (Cohen and Surma, 1981, 1982, 1984; Rohrbach et al., 1982, 1983; Brown et al., 1982;
Parthasarthy and Spiro, 1992; Kanwar et al., 1983). These experimental studies have been
supported by observations in patients with insulin-dependent diabetes mellitus showing a wide-
spread reduction in sulphated proteoglycans in the glomerular basement membrane (Rohrbach,
1986). Thus, as the anionic sites of the glycosaminoglycans within the glomerular basement
membrane regulate the charge-selective nature of the filtration barrier (Kanwar and Farquhar,
1979; Brenner et al., 1978), their reduction may account for the defective functioning of the
filtration barrier (Wu et al., 1987). Similar alterations within other tissues could account for the
altered permeability of many of the vascular beds seen in diabetes. It has been argued that
the increased basement membrane permeability as a consequence of a reduced proteoglycan content

triggers a compensatory synthesis of basement membrane (Rohrbach et al., 1983; Ledbetter et al.,
1987) thus accounting for the increased thickness.
The biochemical disturbances underlying the increased capillary basement membrane thickness
are unclear and both non-enzymatic glycosylation and exaggerated polyol pathway flux have been
suggested (Brownlee et al., 1988; Szarfman et al., 1982; Frank, 1984; Robison et al., 1986b).
Glomerular and retinal capillary basement membrane thickening also occur in galactosemic
animals. Aldose reductase inhibition attenuates the increased capillary basement membrane
thickening in the retina of galactosemic rats (Robison et al., 1983; Frank et al., 1983; Shanks et al.,
1985; Robison et al., 1986b; Dvornik et al., 1986; Robison et al., 1984, 1986a), in the retina
(Chandler et al., 1984; Stribling et al., 1986), glomerulus (Chandler et al., 1984) and muscle (Efimov
et al., 1980) of diabetic rats, in the glomerulus of galactose-fed diabetic rats (Sato et al., 1985) and
in the retina of fructose or sucrose-fed diabetic rats (Kojima et al., 1985; Hotta et al., 1985) thus
implying a role for exaggerated polyol pathway flux in its development. Due to the requirement
of biopsy to assess capillary basement membrane thickness, there are few clinical reports of the
effects of aldose reductase inhibition. In one study, twelve months of sorbinil treatment in patients
with insulin-dependent diabetes mellitus and non insulin-dependent diabetes mellitus produced a
22% decrease in sural nerve endoneurial microvessel basement membrane thickness compared to
a 25% increase in controls (Sima et al., 1991). Although apparently large, these differences were
not statistically significant, but the indicated trend is consistent with at least a small role of
exaggerated polyol pathway flux in this lesion but its precise role requires clarification.
6.2. ENDOTHELIALCELL TIGHT JUNCTIONS

In addition to basement membrane thickening other microvascular abnormalities including
endothelial cell swelling and proliferation and occlusive platelet thrombi have been demonstrated
in sural nerve biopsies and autopsy samples in patients with distal symmetric polyneuropathy
(Leuenberger et al., 1974; Johnson et al., 1986; Sosula et al., 1972; Hori and Mukai, 1980; Klein
et al., 1984a; Williams et al., 1980). Furthermore, an increased number of endothelial nuclei per
capillary and an increased percentage of closed capillaries have been reported in sural nerve biopsies
from diabetic patients (Dyck et al., 1985a). These abnormalities correlated positively with the
degree of peripheral neuropathy. However, others have indicated that such abnormalities are a
consequence of the normal ageing process and that the only endoneurial microvascular abnormality
that is exclusively diabetes-related is a reduced number of endothelial cell tight junctions (Sima
et al., 1991). Such changes may therefore account, at least in part, for the increased capillary
permeability of diabetic capillaries and specifically for the increased vascular permeability to serum
proteins in the endoneurial space of diabetic sural nerves (Graham and Johnson, 1985). However,
increased endothelial cell dysjunction is not affected by aldose reductase inhibition in diabetic rats
(Klein et al., 1984b) or humans (Sima et al., 1991) despite an improvement in nerve fiber
morphometry, biochemistry and function reported in these patients (Leuenberger et al., 1971).
6.3. VASCULARPERMEABILITY
As discussed in other sections, the permeability of new and existing capillaries is increased in
experimental and clinical diabetes. The effect of aldose reductase inhibition, particularly on retinal
vasculature, is also discussed elsewhere. This increased leakiness of new vessels in diabetes and the
effect of aldose reductase inhibition has also been examined by assessing the permeability of plasma
albumin in new granulation tissue formed within polyester implants in the rat (see, for example,
Williamson et al., 1985, 1990; Chang et aL, 1986; Pugliese et al., 1990a,b). Increased albumin
permeation in such new vessels in short-term diabetic rats was prevented by aldose reductase
inhibition.
An increase in collagen cross-linking also occurs in the extracellular matrix in experimental
diabetes in tissues such as tendons and skin resulting in a reduced collagenase digestibility (Hamlin
et al., 1975), increased thermal and mechanical stability (Yue et al., 1983; Andreassen et aL, 1981),
decreased solubility and increased intrinsic viscosity (Yosha et al., 1983; Schnider and Kohn, 198 l;
Golub et al., 1978). It is possible that such increases in cross-linking could play a role in the
increased incidence of atherosclerosis, osteoarthritis, stiffening of arteries, lungs and joints that
occur in diabetes. It is also of interest to note that the severity of joint limitation (Rosenbloom
et al., 1981) and arterial stiffness (Monnier et al., 1986) are correlated with the risk of developing
retinopathy. Cross-linking may be attributable to advanced glycosylation products, due to chronic
elevation of blood sugar, its products are detectable due to their fluorescence. Aldose reductase-
dependent fluorescence formation was found in galactosemic rat lenses (Nagaraj and Monnier,
1990) and the skin of diabetic rats (Suarez et al., 1988; Odetti et al., 1990). However, in tail tendons
of galactosemic rats, the collagen-associated increases in fluorescence were not prevented by aldose
reductase inhibition whereas the increase in tendon breaking time in urea, a likely indicator of total
collagen cross-linking, was prevented (Richard et al., 1991). This suggests that collagen cross-
linking may occur via at least two mechanisms. One is associated with increased advanced
glycosylation product formation, is detected by increased fluorescence, but is not amenable to
aldose reductase inhibitor treatment. Alternatively, a mechanism may exist which is unrelated to
increased glycosylation product formation, does not therefore result in fluorescence but is amenable
to aldose reductase inhibitor treatment. One such possibility is a lysyl oxidase-dependent cross-link
formation. However, other aldose reductase inhibitor-sensitive mechanisms may exist, because
indirect evidence supports a lysyl oxidase-dependent increase in cross-linking in granulation tissue
of diabetic rats, which is insensitive to aldose reductase inhibition (Williamson et al., 1985; Richard
et al., 1991).
There is, therefore, a range of microvascular structural and functional alterations in established
and new blood vessels in experimental and clinical diabetes. However, their contribution to
clinically relevant micro- and macroangiopathy, together with the role of exaggerated polyol
pathway flux in their development remains to be established. Specific possibilities will be considered
in relation to retinopathy, nephropathy and neuropathy.
7. RETINOPATHY
Diabetic retinopathy is a disease of the retinal microcirculation and is identified by structural
changes upon ophthalmoscopic examination. Clinically, retinopathy may be divided into either
'non-proliferative' (background) or 'proliferative' stages. Non-proliferative retinopathy is charac-
terized by the presence of one or more of the following pathologies--venous abnormalities,
microaneurysms, hemorrhages, edema and exudates. Proliferative retinopathy, in addition to the
features of the non-proliferative phase, is characterized by new vessel formation and in advanced
stages, vitreo-retinal traction. Although a brief description of the pathology is given below, the
reader is referred elsewhere (Kohner, 1989; Engerman, 1989) for a more detailed account.
Microaneurysms, particularly of the central retinal region, are the earliest clinically recognizable
feature of diabetic retinopathy. However, fluorescein angiography at this time will also reveal small
areas of non-perfusion which are often associated with areas of capillary dilatation. Microaneu-
rysms always have an area of non-perfusion in their immediate vicinity. However, areas of
non-perfusion may be present without microaneurysms suggesting an earlier development although
the precise relationship between capillary dilatation, non-perfusion and microaneurysms is unclear.
Retinopathic lesions progress with the appearance of hard exudates and hemorrhages. Fluid
extravasation initiates hard exudate formation and this results in retinal thickening. When this
occurs in the foveal and perifoveal regions this represents macular edema and is the commonest
cause of diabetic maculopathy. Although causing visual impairment, diabetic maculopathy in itself
does not result in blindness. However, it is frequently found in patients with non-insulin-dependent
diabetes mellitus and the relatively high number of such patients, compared to those with
insulin-dependent diabetes mellitus, means that maculopathy is a common cause of visual loss in
the diabetic population.
Proliferative retinopathy is, however, an all too common cause of blindness amongst the diabetic
population, particularly those with insulin-dependent diabetes mellitus. At present, treatment of
the proliferative stage is limited to laser photocoagulation and in some cases, where this is
unsuccessful, vitrectomy may help to restore vision. In recognition of the need to treat proliferative
lesions as early as possible, it has been useful to consider those features of the non-proliferative
stage which are prognostic of an impending slide into the proliferative phase. Thus, while a few
discrete 'dot and blot' hemorrhages, usually surrounding microaneurysms, do not suggest early
progression into the proliferative phase, clustered or diffuse large blot hemorrhages indicate
widespread ischemia and become 'pre-proliferative' lesions. Intra-retinal microvascular abnormal-
ities are also designated as pre-proliferative pathologies. These abnormalities are dilated capillaries,
often with microaneurysms and are found in retinas with many non-perfused capillaries. In
addition, multiple 'cotton wool spots' (representing nerve fiber swellings) and venous beading,
reduplication and loop formation within the retinal microvasculature are recognized as being
pre-proliferative.
The vast majority of patients with diabetes, either insulin-dependent or non-insulin-dependent,
will develop at least non-proliferative retinopathy with increasing duration of the disease (Klein
et al., 1984a,b). This non-proliferative phase is a pre-requisite for development of the proliferative
phase. However, the former does not always result in the latter and such progression may be
dependent upon the interaction of factors including genetics, the degree of glycemic control and
other disturbances associated with the diabetic state. Hypertension, for example, seems to be a risk
factor for the development of proliferative lesions thus emphasizing the need for its careful
management. As with other secondary complications, it is difficult to establish beyond doubt that
good glycemic control prevents the development of retinopathy although several studies, in patients
and animals, have indicated that the severity of retinopathy is related to the degree of control and
that improved control may slow or arrest its progression provided that the lesions are not already
too advanced (Klein et al., 1984a,b; Rand et al., 1985; Engerman et al., 1977; Miki et al., 1969;
Pirart, 1978a,b; Job et al., 1976; Engerman and Kern, 1987). It is important to note, however, that
the rapid initiation of improved glycemic control can provoke a deterioration of established
retinopathy (Lauritzen et al., 1983; Kroc Collaborative Study Group, 1984; Dahl-Jorgensen et al.,
1986). The inability of improved control to influence retinopathy once it has passed a particular
point and the possible detrimental effect of rapid improvements in glycemic control are important
considerations in any trials designed to examine the influence of treatment regimes upon the
progress of retinopathy.
The proliferative phase is characterized by new vessel and connective tissue growth initially on
the retina or optic nerve head but often extending into the vitreous compartment. The new vessels
formed are prone to hemorrhage and this produces advancing loss of vision. In addition, traction
between the vitreous and the retina may exacerbate hemorrhage formation and can produce
vitreous and ultimately retinal detachment. Although there are exceptions, in most cases, once
vitreous hemorrhage is present, treatment by means of photocoagulation is difficult and often
unsuccessful. Thus, early treatment of the proliferative condition offers the best prognosis.
However, even at this early stage it is evident that the retinal microcirculation has suffered
considerable insult and prevention of the early events resulting in the lesions of non-proliferative
retinopathy must be seen as the goal. The uncertainty surrounding the ability of good glycemic
control to prevent the development of retinopathy and the impossible task of providing such
control for the entire diabetic population, indicate the need for other therapeutic interventions
which will prevent, arrest or even reverse retinopathic lesions, Ideally, such a strategy would be
based upon an understanding of the pathogenic mechanisms and cellular events underlying the
early changes, but for the present these are far from clear.
Fluorescein angiography has allowed the examination of changes in the retinal vasculature that
are not readily visible upon standard ophthalmic investigation. This has been of particular use in
demonstrating not only the early changes in capillary perfusion, including capillary dilatation and
occlusion, but also the integrity of the blood-retinal barrier. It is clear that leakage from retinal
vessels causes maculopathy and that new vessels formed in the proliferative phase have an increased
permeability. It has been suggested that fluorescein may leak from retinal vessels in the absence
of clinically identifiable retinopathy and may therefore be of predictive value (Cunha-Vaz et al.,
1975, 1985a). However, while fluorescein leakage may be of help in the quantitative assessment of
the severity of early retinopathy, leakage is just one manifestation of retinopathy and indeed does
not always co-exist with early retinopathy (Chahal et al., 1986; Krogsaa et al., 1986). Increased
permeability occurs despite a thickening of the capillary basement membrane which is a
characteristic early feature of retinopathy and indeed diabetic microangiopathy (Rohrbach and
Martin, 1982; Williamson and Kilo, 1977, 1983). However, there is accumulating evidence that
the composition of basement membrane material is affected in diabetes, resulting in a reduced
anionic charge (Rohrbach et al., 1983), which may play a role in altered capillary permeability.
Alternatively, or in addition to this, an increased permeability may be a reflection of the cellular
alterations occurring in the capillary wall. Such cellular changes appear to be centerd upon an
early and preferential loss of pericytes, which are present in capillaries in similar numbers to
endothelial cells in normal retina. Following the loss of pericytes, endothelial cell proliferation
occurs although the function of these cells may be compromised. Ultimately there appears to be
a breakdown of the tight junctions between endothelial cells and endothelial cell death which
results in the appearance of empty basement membrane tubes. The alteration and eventual failure
of endothelial function may result in capillary non-perfusion, a situation which would be
exacerbated by the hemeorheological disturbances such as increased platelet aggregation, reduced
red cell deformability and increased thrombus formation which occur in diabetes. Extensive areas
of capillary non-perfusion appear to be the stimulus for new vessel formation. Although these
new vessels contain both endothelial cells and pericytes they are structurally abnormal and show
exaggerated permeability, so that their proliferation and subsequent hemorrhaging are sight-
threatening unless treated rapidly.
The importance of this relatively lengthy account of the pathology of retinopathy is simple, but
cannot be overemphasized. Any drug intervention must be made early and must impact
significantly upon the progenitive biochemistry. Once the extensive structural changes have
begun, they may not be reversible.
The assessment of the potential effects of aldose reductase inhibition upon the development
and progress of diabetic retinopathy, as with other secondary complications, is difficult for
several reasons. Earlier work examining the influence of improved glycemic control suggested a
point at which the damage to the retinal microcirculation may be too severe to allow reversal or
even prevention of progression. It is unclear at which point this occurs, but the inclusion of
patients with proliferative retinopathy in clinical trials is likely to underestimate the potential
value of the treatment regime. Given the length of time it may take retinopathy to become
apparent and the unpredictability of this period and the rate of progression in a given individual,
it would seem prudent, therefore, to assess drug efficacy using early retinopathic lesions, or
changes associated with retinopathy that are comparatively easily measured and, if possible,
predictive of the prognosis.
Few studies have reported the influence of aldose reductase inhibition on ophthalmological
status in diabetic patients. Based on one and two year studies, epalrestat prevented the
progression of the early stages of retinopathy (Hotta et al., 1988a,b). In a 3 year study the
same group reported a reduction or disappearance of microaneurysms, hemorrhage and
soft exudates in patients with non-proliferative retinopathy indicating significant clinical
benefit of the treatment (Hotta et al., 1990). Even patients with proliferative retinopathy
deteriorated at only one half of the rate seen in untreated controls. In association with
these beneficial effects seen upon ophthalmoscopic observation of retinal structure, epalrestat
also reduced or reversed enhanced retinal vessel fluorescein leakage and improved an abnormal
oscillatory potential of the electroretinogram in patients in the non-proliferative phase. A
large study considering the effects of 41 months of treatment with sorbinil in 497 insulin-
dependent diabetes mellitus patients failed to show such dramatic improvements although
there was some evidence that aldose reductase inhibition slowed the rate of increase in the
number of microaneurysms (Sorbinil Retinopathy Trial Research Group, 1990). Others have
also examined retinas by fluorescein angiography and demonstrated a beneficial effect of 6
months of sorbinil treatment on retinal barrier function (Biersdorf et al., 1988; Pitts et al., 1986).
Similar results have been reported using sulindac a non-steroidal antiinflammatory drug with
aldose reductase inhibitory activity (Cunha-Vaz et al., 1985b). Another study demonstrated that
while comparison between short-term sorbinil treatment and placebo in diabetic patients failed to
show significant effects of the aldose reductase inhibitor on electroretinograms, there was
improvement in patients with low initial retinopathy but not those with more severe retinopathy
(Biersdorf et al., 1988). Thus, although studies with epalrestat have provided promising results
for the effect of aldose reductase inhibition on the clinical manifestations of diabetic retinopathy,
those with sorbinil have been less favourable.
Attempts to assess the precise sequence of events in the development of retinopathy and the
influence of exaggerated polyol pathway flux on the pathology have been hampered by the
unavailability of animal models which develop all of the characteristics of the human condition.
However, certain pathologies, similar to those in the retina of diabetic patients, occur in
experimentally diabetic rats (Musacchio et al., 1964; Toussaint, 1966; Kojima, 1971; Orloff et al.,
1975; Leuenberger et al., 1971, 1974; Sosula et al., 1972; Hori and Mukai, 1980), cats (Mansour
et al., 1990), dogs (Engerman and Bloodworth, 1965) and monkeys (Bresnick et al., 1976) and in
genetically diabetic dogs (Patz and Maumenee, 1962; Patz et al., 1965; Sibay and Hausler, 1967;
Gepts and Toussaint, 1967) and BB rats (Sima et al., 1985). These pathologies consist predomi-
nantly of the early events seen in the clinical condition such as ultrastructural changes, capillary
basement membrane thickening, vasodilatation, areas of non perfusion, microaneurysms, degener-
ation and loss of pericytes and acellularity. Although the retinal lesions in diabetic dogs and
possibly monkeys are morphologically similar to those in patients, there appears to be some doubt
about the changes in rodents, particularly the nature of the microaneurysms (Engerman, 1976).
Despite this, these models have been used to examine the role of increased aldose reductase activity
in the development of retinal lesions in diabetes. Thus, aldose reductase inhibition prevents some
biochemical (Poulsom et al., 1983c) and electroretinogram (Segawa et al., 1988) changes in
streptozotocin-diabetic rats, ameliorates some electroretinogram changes in fructose-fed strepto-
zotocin-diabetic rats (Funada et al., 1987) and prevents capillary basement membrane thickening
in the diabetic cat (Mansour et al., 1990), streptozotocin-diabetic rat (McCaleb et al., 1991) and
in the deep, but not superficial, retinal capillary bed of the diabetic BB rat (Chakrabarti and Sima,
1987, 1989). The use of diabetic animals has been supplemented by the use of sucrose-fed diabetic
rats (Papachristodoulou et al., 1976; Papachristodoulou and Heath, 1977; Boot-Handford and
Heath, 1980) and by galactosemic rats (Robison et al., 1983, 1984; Frank et al., 1983) and dogs
(Engerman and Kern, 1984) in which retinopathic-like pathologies occur. The very fact that such
changes occur in galactosemia implicates a role for exaggerated polyol pathway flux in their
etiology. This is further supported by the observations that in galactosemic rats, aldose reductase
inhibition prevents electroretinogram abnormalities (Segawa et al., 1988), prevents capillary
basement membrane thickening (Robison et al., 1983, 1986b,c, 1988, 1989b, 1990; Das et aL, 1990),
ameliorates ultrastructural and structural changes of the retina (Robison et al., 1986b,c, 1988,
1989a,b, 1990) and prevents blood-retinal barrier failure (Vinores et al., 1990). Structural
alterations, including pericyte degeneration are also prevented by aldose reductase inhibition in
galactosemic dogs (Kador et al., 1988, 1990).
The mechanisms by which aldose reductase activity may influence retinal structure and function
in diabetes or galactosemia are unclear. Levels of polyol pathway metabolites in whole retina are
increased in streptozotocin-diabetic and galactosemic rats and these accumulations are prevented
by aldose reductase inhibition (Segawa et al., 1988; Poulsom and Heath, 1983; Poulsom et al.,
1983a,c; Stribling et al., 1985; Ao et al., 1991a,b). Although there is some confusion over the cellular
distribution of aldose reductase in the retinal capillaries which may arise from methodological or
species differences (see Section 5), immunolocalization studies suggest its presence in pericytes but
not in endothelial cells of the human retinal microcirculation (Akagi et al., 1983, 1984b). This
localization in pericytes, the initial preferential loss of these cells and the apparent ability of
inhibitors to influence the progression of retinopathy or retinopathic changes implicates a
biochemical lesion in pericytes in the etiology of diabetic retinopathy.
Along with the endothelial lining, the retinal pigment epithelium constitutes the blood-retinal
barrier. The neural-derived retinal pigment epithelium forms a monolayer of cells between the
neurosensory retina and circulating blood within the choroid and is essential to the normal
maintenance of retinal cellular and extracellular compartments. Abnormalities of the retinal
pigment epithelium including subtle morphological changes and areas of necrosis have been
described in short-term experimental and genetically diabetic rats (Kirber et al., 1980; Grimes and
Laties, 1980; Grimes et al., 1984; Blair et al., 1984; Tso et al., 1980). The ability of retinal pigment
epithelium cells to exclude fluorescein and to transport it from the vitreous to the choroid is reduced
in diabetics (Kirber et al., 1980; Krupin et al., 1982) and such deficits have been implicated in the
etiology of the edema in diabetic maculopathy (MacGregor and Matschinsky, 1986a). Several
biochemical changes have also been reported in the retinal pigment epithelium of alloxan-diabetic
rabbits including elevated glucose, sorbitol and total Na and reduced myo-inositol and
Na /K -ATPase activity (MacGregor and Matschinsky, 1986a,b; MacGregor et al., 1986). Aldose
reductase has been immunolocalized in retinal pigment epithelium cells of non-diabetic BB rats
(Chakrabarti et al., 1987) and accumulations of sorbitol and fructose with depletion of myo-inositol
occur in human retinal pigment epithelium cells cultured under conditions of elevated glucose (Del
Monte et al., 1991). The changes in sugar and polyol levels in these cells were substantially reduced
by aldose reductase inhibition with sorbinil. In addition, exposure of the cells to high glucose
reduced their ability to phagocytose retinal rod outer segments which is a functional characteristic
of human retinal pigment epithelium cells. This functional deficit was also prevented by sorbinil.
The changes in retinal pigment epithelium biochemistry have been likened to those occurring in
peripheral nerve in experimental diabetes and altered retinal pigment epithelium function has,
therefore, been implicated in the etiology of diabetic retinopathy (MacGregor and Matschinsky,
1986a).
The retinal pigment epithelium hyperpolarizes in response to light-induced activity of the
photoreceptor cells and this forms the c-wave of the electoretinogram. The c-wave amplitude
progressively decreases in experimental diabetes (MacGregor and Matschinsky, 1986a; Paulter and
Ennis, 1980) possibly indicating therefore a deficit in retinal pigment epithelium function. This
progressive deterioration in the c-wave amplitude was ameliorated by sorbinil in streptozotocin-di-
abetic rats suggesting a beneficial effect on the retinal pigment epithelium (MacGregor and
Matschinsky, 1986a). However, the relationship between changes in the retinal pigment epithelium
and clinical retinopathy is unclear.
While the prevention or amelioration of retinal changes in diabetic or galactosemic
animals by aldose reductase inhibition implicates a role for exaggerated polyot pathway flux in
their development, the predictive nature of these changes in terms of the development of
retinopathy in diabetic patients requires some clarification. In addition the role of other pathogenic
factors in the development of retinopathy, including alterations in growth factor status, require
exploration (Frank, 1991). Nevertheless, taken in combination with the limited number of clinical
assessments of aldose reductase inhibitor efficacy in retinopathy, there is room for hope that this
class of drugs may be useful in the future to help to control this condition.
8. KIDNEY
8.1. DISTRIBUTIONOF ALDOSE REDUCTASE
Aldose reductase has been mapped in the kidney by enzyme cytochemistry and immunocyto-
chemistry and its gene transcripts have been localized by in situ hybridization. At the macroscopic
level enzyme activity may be detected in all parts of the kidney, but is highest in the outer medulla
both in rat (Corder et al., 1977) and man (Corder et al., 1979). Under the microscope, the
activity is in the glomerulus, the proximal and distal convoluted tubules and the ascending limb
of the loop of Henl6 (Corder et al., 1977, 1979; Ludvigson and Sorenson, 1980a). The distribution
of sorbitol dehydrogenase is similar (Corder et al., 1977). There is, however, some variability in
reports derived from immunocytochemistry, because some of the antibodies used have been raised
against non-renal sources of aldose reductase (Ludvigson and Sorenson, 1980a; Terubayashi et al.,
1989).
A more selective picture of the cellular distribution of aldose reductase is given by in situ
hybridization and comparison of the results therefrom shows marked differences from the work
described above. Bondy and Lightman (1989) used 2 probes complementary to different regions
of the DNA sequence for aldose reductase. These probes were validated in that they formed a single
hybridization band on Northern blots from rat kidney. Hybridization in situ, in kidneys from
neonatal rats, revealed transcripts only in the papilla. With growth, expression increased markedly,
but only in the inner medulla and none was registered in the outer medulla or cortex. Interestingly,
this study also reported that salt loading of the rats caused a marked increase in the aldose
reductase mRNA expression in the medulla (Bondy and Lightman, 1989). The consequences of this
finding are discussed in the next section.
The kidney also contains an aldehyde reductase, which has been immunocytochemically localized
to the renal cortex, specifically in the proximal convoluted tubule and not in the glomerulus
(Terubayashi et al., 1989).
Clearly, the cytological picture of aldose and aldehyde reductases in the kidney remains to be
fully worked out, but it is possible that the capacity to form sorbitol could be realized almost
anywhere in the kidney. Thus there is plenty of scope for physiological and pathological roles for
the sorbitol pathway in the kidney.
8.2. THE PHYSIOLOGICALROLE OF ALDOSE REDUCTASE IN RENAL OSMOREGULATION

The kidney is one of the few organs in which a physiological role for aldose reductase has been
established. The gradients of extracellular [Na ] and [C1-], which progress through the renal
medulla towards the papilla and form the basis of countercurrent solute exchange, impose osmotic
stress on the cells of the loop of Henl6 and collecting duct. Furthermore, this stress varies with
degrees of diuresis. For example, water deprivation increases medullary interstitial electrolyte
concentration and osmolarity, whilst diuresis has the opposite effect (Beck et al., 1984). As a
compensation, the medullary cells accumulate large amounts of electrically neutral organic
osmolytes to enable maintenance of solute and water transfer without gross distortion of cell
volume (Bagnasco et al., 1986). Sorbitol is an important member of this group of organic osmolytes
and is formed in these cells by the action of aldose reductase on glucose (Bagnasco et al., 1986).
Exposure of cells derived from rabbit renal medulla to raised extracellular [NaCI] caused a
progressive rise in aldose reductase activity and synthesis of sorbitol in amounts related to the
magnitude of the change in extracellular osmolarity (Bagnasco et al., 1988). A return to lower
extracellular [Na ] and [C1-] was associated with a reduction in aldose reductase activity.
However, the phenomenon was clearly complex, because the rate of change of aldose reductase
activity in response to these stimuli had a periodicity of days. Nonetheless, the cells were capable
of adjusting their sorbitol content with a periodicity of hours (Bagnasco et al., 1988). As has already
been stated, salt loading of rats increases the expression of aldose reductase at the transcriptional
level (Bondy and Lightman, 1989; Cowley et al., 1990). This was also shown in a cultured cell line
derived from rabbit inner renal medulla, which expressed increasing amounts of aldose reductase
mRNA with increasing osmolarity of the culture medium (Garcia-Perez et al., 1989). Thus, it
appears that an increase in the osmolarity of the extracellular fluid can trigger expression and
activation of the enzyme in renal medullary cells. This induction of aldose reductase mRNA by
hypertonic stress was also seen in vitro in glomerular endothelial cells (Hohman et al., 1991),
mesangial cells (Kaneko et al., 1990) and in epithelial cells of the lens (Carper et al., 1990).
Other studies have demonstrated further complexity by revealing reciprocity between sorbitol
and betaine as compensatory intracellular osmolytes. Betaine is another organic osmolyte--a
nitro-derivative of glycol--which is widely distributed in animals and plants. Administration of an
aldose reductase inhibitor, sorbinil, to rats halved the medullary sorbitol content. Concomitantly
the medullary betaine content increased and the ratio of total medullary organic osmolytes to Na
was similar in rats with or without sorbinil treatment (Yancey et al., 1990). Inhibition of aldose
reductase in medullary cell lines in culture was shown to compromise cell viability. However,
addition of betaine to the culture medium resulted in its uptake by the cells and an improvement
in their survival (Moriyama et al., 1991). As yet, it is not clear whether betaine is synthesized in vivo
in medullary cells or taken up from extracellular fluid, though the former process is more likely
to afford the necessary control of cellular levels.
This compensation for reduced availability of sorbitol, enforced by aldose reductase inhibitor
treatment, is important in that medullary osmoregulation need not be impaired in patients taking
such drugs.
8.3. DIABETIC NEPHROPATHY

The terminal stage of diabetic nephropathy is renal failure. Progenitive pathology is indicated
by proteinuria and by structural degenerative changes in renal biopsies. The latter have been
characterized by Osterby and her colleagues and their studies form a model of objective
morphometric ultrastructural analysis. The structural abnormalities are concentrated in the
glomerulus and all nephrons are affected in advanced diabetic nephropathy, though glomeru-
lopathy shows some variation in severity (Horlyck et al., 1986). Glomerulopathy is characterized
by a very slow development of basement membrane accumulation, manifested as thickening
of the peripheral basement membrane and increased volume of the mesangial basement
membrane-like material with mesangial expansion (Osterby, 1986). In time, increased deposition
of the two forms of basement membrane leads to the ultimate solidification of the glomerular tuft
with loss of capillary surface. The end-stage is glomerular closure, with elimination of
glomerular function (Osterby, 1986). A related functional variable is increased glomerular
filtration rate and a very close correlation has been found between this and the total remnant
surface area of the glomerular capillaries (Osterby, 1986). The highly significant relationship
between glomerular filtration surface per nephron and glomerular filtration rate was borne
out by another study (Osterby et al., 1988). Along with the classical changes of the diabetic
glomerulopathy, changes in glomerular size are detectable. In early diabetes, during the stages
of glomerular hyperfunction, hypertrophy develops acutely at the onset of diabetes, leading
to an increase in capillary surface corresponding to the increase in filtration rate. In the
advanced stages when glomerular closure involves a proportion of the nephrons compensatory
hypertrophy develops, thereby probably helping to preserve capillary surface for a period of time
(Osterby, 1986). There is also evidence for new vessel formation in the glomerulus, though these
vessels have abnormally-structured endothelium and capsular epithelium (Osterby and Nyberg,
1987).
There is a distinct association between microalbuminuria in diabetes and raised systemic
arterial pressure. Hypertension is also associated with nephropathy, however, causality
remains indistinct. Examination of groups of young, insulin-dependent diabetics without any
renal abnormalities gives systemic arterial pressures which are similar to properly matched
non-diabetic control groups (Tarn and Drury, 1986). A detailed examination of the factors
known to contribute to the variances of systolic and diastolic pressures showed similarity
between matched groups of non-diabetic and insulin-dependent diabetic young adults (Tarn
et al., 1990). Thus, familial indicators for abnormal arterial pressures were the same in both
groups.
Hypertension appears at the same time as microalbuminuria (Mathieson et al., 1984; Wiseman
et al., 1984). Since there are logical arguments for causality in either direction, the precise
timing of these phenomena is critical. One study does indicate that microalbuminuria is the
earlier event (Mathieson et al., 1988), but that does not prove causality. Indeed, there are reasons
to suspect strongly that antihypertensive therapy significantly improves the prognosis for diabetic
nephropathy (Parving and Hommel, 1989).
It is clear, therefore, that an appraisal of the possible impact of aldose reductase inhibition on
diabetic nephropathy must address the possible effect of these drugs on the structural abnormal-
ities of the glomerulus in animal models and should also include hypertensive diabetic animals.
One study examined the effect on glomerular abnormalities of 6 months of streptozotocin-in-
duced diabetes in rats, with and without ponalrestat, given constantly by dietary admixture.
Ponalrestat treatment was associated with a greatly reduced incidence of cataracts, indicating
pharmacological efficacy for the inhibitor. Glomerular basement membrane thickness was signifi-
cantly increased in untreated diabetic rats (174 + 4.5 (SD) nm), compared to that of controls
(154 _ 11.0 nm), but thickening was even more marked in ponalrestat-treated rats (187 + 18.7
nm). However, the ponalrestat-treated rats showed less renal and glomerular hypertrophy than
did untreated diabetic rats. The authors concluded that long-term ponalrestat treatment attenu-
ated diabetic renal and glomerular hypertrophy, but had no effect on diabetic glomerular
basement membrane thickening (Osterby and Gundersen, 1989). This does not augur well for
aldose reductase inhibitors in diabetic nephropathy, though the lack of impact reported above
may be restricted to ponalrestat. This rather assumes that the increase in basement membrane
thickness is a primary etiological factor in altered kidney function. However, there is some
evidence that islet transplantation in diabetic rats reverses albuminuria without attenuating the
thickening of the basement membrane (Steffes et al., 1979).
8.4. FUNCTIONAL EFFECTS OF SORBITOL PATHWAY ACTIVITY AND ALDOSE REDUCTASE INHIBITORS
Flux through the pathway can be demonstrated biochemically in isolated glomeruli from
rat kidney (Beyer-Mears et al., 1984). The aldose reductase inhibitor, sorbinil, binds with realistic
kinetics to isolated glomeruli (Cohen and Klepser, 1985). The activity of the pathway in
glomeruli has been linked to a reduction in ouabain-sensitive (presumptive Na +/K -dependent)
ATPase activity. Thus, this ATPase activity was reduced in glomeruli from diabetic rats unless
they were treated with sorbinil or insulin (Cohen et aL, 1985). Although, this observation
supports the general 'myo-inositol/sorbitol' hypothesis (see Section 9), its relevance to the
functional parameters of nephropathy is unclear.
Of more direct relevance are associations between the sorbitol pathway and microalbuminuria
or disorders of renal hemodynamics. In diabetic rats there is clear evidence of reduced proteinuria
on treatment with aldose reductase inhibitors (Beyer-Mears et al., 1986, 1988). In a 2 year clinical
trial of sorbinil there was a statistically significant increase in albumin secretion rate (of 18.4
(range 2.6 to 64.8) #g/min) in the placebo group. In the group given sorbinil there was no
significant progression in albumin excretion rate ( + 1.3 (range -10.7 to + 20.4) #g/min)
(Jennings et al., 1990).
Renal hemodynamic effects are contradictory. In diabetic rats, one study found no effect
of ponalrestat on the raised glomerular filtration rate, in spite of pharmacological efficacy
as judged by kidney and red cell sorbitol levels (Daniels and Hostetter, 1989). However,
this study also found no effect of the aldose reductase inhibitor on the raised albumin
excretion characteristic of this diabetic model. Since others have reported diminution of
urine protein levels with other, more active inhibitors (see above), it is possible that pharmaco-
logical efficacy was not optimal. Such a postulate is supported by the prevention of a raised
glomerular filtration rate by sorbinil in rats with 2 months of streptozotocin diabetes (Pugliese
et al., 1990a). This study also reported prevention of raised urinary albumin in the sorbinil-
treated diabetic rats.
In complete contrast, a detailed study of renovascular function in long-term diabetic dogs
revealed no effect of sorbinil on the raised renal plasma flow and increased glomerular filtration
rate (Kern and Engerman, 1991).
Galactose feeding to non-diabetic rats also causes raised glomerular filtration rate which is
prevented by either sorbinil or tolrestat (Bank et al., 1989; Pugliese et al., 1990a). There are,
however, clear flaws in the efficacy of galactose feeding as a model of defects related to the
sorbitol pathway in diabetes (Willars et al., 1987b; Pugliese et al., 1990a). Without significant
proteinuria it is difficult to see relevance in the galactose-fed animal as a model of nephropathic
precursive events.
A clinical trial has revealed a modest reduction in glomerular filtration rate (mean values of
140 decreasing to 129 ml/min/L) on patients given ponalrestat, whilst the placebo group showed
no alteration (start value = 142, end value = 141 ml/min/L) (Pedersen et al., 1991).
In synopsis, we can draw a few conclusions. Firstly, it is clear that the sorbitol pathway
does exhibit exaggerated flux in the kidney in both experimental and clinical diabetes. Secondly,
it is clear that this is not the root cause of renal abnormalities. There is enough evidence to
suggest some effect on glomerular structure and function, but not enough attention has been paid
to effects of arterial hypertension. Studies of combined and separate treatments with antihyper-
tensive drugs and aldose reductase inhibitors should be of value.
9. NEUROPATHY
9.1. LOCALIZATION OF ALDOSE REDUCTASE IN PERIPHERAL NERVE

The polyol pathway is clearly active in peripheral nerve of diabetic rats in that sorbitol and
fructose have been found to accumulate (Gabbay et aL, 1966). The pathway has also been
demonstrated in nerves of other animal species (Calcutt et al., 1988; Sekiguchi et aL, 199 l). In nerve
samples obtained at autopsy (Ward et al., 1972; Mayhew et al., 1983) or below knee amputation
(Hale et al., 1987) elevated levels of glucose, sorbitol and fructose have been reported in material
170 D. R. TOMLINSONet al.
from diabetic patients compared to material from non-diabetic sources. Using biopsy material
others have shown wide variability in the levels of these compounds with some, but not all diabetic
patients having levels in excess of the highest levels seen in controls (Dyck et al., 1980, 1988). These
studies failed, however, to demonstrate any diabetes-related depletion of nerve free myo-inositol
characteristic of nerve from rats with acute experimental diabetes. Although significant reductions
in sorbitol (Sima et al., 1988) and sorbitol and fructose (Dyck et al., 1988) have been reported in
sural nerve biopsies following sorbinil treatment of diabetic patients, the location and extent of
aldose reductase activity cannot be determined from such studies.
The cellular location of aldose reductase activity was first sought via experiments in which
activity was measured distal to a crush injury of the sciatic nerve, allowing time for Wallerian
degeneration to remove any axonal enzyme. The lack of reduction in enzyme activity, relative to
uninjured nerve, indicated location of aldose reductase to the Schwann cell (Gabbay and
O'Sullivan, 1968). The same experiments located sorbitol dehydrogenase activity to the axon
(Gabbay and O'Sullivan, 1968). Such an approach is subject to the criticism that changes in the
Schwann cell consequent on axonal degeneration might include up-regulation of aldose reductase
expression compensating for a loss of axonal sources of activity. However, a later experiment
revealed that aldose reductase activity is not subject to anterograde axonal transport (Chandler and
Miller, 1986), implying that it is not expressed in the axon, at least under normal conditions. The
Schwann cell location has now been confirmed by both enzymic histochemistry (Orosz et al., 1981)
and immunohistochemistry (Ludvigson and Sorenson, 1980b). At greater resolution, the enzyme
is present in the paranodal cytoplasm of Schwann cells (Chakrabarti et al., 1987). It is also
demonstrable in pericytes and endothelial cells of endoneurial capillaries in peripheral nerve of BB
rats (Chakrabarti et al., 1987). More recently, intense immunostaining for aldose reductase has
been found in the paranodal region and the Schmidt-Lanterman clefts as well as in the terminal
expansions of paranodal myelin lamelle and nodal microvilli (Powell et al., 1991).
9.2. DEFECTSOF NERVE FUNCTIONIN DIABETESAND DIABETICNEUROPATHY

It is important to distinguish between the very early manifestations of abnormal nerve function
in diabetes and the chronic disorders which constitute diabetic neuropathy. The former may safely
be referred to as sub-clinical neurological dysfunction, if an umbrella heading is required. Thus,
nerve conduction velocity is frequently abnormal in diabetic patients without neurological
symptoms or signs of neuropathy and this conduction deficit may even be present at the time of
diagnosis of diabetes itself (Gregersen, 1967, 1968). These sub-clinical abnormalities are valuable
for the early correlation of disorders of biochemistry with those of function. However, the critical
question is whether they are prognostic for the future development of neuropathic symptoms
(Fedele et al., 1980) and, hence, whether the latter may also be related to the early biochemical
defects. Correlation of symptoms and nerve conduction anomalies with structural pathology in
sural nerve biopsies showed that abnormal vibration sense and conduction velocity showed
predictive value for structural degeneration and, therefore, for diagnosis of neuropathy (Dyck et al.,
1985b).
The slowing of peripheral nerve conduction velocity is clearly of metabolic origin (Gregersen,
1967, 1968) and appears to exhibit similar incidence and metabolic pathogenesis in patients and
in diabetic models (Greene et al., 1975). It is likely that a degree of conduction slowing persists
but evolves, at least in some patients, from a reversible metabolic phenomenon into a structurally-
based dysfunction, which has not proved reversible with interventions currently available. We do
not yet know whether these two superficially similar phenomena--acute and chronic slowing of
conduction--are related.
Another abnormality of nerve function, which appears early in diabetes, is increased resistance
to ischemic or hypoxic conduction block. When the blood supply to the limbs of diabetic patients
is occluded, their nerves continue to function for a significantly longer period than those of
non-diabetic controls (Steiness, 1959; Gregersen, 1968; Seneviratne and Peiris, 1968). The reasons
for this are unclear but an increased availability of glucose-derived energy substrate (Steiness, 1961;
Price et al., 1987) and an adaptation to incipient endoneurial hypoxia (Newrick et al., 1986;
Hampton et al., 1989) are the main candidates in the etiology of this phenomenon.
There is a range of discrete conditions which collectively represent symptomatic neuropathies

attributable to the pre-existence of diabetes. The commonest is distal symmetrical sensory
polyneuropathy, though its prevalence has been documented over a very wide range. This has
resulted from several factors including patient selection, criteria for diagnosis of neuropathy and
sensitivity of detection methods. Pirart (1978a,b) demonstrated clinical evidence of neuropathy in
7.5% of diabetics at time of diagnosis increasing to 45% at 20-25 years of diabetes. A more recent
study showed that even after the exclusion of patients requiring treatment for diabetic somatic or
autonomic neuropathy, 39% of the 278 well characterized Type 1 patients had clinically detectable
peripheral neuropathy (The DCCT Research Group, 1988).
The symptoms of distal symmetrical sensory polyneuropathy begin at the feet and hands and
spread proximally giving the 'stocking and glove' pattern, indicating that longer fibers are more
vulnerable. Numbness, paresthesiae and pain are the most frequent symptoms. Loss of pain and
temperature sensation may lead to foot ulceration and neuropathic joint degeneration. The
symptoms of such 'sensory' polyneuropathy have been found to vary in onset and severity (Pirart,
1978a,b; Thomas and Lascelles, 1966).
Diabetic neuropathy may be prevented or its severity lessened by long term maintenance of blood
glucose within accepted limits (Ziegler et al., 1988, 1991). However, even with the strictest
diet/exercise/treatment regimen and dedicated patient compliance, perfect blood glucose control is
very difficult to attain. Thus, the value of a therapeutic intervention which might attenuate any
metabolic input to the pathogenesis of neuropathy gave the development of aldose reductase
inhibitors a boost and neuropathy has become the prime complication targeted by the developers
of these drugs.
9.3. EXPERIMENTALSTUDIESWITHALDOSEREDUCTASEINHIBITORS
9.3.1. Functional Abnormalities

Aldose reductase inhibition was first demonstrated to prevent the development of slowed nerve
conduction in experimental diabetes in 1982 using the experimental inhibitor, ICI 105552
(Tomlinson et al., 1982). Since that time many studies have examined the effects of different
inhibitors on different defects. The salient findings are grouped here in sections relating to the main
inhibitors studied.
Studies with sorbinil began with the demonstration that it could prevent the development of
deficient motor nerve conduction velocity in diabetic rats (Yue et al., 1982). This was followed by
exploration of the relation between the conduction deficit and biochemical changes in the nerve.
For example, Gillon et al. (Gillon and Hawthorn, 1983; Gillon et al., 1983) carried out two studies
demonstrating effects on nerve polyols and sugars and motor nerve conduction velocity in
streptozotocin-diabetic rats. In the first study, sorbinil treatment at 20 mg/kg/day for 2 and 10
weeks prevented the accumulation of sorbitol and fructose and also the depletion of myo-inositol
in the sciatic nerve. At 10 weeks motor nerve conduction velocity was decreased in untreated
diabetic animals and this decrease was attenuated by sorbinil. In the second, a reversal study, rats
which had been diabetic for six weeks duration were treated with sorbinil at 25 mg/kg/day for 3,
6 and 9 weeks. This dose of sorbinil restored increased sorbitol, decreased myo-inositol and the
decreased motor nerve conduction velocity to control values at 9 weeks. ICI 105552 also prevented
both the reduced myo-inositol and motor nerve conduction velocity in diabetic rats (Mayer and
Tomlinson, 1983). Sorbinil prevented polyol accumulation and myo-inositol depletion in rats with
2 weeks of either diabetes or galactosemia, again implying that myo-inositol loss from nerves is
causally related to sorbitol and galactitol accumulation (Yue et al., 1984).
Thus, the hypothesis emerged that sorbitol accumulation led to decreased myo-inositol content
of the nerve which would, in some way, reduce nerve conduction velocity in diabetes. However,
subsequent work (Cameron et al., 1986) showed that nerve myo-inositol levels in streptozotocin-
diabetic rats were reduced by 45% after three months of untreated diabetes but were normal after
six months. In contrast, tibial motor nerve conduction velocity was significantly lowered in diabetic
rats at six months of diabetes. Sorbinil treatment (25 mg/kg/day) prevented the 10-fold increase
in nerve sorbitol found in diabetes and produced a 60% improvement in tibial motor nerve
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
oo_,
o
20
Control Dlabetl Dlabetlc-ARl

I I I I J
0 10 20 30 40 50
Time (minutes)
FlG. 2. Resistance to hypoxic conduction failure and motor nerve conduction velocity in
control and diabetic rats with and without aldose reductase inhibition. The main graph shows
the decline in compound action potential (CAP) amplitude over 45 min of hypoxia (8% 02)
in sciatic nerves from control rats (squares), diabetic rats untreated (circles) and diabetic rats
given ponalrestat at 40 mg/kg/day by dietary admixture (triangles). These measurements were
made in vitro after 4 weeks of diabetes. The inset bar chart shows sciatic-tibialis motor nerve
conduction velocities (MNCV) measured in vivo in the same animals 3 days previously. Limit
bars indicate + 1 standard deviation. See Calcutt et al. (1991) for full details of experimental
procedures.
conduction velocity. Thus, the m o t o r nerve conduction velocity defect at six months of diabetes
was dissociated from a myo-inositol decrease. Nerve morphometry was carried out in this study
and axon area was reduced in untreated diabetic rats compared to age-matched controls, whereas
sorbinil treated animals showed normal age-related axon growth. Myelin area was increased in
untreated diabetic rats but the increase was prevented by sorbinil. These results indicate that a
failure of normal maturation is a major cause of reduced conduction velocity in the tibial nerves
of diabetic rats of six months duration. However, it must be stressed that although the
morphometric profile of diabetic nerves was normalized at 3 months by sorbinil, conduction
velocity improvements were not substantial until six months. This suggests that other non-morpho-
logical factors contribute to reduced conduction velocity in diabetes. It has been suggested (Greene,
1986a,b) that the mechanism by which sorbinil normalizes the decreased m o t o r nerve conduction
velocity in acute experimental diabetes is by restoration of Na /K -ATPase activity and that this
may be via normalization of decreased myo-inositol content of the nerve. Administration of sorbinil
(20 mg/kg/day) for 8 weeks was also reported to normalize myo-inositol levels and N a + / K -
ATPase activity in streptozotocin rat superior cervical ganglia (Greene and Mackway, 1986). Thus,
sorbinil-responsive defects in diabetes may be seen in both the somatic and autonomic nervous
system.
These results suggest that myo-inositol depletion may be important in the early development of
neurological dysfunction in diabetes but morphological changes become the determining factor as
the disease progresses. Such documented effects on both nerve structure and function in
experimental diabetes have been used to help explain some of the findings of clinical trials with
sorbinil.
Experiments with ponalrestat have demonstrated its effectiveness as an aldose reductase inhibitor
and its ameliorating effect on the decreased m o t o r nerve conduction velocity of the sciatic nerve
in experimental diabetes (see Fig. 2). One study in rats reported prevention of the diabetes-
associated decline in m o t o r nerve conduction velocity with ponalrestat, administered for 10 weeks
at a dose of 3.125 mg/kg/day and this effect was particularly evident from day 42 onwards (Stribling
et al., 1985). A more extensive study assessed the effects of aldose reductase inhibition with
ponalrestat, at a dose of 25 mg/kg/day, on nerve conduction deficits in a variety of nerves in the
diabetic rat (Cameron et al., 1989a). Both prevention studies (2 or 4 months duration) and reversal
studies (2 months untreated followed by 2 months treated) were carried out. Large reductions in
conduction velocity (22-29%) were seen in the fast nerves supplying the calf muscles and sensory
saphenous nerve in diabetes and these were prevented by ponalrestat. In the reversal group,
restoration of conduction varied between nerves ranging from 100% in the sensory saphenous to
25% in the soleus motor branches. It was suggested that axon growth may be limited in diabetes
and that this is corrected by ponalrestat. It was also implied that this ARI-induced conduction
improvement may have resulted from the prevention of hypoxia, which has been implicated in the
etiology of diabetic neuropathy (Dyck, 1989; Dyck et al., 1986; Malik et al., 1990). Further evidence
that ponalrestat may improve the supply of oxygen to the nerve came from the attenuation of the
resistance to ischemic conduction block (after death) by ponalrestat in 4 week streptozotocin-
diabetic rats (Price et al., 1988a). Direct measurement of the rate of decline of the sciatic nerve
compound action potential, under hypoxic conditions in vitro, gave similar results for the effects
of ponalrestat (see Fig. 2). After 40 min of hypoxia, the decrease in compound action potential
amplitude from initial values in ponalrestat-treated diabetic rats resembled more closely that seen
in control nerves than in nerve from untreated diabetic rats. This resistance to ischemic or hypoxic
conduction block may be explained, in part, by the adaptation of diabetic nerves to an incipient
endoneurial hypoxia. Indeed several studies have reported a reduced sciatic nerve blood flow in
diabetic rats (Tuck et al., 1984; Low et al., 1984; Monafo et al., 1988). Although ponalrestat may
have reduced the resistance to ischemic or hypoxic conduction block by improving oxygen delivery
to the nerve during the period of diabetes, little is known of the effects of aldose reductase inhibition
on nerve blood flow. To date there is only one study in this area (Yasuda et al., 1989) and this
demonstrated that epalrestat prevented reduced sciatic nerve blood flow in diabetic rats.
Of perhaps more relevance to the development of distal axonopathy are effects of aldose
reductase inhibitors on the delivery of vital components of axoplasm to the distal axon by axonal
transport. The effects of ponalrestat on these phenomena have been addressed in several studies
(Tomlinson et al., 1986; Mayer et al., 1984; Whiteley et al., 1986; Willars et al., 1987a, 1988;
Ekstrrm and Tomlinson, 1989). Ponalrestat did not affect the altered slow anterograde axonal
transport of radiolabelled protein in sciatic motoneurons of rats with short-term experimental
diabetes (Tomlinson et al., 1986). Again, ponalrestat had no effect on the defect in accumulation
of both anterograde and retrograde axonal transport of phosphofructokinase in short-term
streptozotocin-diabetes in the rat (Willars et al., 1987a). However, six months of treatment with
ponalrestat to diabetic rats did prevent the diabetes-associated reduced accumulation of choline
acetyltransferase proximal to a ligature (Willars et al., 1988). Five weeks of ponalrestat treatment
was without effect on the impaired nerve regeneration after a crush in diabetic rats (Ekstrrm and
Tomlinson, 1989). These results imply that any effects of ponalrestat on axonally transported
proteins must be selective. Indeed, we are inclined to the view that the process of axonal transport
per se is probably not markedly disordered in experimental diabetes (Tomlinson et al., 1988).
Rather, the deficiencies in axonally transported components of axoplasm referred to above may
well be reflections of altered protein expression in the nerve cell body (Tomlinson et al., 1990).
Axonal regeneration from a crush site certainly requires competent anterograde axonal transport
(Singer et al., 1982; Ochs, 1984; Skene, 1984), but a failure of the process may derive from other
defects.
The use of auditory-evoked brain stem response has been employed as an alternative method
of measuring nerve conduction, as it can be used in conscious unrestrained animals. Streptozotocin-
diabetes of four weeks duration causes an increase in distal nerve transmission time of the auditory
pathway, which includes conduction along the eighth cranial nerve (Notvest and Inserra, 1987).
Oral administration of tolrestat at 20 mg/kg twice daily for four weeks prevented this diabetes-in-
duced phenomenon (Notvest and Inserra, 1987).
The Japanese aldose reductase inhibitor, epalrestat (ONO 2235) was given to streptozotocin
diabetic rats in a 14 day trial. The compound significantly improved motor nerve conduction
velocity (treated = 28.8 + 0.5 m/sec versus untreated 23.2 _ 4.7 m/sec, p < 0.01, mean + SD)
and reduced the sorbitol content of both sciatic nerve and red blood cells (Kikkawa et al., 1983).
It also improved decreased motor nerve conduction velocity in streptozotocin-diabetes of 4 to 6
weeks duration (Yasuda et al., 1989). Concomitantly it restored the decreased nerve blood flow
associated with diabetes towards normal values. These data again suggest that in short-term
JPT 54/2 D
174 D.R. TO/dLINSONet al.
experimental diabetes in the rat, aldose reductase inhibitors may increase motor nerve conduction
velocity via an improvement of the blood supply to the nerve. Epalrestat was also used to examine
the effects of aldose reductase inhibition on reduced sympathetic nervous activity and reduced
motor nerve conduction velocity in streptozotocin-diabetic rats (Yoshida et al., 1987).
Noradrenaline turnover was taken as an indicator of sympathetic nervous activity in interscapular
brown adipose tissue, heart and pancreas and motor nerve conduction velocity was measured
in the tail nerve. Sympathetic nervous activity in all tissues and motor nerve conduction
velocity in the tail nerve were significantly reduced in untreated diabetic rats when compared with
controls. All reductions were partially prevented by treatment with epalrestat.
9.3.2. S t r u c t u r a l A b n o r m a l i t i e s
Clinical features of diabetic neuropathy encompass a wide variety of presentations but it was
suggested that neuronal segmental demyelination may be a common causative link (Thomas and
Lascelles, 1966). However it is not clear whether such demyelination, observed in sural or radial
nerve biopsies, is a direct effect of the diabetic state on Schwann cell function or secondary to
axonal abnormalities. Sharma and Thomas (1974) could not demonstrate segmental demyelination
in streptozotocin or alloxan-treated rats. However, more modern studies have questioned the
validity of long-term, unrestrained diabetes due to unacceptable morbidity and mortality (Willars
et al., 1988). It is not possible to determine whether it is the sub-group of rats which survive, or
the rats which do not, that are the most representative of the condition aimed at by the model.
Furthermore, secondary morbidity is probably unrepresentative of the human condition (Willars
et al., 1988). Certainly, clear indication of distal axonai degeneration has been demonstrated in
streptozotocin-diabetic rats (Chokroverty et al., 1980, 1988).
The majority of studies, both human and experimental, have focused on the sciatic nerve as the
'model' diabetic nerve. It is the largest in diameter, making it the most easily accessible and
innervates the lower limbs, where sensory neuropathy primarily occurs. The sural nerve which
branches from the tibial nerve (the larger terminal division of the sciatic) has been used extensively,
via biopsy, for analysis of both biochemical and morphological abnormalities in diabetic humans
(Sima et al., 1988).
The most important studies have focused on the effects of aldose reductase inhibition on
ultrastructural abnormalities seen in long-term experimental diabetes. The reduced fiber occupancy,
seen in sural nerves of untreated rats with long-term streptozotocin-induced diabetes, was
prevented by ponalrestat treatment (0.023% wt/wt) for 28 weeks (Yagihashi et al., 1990).
Axon-fiber size ratio was also preserved in the ponalrestat-treated group. However, ponalrestat had
no effect on unmyelinated nerve fiber structure revealed by morphometry. Others have examined
the effects of ponalrestat treatment at 25 mg/kg/day for both four and six months on sural nerve
structure in the diabetic BB rat (Sima et al., 1990). Motor nerve conduction velocity was also
determined at these time points in the sciatic nerve and the reduction associated with diabetes was
prevented at four months but only attenuated at six months. Treatment for either duration
prevented structural abnormalities at the node of Ranvier but only partially preserved axonal
integrity. Four and six months of treatment resulted in an increase in regenerating nerve fibers in
diabetic BB rats but ponalrestat treatment of non-diabetic rats also caused increased myelinated
fiber regeneration, when evaluated using teased fiber preparations. These results suggest that
ponalrestat may have direct effects on nerve fibers as well as those mediated via inhibition of aldose
reductase. Another study from the same laboratory investigated the effects of ponalrestat treatment
for the same duration on autonomic neuropathy and vagus nerve structure in the BB rat (Zhang
et al., 1990). At four months, ponalrestat completely prevented the characteristic decrease in
heart-rate variability (an index of autonomic neuropathy) and also prevented axonal atrophy in
the vagus nerve. However, after six months of treatment, the preventative effect on heart rate
variability was only partial and the vagus nerve demonstrated an increase in regenerating
myelinated and unmyelinated fibers. The effect of ponalrestat at six months in both these studies
is difficult to explain.
The frequency of neuroaxonal dystrophy in the superior mesenteric sympathetic ganglia of rats
with 8 months of streptozotocin diabetes was increased sevenfold compared with age matched
controls (Schmidt et al., 1989). Sorbinil administration decreased, but did not completely normalize
this neuroaxonal dystrophy. Again, this suggests that sorbinil may have an effect on nerve structure
in the autonomic system.
9.4. CLINICALSTUDIESWITH ALDOSEREDUCTASEINHIBITORS

Judzewitsch et al. (1983) performed a randomized, double-blind crossover trial of sorbinil
(250 mg/day) in 39 patients without symptoms. A six week baseline period was followed by nine
weeks of sorbinil or placebo treatment, then another nine week crossover period with a three week
washout period. Conduction velocity in the peroneal motor nerve was found to be significantly
higher during the sorbinil treatment period than during placebo, though the mean difference was
small. When 20 patients were switched from placebo to sorbinil there was an increase in peroneal
nerve conduction velocity from 40.6 to 42.1 m/sec (p < 0.05). Cessation of sorbinil treatment was
associated with a decline in peroneal conduction velocity. Significant increases in nerve conduction
velocity were also seen in the median motor and sensory nerves during sorbinil treatment. These
effects of sorbinil were not related to glycemic control and washout of the drug was associated with
a decline in nerve conduction velocity in all three nerves. These results suggested that sorbinil may
improve nerve conduction velocity, but it is difficult to assess to what extent as values from control
subjects were not measured.
A sub-cohort of the patients studied in the multi-center trial reported above were examined to
determine whether sorbinil treatment affected the pain sometimes associated with diabetic
neuropathy (Jaspan et al., 1983). They found that sorbinil, given at a daily dose of 250 mg for
between 10 days and five weeks, relieved pain in eight out of eleven patients. It is, however,
notoriously difficult to demonstrate unequivocal effects of agents on pain and the trial design was
not perfect (see Jaspan et al., 1983). Pain associated with diabetic neuropathy can remit
spontaneously, so that trial design must enable demonstration of a return of painful symptoms on
crossover from drug to placebo.
A double-blind, randomized placebo-controlled crossover trial studied sorbinil (200 mg/day) and
placebo treatment for four weeks (Young et al., 1983). Baseline and washout periods were also for
four weeks, whilst placebo was administered. Treatment was evaluated by subjective pain
responses, clinical examination, vibration perception threshold, motor and sensory nerve electro-
physiology and cardiovascular reflex tests of autonomic nerve function. Only pain, tendon reflex
scores and sural nerve action potential amplitude improved significantly during sorbinil adminis-
tration.
Lewin et al. (1984) were not able to demonstrate a beneficial effect of sorbinil treatment (200 mg
daily for four weeks) on conduction velocity (both sensory and motor) in many nerves tested.
Abnormal autonomic function, subjective pain scores and duration of sleep were not improved by
sorbinil. The patients included in this study (n = 13) had symptomatic diabetic neuropathy and
sorbinil did not reverse any of these potential indicators of neuropathy over four weeks. Again,
Christensen et al. (1985) failed to demonstrate any objective neurophysiological improvements after
four weeks treatment with sorbinil at doses of 200 mg or 50 mg/day.
By this stage it was obvious that longer trials were needed to examine the efficacy of sorbinil
in treating abnormal nerve function in diabetes. Increasing the duration of treatment to 24 weeks,
however, showed that sorbinil was only of limited benefit in the treatment of diabetic nerve
dysfunction in patients with symptomatic symmetrical polyneuropathy (Fagius et al., 1985). In only
three out of nine neurophysiological tests (motor nerve conduction velocity of the posterior tibial
nerve, F-wave latency and sensory distal latency of the ulnar nerve) and one out of five autonomic
nerve function tests (heart rate variability during deep breathing) was there any significant effect
of sorbinil treatment. Again, non-diabetics were not used in this trial as controls. Any beneficial
effects that were observed were no greater than those seen in previous trials of considerably shorter
treatment periods.
A six month trial with sorbinil (125 mg/day) studied 22 diabetic patients with sub-clinical
abnormalities of nerve function (Martyn et al., 1987). Measurement of erythrocyte sorbitol
concentrations demonstrated very significant inhibition of aldose reductase activity with sorbinil
treatment but no concomitant improvement in either peripheral or autonomic nerve function was
observed.
Sundkvist et al. (1987) investigated the influence of sorbinil on both autonomic and peripheral
nerve function in male diabetic patients with decreased motor nerve conduction velocity in both
legs and arms but no symptoms of peripheral neuropathy. They used a double-blind crossover
study with a four week treatment protocol with 300 mg/day sorbinil and a four week washout
period between treatment and placebo periods. Thirteen out of 29 Type I patients had autonomic
neuropathy, as assessed by heart rate reaction to deep breathing and to tilt. Only sural nerve
function tests were found to correlate with autonomic function. Sorbinil treatment had no effect
on peripheral nerve function but did improve the capacity of lung inflation/deflation to alter heart
rate via reflex changes in vagal efferent tone. This finding implies a possible effect of aldose
reductase inhibition on vagal dysfunction. Such an effect is supported by animal work (Tomlinson
et al., 1985), but it is difficult to see how the drug could achieve differential efficacy, with vagal
improvements not supported by changes in conduction velocity. Two trials (Guy et al., 1988;
Stornello et al., 1992), both of one-year duration, showed no beneficial effect of sorbinil treatment
(250 mg/day) in clinical and neurophysiological studies.
These functional studies with sorbinil had employed all available expertise in 'classical' trial
designs but had produced little to indicate real efficacy. It is clear that acceptable functional
end-points with proven prognostic value for neuropathy will change measurably only in very long
trials. Another strategy to indicate efficacy and secure product registration was indicated with the
ultrastructural approach of Sima et al. (1988). This came with a randomized, placebo-controlled,
double blind trial of sorbinil at 250 mg/day in diabetic patients, in which the major assessment was
ultrastructural examination of sural nerve biopsies, taken at the start and after one year of the trial.
Nerves were analyzed morphometrically and sorbinil treated patients showed a 42% decrease in
nerve sorbitol and a 3.8 fold increase in the percentage of regenerating myelinated nerve fibers,
when compared with placebo treated diabetics. These sorbinil-treated patients also had quantitative
improvement in terms of paranodal demyelination, segmental demyelination and myelin wrinkling.
Thus, it was suggested that sorbinil could improve the lesions of diabetic neuropathy.
Evidence for a significant improvement of nerve function or structure by sorbinil treatment in
clinical trials was scant. Any effect of sorbinil seen on nerve function was small and seemed to be
of a similar degree in trial durations from four weeks to six months. However, sorbinil did seem
to improve the pain associated with diabetic neuropathy and this may be associated with effects
on nerve regeneration. The value of these sorbinil trials has been to pioneer study design with a
pharmacologically effective aldose reductase inhibitor. Hypersensitivity reactions to sorbinil had
been reported in most of the studies referred to above. Incidence varied, but in one study four out
of fifteen patients experienced 'an idiosyncratic reaction' (Young et al., 1983). The severity of some
of these reactions led to withdrawal of sorbinil from clinical evaluation as a systemic agent, leaving
it as a topical drug directed at corneal lesions (Datiles et al., 1990).
Ponalrestat had been introduced through experimental studies as a potent inhibitor of aldose
reductase (Forsgren et al., 1992) and clinical trials began on the basis of satisfactory toxicity studies.
Clinical studies with ponalrestat were based on monitoring of erythrocyte sorbitol levels as an index
of pharmacological efficacy and studies showed that the drug suppressed flux through the sorbitol
pathway in the human red cell (Price et al., 1988b; Airey et al., 1989). Furthermore, altered red
cell distensibility (Rillaerts et al., 1988) and ATPase activity (Umeda et al., 1989)--parameters of
sorbitoi pathway flux--were also normalized by clinical dosage with the drug.
In spite of this, trials published to date have indicated no significant efficacy against functional
end-points of neuropathy. A study by Bloom's group indicated little effect (Gill et al., 1990). A
more recent study by Florkowski et al. (1991) examined effects of six months of ponalrestat
treatment (300 mg or 600 mg daily) on both symptoms and neurophysiological parameters in
chronic symptomatic diabetic peripheral neuropathy. This longer duration of treatment had no
effect on pain, numbness or paresthesia, estimated using visual analog scores. Vibration perception
threshold and thermal threshold of various sites and posterior tibial nerve conduction velocity were
not affected by ponalrestat treatment at either dose. Autonomic neuropathy was similarly
refractory to any effects of ponalrestat. Bertelsmann et al. (199l) carried out a randomized
double-blind study with ponalrestat for 28 weeks on diabetic patients with proven autonomic
neuropathy. No significant improvements in symptom scores, neurological examination, cardio-

vascular reflexes, spectral analysis of spontaneous heart rate, pupillary light reflexes, skin
vasomotor control of the leg and sensory thresholds of the foot occurred with ponalrestat
treatment.
Direct assessment of sural nerve sorbitol levels (Greene et al., 1990) showed that the aldose
reductase inhibitor, tolrestat, was effective at clinical dosage against peripheral nerve aldose
reductase. However, functional studies using 'classical' trial design have produced no more clear
indicators for tolrestat than they did for sorbinil. No direct demonstration of reduced sural nerve
sorbitol levels has been published for ponalrestat treatment in man.
A multi-center trial of tolrestat used patients with symptomatic diabetic neuropathy (Boulton
et al., 1990). Of the four doses tested only the highest dose 200 mg/day showed any improvements
over baseline and placebo. Improvement in paresthetic symptoms were seen at one year but painful
symptoms improved in both placebo and active therapies. Both tibial and peroneal motor nerve
conduction velocities improved significantly with one year of treatment with this high dose of
tolrestat. Long term benefit (improvement at 24 weeks maintained until 52 weeks) was seen in 28%
of treated patients compared with 5% on placebo. Tolrestat was well tolerated in this study but
11% of patients taking 200 mg/day did experience dizziness.
Another study (Giugliano et al., 1991) assessed the effects of six months treatment with tolrestat
on various abnormalities of autonomic nerve function. The results were more encouraging, with
significant improvements in heart-rate responses to deep breathing, lying-to-standing and in
vibration perception threshold.
The most encouraging evidence to date of any effect of an aldose reductase inhibitor on
symptomatic peripheral neuropathy has come from a recent withdrawal study with tolrestat
(Gonen et al., 1991). Patients who had been receiving tolrestat for 4-5 years were either continued
on tolrestat (200 or 400 mg/day) or placebo for 52 weeks. Pain was found to worsen significantly
in placebo-treated patients compared to those taking tolrestat. Sensation in the finger and great
toe were improved for patients remaining on tolrestat and the effect reached significance for the
great toe at 36 weeks. Motor nerve conduction velocity, measured in the median and peroneal
nerves, worsened in the placebo-treated group by l m/sec whilst remaining unchanged in the
tolrestat-treated group.
The functional findings in this ingenious trial design were supported by a study which showed
tolrestat treatment to be associated with a reduction in structural signs of axonal degeneration,
reduced incidence of myelin abnormalities and an increase in the ultrastructural evidence of
regeneration in sural nerve biopsies (Greene et al., 1990).
10. POTENTIAL ROLE FOR ALDOSE REDUCTASE IN OTHER

DIABETES-RELATED DISORDERS
The potential use of aldose reductase inhibitors has been explored both experimentally and
clinically in areas representing the major clinical manifestations of the secondary complications of
diabetes, namely: cataracts, nephropathy, retinopathy, neuropathy and vasculopathy. However,
aldose reductase inhibitor treatment has been employed clinically in the treatment of limited joint
mobility, where the results have been equivocal (Eaton et al., 1985; Buithieu et al., 1990) and
experimentally in the prevention of some skeletal muscle functional changes in diabetic rats
(Cameron et al., 1990). In addition, the inclusion of an aldose reductase inhibitor with cultured
fibroblasts prevented a range of alterations associated with high glucose levels including reduced
proliferation, reduced thymidine incorporation in response to platelet-derived growth factor and
diminished replicative life span (Sibbitt et al., 1989). The importance of the above observations
awaits further investigation.
11. SYNOPSIS AND CONCLUSIONS

In this review we have attempted to cover most of the studies on the effects of aldose reductase
inhibitors on diabetic complications. As stated at the outset, our account is not exhaustive and we
apologize to authors who feel that their work has been neglected without justification. Our purpose
has been to amass sufficient evidence to determine whether, at the time of writing, it is possible
to ascribe efficacy to these drugs.
Studies on experimental diabetes give a picture which is reasonably clear. The value of this is
hampered by questions of validity of the experimental models, with respect to complications in
humans. However, progress on the latter has been significant by virtue of a switch from attempts
to model entire syndromes, to a concentration on specific aspects of relevant pathology.
In this way, we know that aldose reductase inhibitors attenuate or prevent slowed nerve
conduction, reduced free myo-inositol content, some deficits of axonal transport--particularly for
neuropeptides--and some aspects of distal degeneration in nerves of diabetic rats. In the same
model they prevent cataracts and keratopathy and they reduce retinal basement membrane
thickening. These occur in association with appropriate reductions in lens and retina sorbitol and
fructose, together with an attenuation of myo-inositol depletion. In the kidney there are reductions
in albuminuria, in glomerular hypertrophy and basement membrane thickening, although these
changes have little impact on disorders of renal hemeodynamics and glomerular filtration rate. In
the cardiovascular system there is clear antagonism of polyol accumulation in blood cells, but the
functional aspects of this effect on erythrocytes, neutrophils and platelets remain unclear. As
regards vascular tone and hypoperfusion, more work is needed to enable firm conclusions.
Clinically, there has been a massive concentration of effort on neuropathy. Many of the early
trials were inconclusive and the assessment of these drugs has been bedeviled by a perceived need
to begin with symptomatic patients, whose deficits may prove to be irreversible even when agents
of clearly demonstrable efficacy are available. More recently we have exciting demonstrations of
efficacy for tolrestat against ultrastructural signs of degeneration in sural nerve biopsies with
concomitant signs of stimulation of regeneration. There has also been an encouraging withdrawal
study, in which replacement of tolrestat with a placebo after 4-5 years treatment was associated
with an accelerated decline of function relative to the patients who remained on the drug. There
are few positive outcomes in other areas, though there was an impressive retinopathy trial with
epalrestat, in which drug-treated patients showed improvements of retinal structure and in the
electroretinogram.
It appears that these drugs will offer some benefit, but how extensive this will be remains to be
determined. Two are now available for clinical use in several countries and may therefore be
evaluated in the clinic. Time will tell whether the hopes that many of us hold for these agents are
to be fulfilled.
Acknowledgements--During the writing of this review, Dr G. B. Willars was a Senior Scientist of the William
Harvey Research Institute, funded by ONO Pharmaceuticals and Dr A. L. Carrington was in receipt of a
British Heart Foundation Postdoctoral Fellowship. We also thank Carol Brown for committing our scribbles
to word processor.
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Aldose Reductase Inhibitors

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Pharmac. Ther.Vol. 54, pp. 151-194,1992 0163-7258/92$15.

Specialist Subject Editor: B. L. FURMAN

ALDOSE REDUCTASE INHIBITORS A N D

DAVID R. TOMLINSON, GARY B. WILLARS a n d ANNE U CARRINGTON

Abstract--Aidose reductase inhibitors impede flux of glucose through the sorbitol

followed by conversion of the sorbitol to fructose by sorbitol dehydrogenase (L-iditol:NAD

D-GLUCOSE SORBITOL D-FRUCTOSE

4.2. WHITE BLOOD CELLS AND PLATELETS

5.2. VASCULAR AND CARDIAC FUNCTION

Although disturbances of the autonomic nervous system influence aspects of cardiovascular

the increased basement membrane permeability as a consequence of a reduced proteoglycan content

6.2. ENDOTHELIALCELL TIGHT JUNCTIONS

8.1. DISTRIBUTIONOF ALDOSE REDUCTASE

8.2. THE PHYSIOLOGICALROLE OF ALDOSE REDUCTASE IN RENAL OSMOREGULATION

8.3. DIABETIC NEPHROPATHY

9.1. LOCALIZATION OF ALDOSE REDUCTASE IN PERIPHERAL NERVE

9.2. DEFECTSOF NERVE FUNCTIONIN DIABETESAND DIABETICNEUROPATHY

There is a range of discrete conditions which collectively represent symptomatic neuropathies

9.3.1. Functional Abnormalities

Control Dlabetl Dlabetlc-ARl

9.4. CLINICALSTUDIESWITH ALDOSEREDUCTASEINHIBITORS

neuropathy. No significant improvements in symptom scores, neurological examination, cardio-

10. POTENTIAL ROLE FOR ALDOSE REDUCTASE IN OTHER

11. SYNOPSIS AND CONCLUSIONS

DURANTE,W., SEN,A. K. and SUNAHARA,F. A. (1988) Impairment of endothelium-dependent relaxation in

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