TRANSACTIONS OF THE AMERICAN CLINICAL AND CLIMATOLOGICAL ASSOCIATION, VOL.

118, 2007

REGULATION OF KIDNEY FUNCTION AND METABOLISM:

A QUESTION OF SUPPLY AND DEMAND

ROLAND C. BLANTZ, M.D. and (by invitation) AIHUA DENG, M.D.,

CYNTHIA M. MIRACLE, M.D., SCOTT C. THOMSON, M.D.

LA JOLLA, CALIFORNIA

ABSTRACT
Kidney blood flow and glomerular filtration rate (GFR) are maintained
relatively constant by hormonal influences and by efficient autoregulation.
However, the kidney remains at risk for ischemia and acute kidney injury.
Increases in kidney blood flow cause parallel increments in GFR, thereby
dictating tubular reabsorption and increased oxygen/metabolic demands.
Coordination between kidney blood flow and GFR with tubular reabsorp-
tion is maintained by the tubuloglomerular feedback (TGF) system
whereby delivery of NaCl to the macula densa varies inversely with
nephron GFR. Metabolic products, ATP and adenosine, are the mediators
of TGF via afferent arteriolar vasoconstriction, and nitric oxide; COX-2
products and angiotensin II are modulators of acute TGF responses and
temporal adaptation of TGF. Oxygen requirements and metabolic effi-
ciency of Na transport in the kidney are significant variables that are
regulated by both mediators and modulators of TGF. These metabolic and
hormonal substances efficiently regulate both kidney supply and demand.

Introduction
The kidney must be normally viewed as the champion among organs
of the body when it comes to stability of organ blood flow and, in the
case of the organ itself, stability of glomerular filtration rate. This
regulation is normally attributed to a) highly efficient autoregulation
of kidney blood flow in response to normal variations in systemic blood
pressure (1), b) a multiplicity of complex interacting humoral vasocon-
strictor and vasodilator influences (2,3) and, c) tubuloglomerular feed-
back system, an intrinsic system which interrelates tubular reabsorp-
tion and the rate of glomerular filtration via regulation of kidney
vascular resistances (4 – 6). In spite of this plethora of regulatory
devices, the kidney remains at risk of ischemia and persistent high
likelihood of clinical acute kidney injury (AKI) or acute renal failure.
From the University of California, San Diego and VASDHS, La Jolla, CA.
Correspondence: Roland C. Blantz, M.D., UCSD & VASDHS, 3350 La Jolla Village Drive
(111-H), San Diego, CA 92161. 858-552-7528; 858-552-7549 fax; rblantz@ucsd.edu
23
24 ROLAND C. BLANTZ, M.D. ET AL

This clinical observation raises further questions regarding the ade-

quacy of regulation of oxygen and substrate delivery and the utiliza-
tion of oxygen/substrate or the metabolic demands of the kidney (7,8).
These apparent conflicting observations can be resolved when cer-
tain facts are considered. Glomerular filtration rate (GFR) is highly
plasma flow dependent, changing more or less in parallel in normal
conditions (3,9). This linkage means that maintenance or increases in
renal blood flow (RBF) not only satisfy the supply of oxygen and
substrate but also dictates maintenance or increases in GFR, which is
largely reabsorbed by kidney tubules, a process which requires ATP,
oxygen and substrate. This makes the relationship highly complex,
since restoration or increases in kidney blood flow not only augment
metabolic supply but also increase metabolic demands required for
increased tubular reabsorption (7). Normally, the arterio-venous oxy-
gen difference in the kidney is small, implying a significant oxygen and
substrate reserve for prevention of ischemia. However, the presence of
a preglomerular countercurrent oxygen diffusion shunt produces a
kidney environment which is relatively hypoxic (10); the pO2 in the
kidney cortex is normally low at 40 – 45 mmHg (11) and progressively
even lower in the medulla and papilla (8,12). In certain experimental
conditions, diabetes and hypertension, the pO2 in the kidney cortex
may be even lower (11,13). Therefore, any consideration of the risk of
kidney ischemia must take into account both oxygen and substrate
delivery and adequacy of kidney blood flow and the glomerular filtra-
tion rate, but also the metabolic demands for tubular reabsorption.
In the kidney there is a unique linkage between a) blood flow and
GFR (3,9) and, b) tubular reabsorption provided in major part by the
tubuloglomerular feedback (TGF) system and by glomerulo-tubular
balance (5,6). The TGF relationship dictates a negative feedback sys-
tem which predicts that increases in delivery and reabsorption at the
macula densa (MD) dictate a reduction in the filtered load via reduc-
tions in kidney blood flow (Figure 1). The mediators of TGF, ATP and
adenosine, a product of AMP, are metabolically linked (5,14 –16), and
the modulators of TGF activity, nitric oxide (NO) from nitric oxide
synthase-1 (NOS-1) (17–19), angiotensin II (AII) (20) and, potentially,
cyclooxygenase-2 (COX-2) products (21) also exert major metabolic
impacts (Figure 2). Prior investigations have shown a linear relation-
ship between kidney oxygen consumption (QO2) and tubular reabsorp-
tion, (22,23) and, therefore, GFR, with a positive y intercept which
defines the metabolic demands of the non-filtering kidney. However,
more recent observations have suggested that this relationship be-
tween kidney Na reabsorption and oxygen consumption is highly vari-
 KIDNEY FUNCTION AND METABOLISM 25

FIG. 1. Schematic operation of the tubuloglomerular feedback system (TGF). In-

creases in delivery and reabsorption of NaCl at the macula densa cell elicits vasocon-
striction of the afferent arteriole resulting in a reduction of renal blood flow and the
glomerular hydrostatic pressure as well as reductions in the glomerular ultrafiltration
coefficient, Kf. The net effect of this TGF response is to reduce glomerular filtration rate.
This reduction constitutes a negative feedback system which returns tubular fluid
delivery and reabsorption of NaCl at the macula densa to control levels. The net effect of
this TGF system is to stabilize late proximal flow rate, augment autoregulation of renal
blood flow (RBF) and prevent major urinary losses of NaCl and water by overwhelming
distal tubular reabsorptive capacities.

able, affected, in major part, by hormonal influences which also regu-

late kidney blood flow and TGF (11,12,19,21,24 –27). Mediators and
modulators of TGF function are either critical metabolic byproducts or
regulators of metabolism (5). Therefore, kidney ischemia is dictated by
regulation of both metabolic supply and demand, and the regulatory
factors may be similar hormonal/metabolic systems, which greatly
simplifies these biologic processes.

Methods
Studies were performed in Wistar and Wistar Frömter rats, the
latter with surface glomeruli and raised in a colony at the San Diego
VA Animal Research Facility. These studies were conducted in con-
formance with the local VA/UCSD IACUC protocol (A3659-01) dated
November 5, 2005.
General micropuncture. Under terminal Inactin anesthesia, the kid-
ney is placed in a cup and covered with saline solution, and the
configuration of distal tubules is identified by inserting a micropipette
(1–3 ␮m tip) into urinary space of surface glomeruli to inject small
boluses of stained artificial tubular fluid (ATF). A pipette is inserted
into the last proximal or first distal accessible tubular loop to perform
26 ROLAND C. BLANTZ, M.D. ET AL

FIG. 2. The signal transduction mechanism for transmission of the macula densa
tubuloglomerular feedback signal to the afferent arteriole and glomerulus. Increased
delivery of fluid to the macula densa promotes increased NaCl reabsorption. NaCl
reabsorption causes the immediate release of ATP, which is degraded extracellularly to
AMP and, specifically, further degraded by ecto-5⬘-nucleotidase (e-5⬘-NT) and to aden-
osine, a substance which is a vasoconstrictor at the afferent arteriole. Increased NaCl
reabsorption in the macula densa cell also causes generation of nitric oxide and may
release prostaglandins via COX-2. In addition to acting as primary vasodilators which
counteract the effects of adenosine and ATP, NO also inhibits e-5⬘-NT thereby reducing
the generation of adenosine. NaCl transport at the macula densa also modifies the
generation of angiotensin II (AII) via renin. NO and COX-2 are also modulators of AII
generation. ATP and adenosine mediators of the acute tubuloglomerular feedback re-
sponse, and NO, prostaglandins from COX-2, and AII are further modulators of the
tubuloglomerular feedback response and contribute to resetting or adaptation of tubu-
loglomerular feedback.

timed (2–3 min) collection of tubular fluid under free-flow conditions,

using a short mineral oil block (28). Tubular fluid collections are
analyzed for tubular flow rate and Na⫹, K⫹, and Cl⫺ concentrations as
well as 3H-inulin concentration to determine single nephron glomeru-
lar filtration rate (SNGFR). Absolute and fractional reabsorption of
fluid or Na⫹, K⫹, and Cl⫺ between the glomerulus and the late prox-
imal or early distal tubule are calculated as previously described
(28,29).
Analysis of proximal reabsorption. When proximal reabsorption
(Jprox) changes due to a change in SNGFR, this is normal glomerulo-
tubular balance (GTB). When Jprox changes due to a change in the
avidity of the tubule, this constitutes a “primary” change in tubular
reabsorption. To test for primary changes in tubular reabsorption it is
 KIDNEY FUNCTION AND METABOLISM 27

necessary to control for the influence of GTB (29,30). We accomplished

this by using TGF as a tool for manipulating SNGFR. SNGFR will be
manipulated by perfusing the nephron downstream from a late prox-
imal wax block. SNGFR and Jprox will be determined by late proximal
collections during perfusion at 8 and 40 nl/min and Jprox expressed as
a function of SNGFR.
Micropuncture analytic methods. Late proximal flow (VLP) and early
distal tubular flow rate (VED) are determined after transferring the
tubular fluid collections to a constant bore capillary. We also examined
changes in electrical conductivity of the early distal nephron fluid
(TED) measured continuously as a legitimate surrogate for the ionic
content of fluid flowing past the macula densa (MD). TED is determined
from the electrical conductance across the tip of a micropipette (3–5
␮M diameter filled with 1.2 M NaCl). Tubular fluid is allowed into the
tip by gentle suction and pipette is wired to an electrical circuit such
that the rat and pipette in series constitute a segment of a voltage and
prior divider (31). Measurements of [Na⫹] and [K⫹] are expressed as a
fraction of the conductance of artificial proximal tubular fluid and
determined as described previously (29), using a micro flame photom-
eter. [Cl⫺] is measured in samples with a micro adaptation of the
electrometric titration method (Microtitre ET-1; World Precision In-
struments, Sarasota, FL) (29). Concentrations of 3H-inulin in plasma,
tubular collections, and urine are measured by liquid phase scintilla-
tion counting.
Glomerular hemodynamics. In certain micropuncture studies, glomer-
ular hemodynamics were evaluated by measuring glomerular capillary,
urinary space, and efferent arteriolar hydrostatic pressure (32,33). In
conjunction with efferent arteriolar protein samples (approximately
three per period) and SNGFR, we can compute nephron plasma flow, the
ultrafiltration coefficient (LpA) and effective filtration pressure.
Micropuncture analysis of tubuloglomerular feedback and glomeru-
lotubular balance Conceptual framework for TGF. Briefly, the TGF
system operates as a negative feedback loop in which two parameters
(SNGFR and VLP) are linked by TGF and glomerulotubular balance
(GTB). In the first type of experiment, SNGFR and VLP are measured
during orthograde perfusion of Henle’s loop in wax-blocked nephrons.
Measurements are made in each nephron during high and low rates of
loop perfusion. These data establish a range for the TGF response. The
second type of experiment involves measuring the fractional compen-
sation for flow perturbations in free-flowing nephrons in order to
reveal the behavior of TGF near the natural operating point. Data from
these two sets of experiments are combined to calculate the slope of the
28 ROLAND C. BLANTZ, M.D. ET AL

TGF curve to generate complete TGF curves, and to predict the impact
which a primary change in tubular reabsorption will have on SNGFR
by altering the TGF stimulus.
TGF range and operating point SNGFR. SNGFRmax refers to
SNGFR during perfusion of Henle’s loop at 8 nl/min. SNGFRmin refers
to SNGFR during perfusion at 40 nl/min. SNGFRmax minus SNG-
FRmin equals the range. SNGFR at the TGF operating point is deter-
mined by collection from the early distal tubule and is referred to as
SNGFRd. SNGFR at the inflection point of the sigmoid TGF curve is
simply the average of SNGFRmax and SNGFRmin or SNGFRmid. As
an index of TGF activation, SNGFRd is compared with SNGFRmid.
SNGFRd-SNGFRmid will increase when conditions external to the
juxtaglomerular apparatus (JGA) influence SNGFRd to increase or
when conditions within the JGA influence TGF to reset downward.
Measurement of TGF efficiency in free-flowing nephrons. Ambient
proximal flow and the fractional compensation for perturbations in
ambient flow are measured in free-flowing late proximal nephrons
(6,34 –36). Tubular flow is measured by a non-invasive optical tech-
nique videometric flow velocitometry (VMFV). A Hample nanoliter
pump is employed to perturb flow. Tubular flow, VM, is measured just
upstream from the pump pipette. Perturbations, VH, are applied by
adding or subtracting fluid in the following sequence: VH(nl/min) ⫽ 0,
⫺4, ⫹4, ⫺8, ⫹8, and 0. Each perturbation is for three minutes and
data are collected over the last 60 seconds. VM is expressed as a
function of VH by polynomial curve fitting or linear regression and
fractional compensation at the operating point is calculated from C ⫽
(⫺dVM/dVH) for VH ⫽ 0.
Videometric flow velocitometry (VMFV). Small boluses of artificial
tubular fluid containing rhodamine B dextran as a fluorescent marker
are injected into the proximal tubule by a pneumatic microinjection
pump. The dye is excited by a laser reflected onto the kidney surface.
The image of the kidney surface is filtered to maximize resolution of
emitted fluorescence and monitored with a video microscope. The
intensity of the video image is monitored at two points downstream
along the nephron. The time required for fluid to traverse the two
points is calculated by cross-correlating the video densities. Geometry
of the tubular segment is measured from digitized images. Tubular
fluid flow rate, VM, is calculated from the cross-correlations and the
geometry of the tubule.
Enzyme assays for 5⬘-NT. A sieving technique is used to separate the
glomeruli from the tubules by gently pressing the cortex through a 106
␮m stainless steel sieve. The passed through suspension is forced
 KIDNEY FUNCTION AND METABOLISM 29

through an 18 gauge needle to decapsulate the glomeruli and then

passed through a 75 ␮m sieve. The glomeruli are trapped on the top
while the tubules pass through. 20 ␮l of each sample are added to a
tube containing 20 ␮M Dipyridamole, 10 ␮M EHNA, and either buffer
or 50 ␮M, aß-methylene adenosine diphosphate (MADP). MADP is
supposed to inhibit plasma membrane bound ecto- and endo-5⬘-NT but
not AMP-specific cytosolic 5⬘-NT. This mixture is incubated for 10
minutes at 37°C in a shaking water bath. 14C AMP and 10 ␮M AMP or
IMP were added and the incubation continued for 15 minutes. 5 ␮l of
17 N Formic acid is added to stop the reaction. The reaction tubes are
spun down and a portion of each reaction supernate is separated by
thin layer chromatography on cellulose sheets with fluorescent indica-
tor in water: methanol (1:1). The reaction product (adenosine) and
substrate (cold AMP) are located by nonradioactive markers. The spots
are marked, cut out and quantitated by liquid scintillation counting.
Plasma membrane bound ecto- and endo-5⬘-NT activity represents the
MADP-inhibitable phosphatase activity (37).
Whole kidney clearance and O2 consumption studies. The rats are
prepared for surgery as previously described (6,35), and animals anes-
thetized with Inactin (100 mg/kg i.p.). Rats are maintained with sys-
temic infusion of 1.5 ml/hr of isotonic NaCl/NaHCO3 containing appro-
priate amounts of 3H inulin 5 ␮Ci/hr for clearance studies and 60 – 80
␮Ci/hr for micropuncture evaluation of single nephron filtration rate
(SNGFR) (6,14). After anesthesia, rats are placed on a thermostatically
controlled surgical table to maintain body temperature at 37°C. After
surgery, the left renal artery is then separated from the renal vein
through a midline incision plus a flank extension. The left kidney blood
flow (ml/min) is monitored with a perivascular ultrasonic transit time
flow probe (Transonics T206, Ithaca, NY, USA) connected to a com-
puter for continuous recording (26,27). A 23 gauge needle is bent at 90°
and inserted into the proximal left renal vein for sampling of venous
blood (19). GFR (ml/min) is measured by clearance of 3H-inulin or
calculated from RBF, hematocrit (HCT) and filtration fraction (FF)
where FF is calculated according to the Fick principle from 3H-inulin
content of arterial (RA) and renal venous (RV) plasma: FF ⫽ 1 ⫺
(RV/RA). Oxygen consumption is computed from A-V difference in O2
multiplied by RBF. The cost of sodium transport (QO2/TNa) is the ratio
of total amount of renal QO2 over the total amount of sodium reab-
sorbed (TNa). TNa is equal to the total amount of Na filtered minus the
amount of Na excreted. In certain studies we examined GFR and renal
plasma flow (PAH clearance) in awake, chronically catheterized rats as
previously described.
30 ROLAND C. BLANTZ, M.D. ET AL

In vitro assessment of proximal tubules. Isolation and purification of

rat renal proximal tubules are performed according to previously pub-
lished methods (26,27,38) with some modifications. The rat kidneys
are first flushed with 40 ml cold oxygenated perfusion buffer contain-
ing (in mM, pH 7.1) NaCl: 112, NaHCO3: 20, KCl: 5, CaCl2: 1.6,
Na2HPO4: 2, MgSO4: 1.2, glucose: 5, HEPES: 10, mannitol: 10, glu-
tamine: 1, sodium butyrate: 1, and sodium lactate: 1 and then perfused
with 30 ml warm collagenase containing buffer (2 mg/ml in perfusion
buffer, Collagenase Type II, Worthington) for in situ digestion within
10 min. The dissected and chopped cortex were submitted to another 5
min enzyme digestion. After straining through a sieve (pore size 125
␮m), the tubules are washed and purified by 50% Percoll centrifuga-
tion. Oxygen consumption (QO2, pmole/min/␮g protein) is measured
polarographically in a 0.6 ml chamber (Instech Laboratories, Inc.) with
a water jacket maintained at 37°C by using a Clark-type oxygen
electrode and an YSI model 5300 oxymeter (Yellow Spring Instru-
ment). The reaction chamber is filled with chamber buffer and a
baseline recorded. Then 0.1 ml of the tubular suspension is added to
the chamber for QO2 measurements. Once a stable recording (basal
QO2) is achieved various reagents were added into the chamber via a
port on the upright side of the chamber using an extra long tip to allow
the deposition of the drugs near the bottom of the chamber with
continuous tracing recording. QO2 is calculated assuming that the
concentration of O2 in the buffer under saturating conditions at 37°C is
544 nmol/ml as described in a recent publication (27).
Radioimmunoassay of Ang II. Plasma is extracted using a Bondelut
C18 column (Varian, Harbor City, CA) previously washed with meth-
anol and a triethylamine and formic acid buffer. The Ang II is eluted
with acetonitrile:triethylamine/formic acid (70/30), lyophilized on a
Speed-Vac (Savant, Farmingdale, NY) overnight, and kept at ⫺20°C
until assayed. This plasma extraction yielded 98.5% recovery of Ang II.
Details of RIA are provided in recent publications (20,39). In the case
of nanoliter volumes (100 –200 nl) of tubular fluid, no extraction is
required and the sample is directly analyzed as described (20). Ang II
concentrations were calculated using a computer-aided logit/log trans-
formation of the standard curve. Cross-reactivity of this Ang II anti-
body with angiotensin I is 0.33% and with Ang III is 69%.

Results and Discussion

The kidney exhibits extraordinary efficiency of blood flow autoregu-
lation and a multiplicity of overlapping neurohormonal factors that
 KIDNEY FUNCTION AND METABOLISM 31

combine to regulate kidney blood flow and glomerular ultrafiltration

(1–3,5,6), but in spite of these attributes, acute kidney injury or acute
renal failure remains a major clinical problem. Such a finding may
imply a breakdown in the balance of supply and demand of oxygen and
substrates (7). Experimental findings would suggest that the tubulo-
glomerular feedback system normally provides exquisite coordination
between a) kidney blood flow and load of glomerular filtrate and, b)
tubular reabsorption (4 – 6,14). The TGF system adapts over time using
complex hormonal modulators (17–21). The mediators and modulators
of temporal adaptation of TGF are either substances critical to the
metabolic processes or regulators of metabolic rate (7,24 –26).

Tubuloglomerular feedback system: The mediators and the

modulators
Increases in flow rate to the macula densa (MD) and NaCl reabsorp-
tion at the MD elicits reduction in the filtered load to that nephron unit
(4 – 6), mediated primarily by constriction of the afferent arteriole
(40,41). This TGF effect tends to stabilize late proximal tubular flow
rate, prevents overload of distal tubular reabsorptive capacities and
contributes to autoregulation (Figure 1). Such a linkage between tu-
bular reabsorption and filtered load had been demonstrated repeatedly
both in vitro and in vivo, the latter via alterations in the MD flow at the
single nephron level and with whole kidney studies in which proximal
tubular reabsorption is inhibited with application of agents such as
carbonic anhydrase inhibitors (17,19,21,27,42,43). The mediators of
this acute TGF response have been investigated extensively, both in
vitro, in the perfused MD, glomerulus and afferent arteriolar prepara-
tion, and in vivo using micropuncture and pharmacologic blockade.
Concurrent with increases in MD NaCl delivery and reabsorption is
the release of ATP producing afferent arteriolar vasoconstriction via
purinergic receptors from both in vitro and in vivo studies (44). How-
ever, ATP is rapidly metabolized extracellularly to AMP by nucleotide
triphosphate diphosphohydrolases to AMP. This compound is then
converted to adenosine by abundant kidney ecto-5⬘-nucleotidase en-
zyme in glomeruli, mesangial, and tubular cells (37,45) (Figure 2).
Adenosine can then produce afferent arteriolar constriction via the
adenosine A-1 receptor. Clearly, ATP release is required and may
contribute to acute TGF responses, but evidence has accumulated that
adenosine may maintain TGF elicited vasoconstrictor responses over a
longer time period (Figure 2). In vivo micropuncture experiments uti-
lizing adenosine “clamp” constraints, in which adenosine levels cannot
32 ROLAND C. BLANTZ, M.D. ET AL

change, completely eliminate TGF responses (14). In support of the

viewpoint that adenosine is a critical mediator are the findings that
mice lacking ecto-5⬘-nucleotidase exhibit greatly diminished acute
TGF responses (44,45) and mice minus the adenosine A1 receptor also
demonstrate nearly absent TGF responses (15,46). What is critical to
this discourse on ATP versus adenosine as primary mediators is the
fact that the candidate substances are major products of normal me-
tabolism, suggesting that TGF function is linked to the kidney meta-
bolic requirements for NaCl reabsorption.
TGF is a negative feedback system which exhibits a rapid response
to maintain homeostatic efficiency for the process and preserves sta-
bility of tubular flow rates. Acute TGF responses are accompanied by
generation and release of NO, primarily derived from NOS-1, located
primarily within the MD but also other renal cells (47– 49). NO, a
vasodilator, has been shown to ameliorate the intensity of the acute
TGF response. However, the TGF system cannot be truly relevant to
the control of kidney function if it remains static in spite of changes in
physiologic conditions. In order to maintain homeostatic efficiency, the
TGF system must be capable of “resetting” or temporally adapting to
new physiologic conditions. The nature of this TGF temporal adapta-
tion has been examined in vivo after inhibition of proximal reabsorp-
tion over periods from one to 24 hours (17,19,21,34,35) and in the in
vitro perfused MD/glomerulus/afferent arteriolar (AA) preparation
(36). In normal conditions, acute TGF responses persist for up to
30-minutes but then nephron blood flow and AA diameter return to
normal control values, permitting a readjustment of the relationship
between MD NaCl delivery and filtered load or afferent arteriolar
constriction, and consequent restoration of homeostatic efficiency of
TGF and the capacity to respond to further perturbations. In practical
clinical terms, this would imply that tubular injury should not lead to
permanent kidney vasoconstriction or pre-renal conditions, but that
kidney hemodynamic responses should be transient and temporally
adapt (2,17). In vitro, TGF induces afferent constriction followed
within 30 minutes by a full restoration of AA diameter (36). Studies in
vivo have addressed the factors or mediators contributing to temporal
adaptation of TGF. We have shown earlier that application of benzol-
amide, a carbonic anhydrase inhibitor which reduces proximal tubular
reabsorption by ⬃50% and activates TGF for a period of 18 –24 hours,
produces a rebound increase in kidney GFR (17) after withdrawal of
this agent, generating temporal adaptation of TGF or resetting of the
relationship of MD NaCl delivery and GFR. Application of agents
which inhibit kidney NOS-1 prevented this hyperfiltration response
 KIDNEY FUNCTION AND METABOLISM 33

and, in fact, caused a sustained reduction in GFR which is character-

istic of the acute TGF response to benzolamide. More acute studies
demonstrated temporal adaptation of kidney blood flow after benzol-
amide within 30 – 60 minutes, and this return of RBF to control values
was prevented by concurrent application of SMTC, a selective NOS-1
inhibitor (19). In a pattern identical to NOS-1 inhibition, concurrent
administration of COX-2 inhibitors also completely prevented tempo-
ral adaptation (21). It is of interest to speculate that this physiologic
effect of COX-2 inhibition to prevent normal temporal adaptation of
TGF may contribute to the NaCl retention and the hypertension fre-
quently observed with administration of COX-2 inhibitors.
The most physiologically relevant example of normal temporal ad-
aptation is the transition between low and high NaCl intakes. This
adaptation requires an adjustment of the relationship between MD
NaCl delivery and kidney GFR. In spite of this adjustment and con-
trary to earlier speculations and publications, we found in nephron
micropuncture studies that TGF maintained homeostatic efficiency
and normal acute TGF responses while rats were maintained on high
NaCl intakes (20). Reports have suggested that kidney adenosine
content is actually increased on high NaCl intake (50,51). We have
developed assays for ecto-5⬘-nucleotidase (ecto-5⬘-NT) and found that
glomerular enzyme activity increases in parallel with NaCl intake in
rats. We also observed that NO donors significantly inhibit glomerular
enzyme activity, which might provide a further mechanism whereby
NO, derived from NOS-1 could cause temporal adaptation of TGF by
decreasing adenosine formation (36).
AII is also a modulator of TGF function whereby increased AII has
been shown to increase TGF responses. AII in physiologic concentra-
tions also increases proximal tubular reabsorption (33). Multiple stud-
ies have shown that the traditional RAAS system is suppressed by
high NaCl intakes. Studies in rats on high NaCl intakes from our
laboratory also demonstrated lower plasma and kidney values for AII
by RIA (20). However, when we evaluated AII levels in proximal
tubular fluid samples we noted a 50% increase in AII in rats on high
NaCl intake (8 mEq/day) in comparison with rats on normal NaCl
intake (⬃2 mEq/day). This increase in AII in the intrarenal microen-
vironment exerts unexpected physiologic impacts in that administra-
tion of AT-1 receptor blockers produced major reductions in proximal
tubular reabsorption and significant inhibition of acute TGF responses
(20). The net effect was significantly elevated late proximal tubular
flow rates in high NaCl intake rats receiving blockers of AII activity.
These unexpected results regarding changes in AII within the proxi-
34 ROLAND C. BLANTZ, M.D. ET AL

mal tubule may help explain why patients receiving inhibitors of AII as
therapy, but are noncompliant with dietary NaCl restriction, still
appear to benefit from this therapy in terms of both natriuresis and
blood pressure control. AII also functions as an important modulator of
TGF by both influencing the magnitude of TGF during temporal ad-
aptation and the stability of proximal reabsorption and tubular flow
rates.

The metabolic costs of kidney tubular reabsorption

Oxygen consumption by the kidney has been noted to relate directly
and predominantly to Na reabsorption (22,23). Recent studies have
supplied evidence that the slope of this relationship is variable among
physiologic and pathophysiologic conditions and affected by hormonal
factors (11–13). Given the finding of temporal adaptation of TGF (17),
one might postulate that during this period there are metabolic adap-
tations and changes in tubular reabsorption that permit or justify
“resetting” of the relationship between GFR and MD NaCl delivery/
reabsorptive rate. NOS-1 is a major modulator of TGF and is critical to
temporal adaptation of TGF (17,19). NO exerts several chemical inter-
actions which influence oxidative metabolism. NO suppresses the citric
acid cycle enzyme, aconitase, and inhibits mitochondrial pyruvate
uptake, but the major “braking” effect of NO on oxidative metabolism
is accomplished via inhibition of cytochrome c oxidase (52–56). Al-
though effects are more complex than we will describe in detail, NO
can inhibit mitochondrial respiration in vitro by up to 85%. Therefore
it is possible that NO generated during temporal adaptation of TGF
subsumes functions which reduce epithelial metabolism or increase
metabolic efficiency of tubular reabsorption during this period of TGF
adaptation. There is evidence that epithelial NOS-1 and possibly NO
generated within mitochondria constitutively act as a “metabolic
brake” on tubular oxygen consumption (25,26).
Earlier studies have observed a reduction in the metabolic efficiency
of kidney Na reabsorption as well as cortical pO2 in the spontaneously
hypertensive rat (SHR) (11,57). The authors attributed the increased
QO2/TNa in SHR to a deficiency of NO generation. Laycock also dem-
onstrated in the dog that application of non-selective NOS inhibitors
produced major increases in kidney QO2 (24). Koivisto et al., also
demonstrated an effect of NO on oxygen consumption in isolated kid-
ney proximal tubules (25). We have recently demonstrated a major
increase in kidney QO2 and QO2/TNa after NOS inhibition, and that the
NO which influences QO2 is entirely derived from NOS-1, an isoform
 KIDNEY FUNCTION AND METABOLISM 35

which may be located within mitochondria (Figure 3) (26). The effects

of NOS inhibition were not influenced by blockade of AII activity via
the AT-1 receptor. There were also major increases in oxygen consump-
tion in freshly harvested proximal tubules after SMTC, an NOS-1
inhibitor, studied while in motion in a specially designed metabolic
chamber (Figure 3). These collective results raise the possibility that
NO derived from NOS-1 may function as a “metabolic brake” in kidney
epithelial cells. The increase in NOS-1 activity after TGF activation
and temporal adaptation or resetting of TGF may further amplify
these influences acting to suppress oxygen consumption and enhance
metabolic efficiency of Na reabsorption, thereby influencing both met-
abolic “supply” and “demand”.
Alterations in selective kidney transport processes, particularly in
the proximal tubule, can also change the metabolic efficiency of Na
reabsorption. The metabolic cost of Na transport in the in vivo proxi-
mal tubule is less than the more distal nephron because of the large
component of passive NaCl reabsorption set up by proton secretion and
NaHCO3 reabsorption in the early S-1 segment (58,59). Several of the
mediators and modulators of TGF also regulate components of kidney
tubular reabsorption, particularly NHE3 and NHE2 by adenosine, AII
and NO, respectively.

FIG. 3. Effects of NO and NOS-1 blockers on oxygen consumption in freshly har-

vested isolated proximal tubules. Panel A demonstrates the profile of declining O2 % in
a metabolic chamber containing proximal tubules. When the NOS-1 inhibitor, SMTC, is
added, oxygen consumption increases, designated by the increase in slope of decline.
When an NO donor, NONOate is applied, oxygen consumption decreases dramatically.
In Panel B absolute oxygen consumption is depicted in control tubules and after SMTC,
the NOS-1 blocker. The increase in oxygen consumption after NOS-1 blockade is totally
reversed by application of the NO donor, suggesting NO specificity to the phenomenon.
These data suggest a major role for intracellular NOS activity in regulation of oxygen
consumption, probably at the mitochondrial level.
36 ROLAND C. BLANTZ, M.D. ET AL

We have recently published surprising results on the in vivo and in

vitro effects of benzolamide, a carbonic anhydrase inhibitor, which
exerts its primary effects in the proximal tubule (43). This agent
reduces proximal tubular reabsorption by at least 50%, yet we ob-
served a 50% increase in kidney oxygen consumption (QO2) and QO2/TNa
increased by approximately 80% (27). We postulated that the resulting
decrease in luminal pH which accompanies carbonic anhydrase inhi-
bition somehow promotes compensating active NaCl reabsorption
while passive NaCl reabsorption is essentially eliminated. This in vivo
phenomenon was duplicated in freshly harvested proximal tubules in
vitro since NaCl transport persists in this preparation. Of interest to
this discussion, application of adenosine A-1 receptor antagonists and
NHE3 blockers of proton secretion completely normalized QO2 and
QO2/TNa. These results demonstrate again that selective alterations in
transport mechanisms can markedly influence overall oxygen costs of
kidney function, dominantly within the proximal tubule, by either
eliminating passive or promoting active transport (27,60,61). Adeno-
sine, AII, and NO have major influences on TGF function and adap-
tation and all exert major potential impacts on kidney oxygen con-
sumption and metabolic efficiency of kidney function.

Kidney oxygen consumption in pathophysiologic states

There are several pathophysiologic conditions in which increased
QO2/TNa has been observed, including models of hypertension, experi-
mental diabetes and models of chronic kidney disease, created by
reduction in renal mass (the 5/6th nephrectomy model) (11,13,57,62).
Factoring QO2 by total Na reabsorption may not be a reasonable as-
sessment of metabolic oxygen utilization by the kidney in pathophys-
iologic states, since one should also consider other uses of oxygen by a)
chemical oxidation reactions, b) gluconeogenesis, c) inefficiencies of Na
reabsorption and, d) reabsorption of other filtered molecules such as
albumin and other “middle molecules” as a result of increased glomer-
ular leak of these substances. Nevertheless such phenomena will in-
crease in demand for oxygen in pathophysiologic conditions.
In experimental diabetes, QO2 and QO2/TNa are increased in hyper-
glycemic rats (13,63). We and others have also observed increased
kidney NO generation which derives primarily from NOS-1 (64).
NOS-1 is derived from cells other than the macula densa, the TGF
sensing element or afferent limb. Blockade of NOS-1 in the diabetic
kidney produces major reduction in renal blood flow (RBF) and GFR in
contrast with little effects in control, non-diabetic rats. In addition, we
 KIDNEY FUNCTION AND METABOLISM 37

have observed a significantly greater increase in kidney QO2 after

SMTC, a NOS-1 inhibitor, in diabetic rather than non-diabetic rats
(65). Such findings suggest that QO2 and QO2/TNa are increased in
diabetic kidneys, for reasons yet to be explained, and that NOS-1
activity is increased possibly as a compensatory “braking” mechanism
in an attempt to maintain metabolic efficiency in the diabetic kidney.
In models of chronic kidney disease, pathogenetic roles for both
reactive oxygen species (ROS) and kidney hypoxia have been postu-
lated as critical to progression of disease (62). We have examined
kidney oxygen consumption in a model of chronic kidney disease, the
5/6 nephrectomy model. In this model at one week we observed a
remarkable increase in both absolute oxygen consumption (QO2) and
QO2/TNa (66). The kidney exhibited significant proteinuria at this early
stage. We have examined the effects of chronic application of Losartan,
an angiotensin II AT-1 receptor blocker. Losartan nearly doubled GFR
and RBF, implying a major increase in AII activity in this early phase
of this model of CKD. In spite this change in RBF or “supply”, oxygen
consumption remained constant and Losartan essentially normalized
QO2/TNa. In normal rats, we have found no effect of AII, AT-1 receptor
blockade, on kidney oxygen consumption (26). In the 5/6 nephrectomy
model, Losartan therapy also eliminated proteinuria. Proximal tubu-
lar reabsorption of albumin and other proteins requires expenditure of
ATP for proteasomal degradation of reabsorbed proteins. These obser-
vations raise questions as to whether some of the appreciable benefits
of inhibition of the renin-angiotensin system in CKD, particularly with
significant proteinuria, could be mediated by altering the metabolic
efficiency and increased oxygen consumption in chronically diseased
kidneys (66).
A balance between glomerular filtration and tubular reabsorption
must be maintained by the kidney. Tubuloglomerular feedback sys-
tems exert acute and temporally adapted control over the relationship
of kidney blood flow and filtered NaCl and tubular reabsorption. The
mediator and modulators of TGF have been well-defined (5,14,17). The
kidney must also maintain a proper balance between metabolic supply
and demand in order to prevent kidney hypoxia and ischemia. The
metabolic costs of kidney reabsorption are also highly regulated such
that the metabolic demands of epithelial cells become an important
variable that contributes to the threshold for ischemia. We have ob-
served that hormonal and metabolic products that regulate tubuloglo-
merular feedback systems play significant roles in the regulation of
oxygen consumption in the kidney (26,27,65,66). (Figure 4). In fact, the
critical operative substances tend to individually affect metabolic sup-
38 ROLAND C. BLANTZ, M.D. ET AL

FIG. 4. The hormonal control of kidney oxygen supply and demand: Prevention of
ischemia. Both oxygen and substrate supply and oxygen and substrate utilization or
demand are controlled by a variety of hormonal and metabolic factors. There is a general
pattern that substances which decrease supply tend to increase demand at the level of
tubular epithelium, either by increasing reabsorption or increasing oxygen required for
sodium transport. Such a relationship is logical from the standpoint of evolutionary
biology in that metabolic and hormonal regulators will modify both supply and demand.
The balance of these forces determines the threshold of kidney ischemia.

ply and demand in opposite directions, as noted with a) ATP/adeno-

sine, b) NO from NOS-1, and, c) angiotensin II, and, d) possibly COX-2
products (Figure 4). Such regulatory efficiency makes sense from the
standpoint of evolutionary biology in that regulatory substances
should control both metabolic supply and demand.

ACKNOWLEDGMENTS
These studies were supported through funding supplied by the National Institutes of
Health (DK 28602, DK 56248, T32HL 007261) and from funds derived from the Research
Service of the Dept. of Veterans Affairs.

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DISCUSSION
Mitch, Houston: Thank you. I was just wondering if you factored the increase in
oxygen consumption with diabetes or even with the remnant kidney, which is growing
rapidly in the proximal tubule. What is the contribution of protein synthesis and/or
breakdown in those organs in those parts of the kidney?
Blantz, La Jolla: We factored it both by sodium transport, and as you have percep-
tively picked up, there are other functions that may be reabsorptive functions, such as
protein absorption and things like that. Whether we factor it by sodium reabsorption or
whether we factor it by protein content or even DNA, it is still elevated and there is quite
a bit of variation from animal to animal, but the net average is high. As I remember, one
of your ex-faculty, Harold Franch, did some elegant studies about protein synthesis in
early diabetes. As I recall, protein synthesis actually was only amplified for the first few
days after creating diabetes and the rest of the increase in protein was due to decreased
degradation. In other words, there were lysosomal disposal problems of protein in
diabetes that I think accounted for the extra protein. Right now, I’d say we don’t know,
and it is, I’m sure, of interest to the diabetologists as well as why the kidney as a peculiar
organ seems to be inefficient in oxygen utilization. We’ve also noted, as Sharon Anderson
at University of Oregon, the dominant NOS isoform in the diabetic kidney is actually
brain NOS or NOS 1, which is not the case in normal kidney. Interestingly, the diabetic
kidney does not allow for resetting or TGF adaptation. So, it already has a peculiar
behavior when one changes salt intake as we’ve observed in other publications.
Luke, Cincinnati: I think you’re suggesting a role, in that there have been a lot of
papers recently saying that even a 0.3 or 0.4 increase in serum creatinine in the ICU
after an MI or after an invasive procedure confers greater, or is associated with greater
morbidity and mortality subsequently. Is the kidney just acting as a sensor for reactive
oxygen species in that setting? I mean, you’ve just eluded to that.
Blantz: Well your statement is certainly true and we’ve got some studies that I didn’t
have a chance to mention that even cytokinemia or having more cytokines around, early
sepsis before your kidney actually fails, the oxygen consumption by the kidney almost
doubles, and this is not related to NO. We are trying to figure why in the world that
occurs, but certainly, several of the acute kidney injury centers that are developing and
sponsored by NIH are now interested in basically why this phenomenon of having a prior
hit seems to set you up for progressive renal disease. I think that what we haven’t taken
serious consideration, is there a metabolic component that causes transformation or
rather epithelial mesenchymal transformation of cells that derives from some kind of
insult that led to a metabolic defect? All I am trying to say is that nephrologists, for 40
or 50 years, have been concerned about supply of blood to the kidney. I think now we
ought to start thinking about what are the regulators that dictate the oxygen demands
of the kidney, and are they factors that we can predict in particular problem patients as
a consequence of the ICU setting.
Willerson, Houston: As a cardiologist, Roland, where is endothelin’s role in the
 KIDNEY FUNCTION AND METABOLISM 43
vasoregulatory cascade you’ve shown us; and Robin, I wondered where it was on your
slides too?
Blantz: Well this wasn’t a question of lack of loyalty. We just don’t have a grant on
endothelin. But endothelin is obviously one of the more potent vasoconstrictors, but it
becomes a really complicated issue to study, and we haven’t studied it yet because, as
you well know, endothelin has a couple of different receptors, one of which liberates
nitric oxide; and the question is: is all nitric oxide created equal? No, it isn’t! It depends
on which nitric oxide you are generating, and we also have some interest in what role the
NMDA receptor has in the proximal tubule in regulating NO. It’s hard to imagine that
NO was primarily a metabolic regulator, but I think that nitric oxide and endothelin may
have preceded the cardiovascular system, and that means preceded cardiologists, I
presume. So, I don’t know whether the biologic functions for having nitric oxide and
endothelin might have derived from metabolic origins. Certainly, these substances,
adenosine and NO, occurred before kidneys and cardiovascular systems.
Luke: Perhaps the cardiologists and nephrologists can continue this debate outside.
The President’s address is not allowed to be questioned, Dr. Willerson.