JOURNAL OF ENDOUROLOGY

Volume 22, Number 3, March 2008

A4
© Mary Ann Liebert, Inc.
DOI: 10.1089/end.2007.0295

Pyrolidium Dithiocarbamate Prevents Shockwave

Lithotripsy-Induced Renal Injury Through Inhibition of Nuclear
Factor-Kappa B and Inducible Nitric Oxide
Synthase Activity in Rats

VULCAN TUGCU, M.D.,1 MUZAFFER BAS, M.D.,1 EMIN OZBEK, M.D.,2 EMAY KEMAHLI, M.D.,1
YASAR VOLKAN ARINCI, Ph.D.,3 MEHMET TUHRI, M.D.,4 TUNCAY ALTUG, M.D.,5
and ALI IHSAN TASCI, M.D.1

ABSTRACT

Purpose: Extracorporeal shockwave lithotripsy (SWL) is commonly used for treatment of renal stones. Free
oxygen radicals are involved in the pathophysiology of renal injury due to SWL. We investigated the protec-
tive effect of pyrolidium dithiocarbamate (PDTC), an antioxidant and nuclear factor -B (NF-B) inhibitor,
against renal injury.
Materials and Methods: Forty-eight rats were divided into three groups: group 1, controls; group 2, SWL
(15 kW, 1500 pulses); and group 3, SWL  pyrolidium dithiocarbamate (PDTC) (100 mg/kg/day given in-
traperitoneally 1 day before and 5 days after SWL). The rats were sacrificed and their kidneys were removed
on days 7 and 35 after SWL. Samples were fixed in formaldehyde solution, and renal tissues were examined
for proximal tubular injury under light microscopy. iNOS activity and active subunit of NF-B, p65, were
evaluated immunohistochemically using rat monoclonal antibodies interpreting results semiquantitatively.
Results: There were significant differences between SWL and control groups on days 7 and 35, consider-
ing histological changes under light microscope (p  0.02). There was significant decrease in necrosis and fi-
brosis in PDTC group compared to SWL group. Expression of inducible nitric oxide synthase (iNOS) and p65
on days 7 and 35 were at basal levels as seen with immunohistochemical staining. There were high concen-
trations of iNOS and p65 in the SWL group (P  0.02). No significant difference in concentrations was seen
between the control and the PDTC groups (P  0.02).
Conclusion: We found that curcumin decreased the expression of iNOS, p65, and serum nitric oxide levels,
and helped prevent interstitial, glomerular, tubular epithelial, and endothelial cellular injury. We believe that
PDTC could be used, particularly in high-risk patients, as a protective agent against renal injury due to SWL.

INTRODUCTION
E XTRACORPOREAL SHOCKWAVE LITHOTRIPSY
(SWL) is currently the first-line treatment for upper uri-
nary tract calculi. This treatment is not without side effects, and
kidney damage during SWL is a clinically significant problem.1
The mechanisms underlying shockwave-induced renal tubular
injury are not completely understood, though shear forces, ther-
mal and cavitation effects, and free radical formation have been
postulated.2–6
Morgan and associates5 adapted the Fricke ferrous sulfate ra-
diation dosimeter to examine the chemical effects of high-en-
ergy shockwaves, and significant free radical production was
documented.7,8
Serel and colleagues9 presented intracellular and extracellu-
lar in vitro measurements of free radicals and investigated cell
viability after shockwave treatment. They demonstrated an el-
evated concentration of intracellular free radicals during such
treatment in suspended cells in vitro.
Nuclear factor kappa-B (NF-B) regulates host inflamma-

1Department of Urology, Bakirkoy Research and Training Hospital, 2Department of Urology, Vakyf Gureba Research and Training Hospital,
3Departmentof Chemical Engineering, Istanbul Technical University, 4Department of Pathology, Bakirkoy Research and Training Hospital, and
5Animal Research Laboratory, Istanbul University, Cerrahpasa Medical Faculty, Istanbul, Turkey.

559
560
tory and immune responses10,11 and cellular growth properties12
by increasing the expression of specific cellular genes. These
include genes encoding at least 27 different cytokines and
chemokines, receptors involved in antigen presentation, and re-
ceptors required for neutrophil adhesion and migration.13 Cy-
tokines that are stimulated by NF-B such as interleukin-1
and tumor necrosis factor-, can also directly activate the NF-
B pathway, thus establishing a positive autoregulatory loop
that can amplify the inflammatory response and increase the
duration of chronic inflammation.
It has been suggested that reactive oxygen species such as
hydroxyl radicals and superoxide anions act as mediators of
NF-B activation by F-B degration.14,15
Excessive nitric oxide (NO) production due to elevated ex-
pression of inducible nitric oxide synthase (iNOS) may cause
cytotoxic effects on various organs, including the kidney.16,17
At high concentrations the nitric oxide can rapidly react with
superoxide anion to yield a potent oxidant, peroxynitrite, that
in turn causes extensive protein tyrosine nitration.18 The ex-
pression of iNOS is primarily controlled by the activation of its
transcriptional factors, including NF-B.18
Antioxidants are well known to enhance the biological ac-
tions of NO against oxidative destruction by reactive oxygen
species.19 We used superoxide dismutase, malondialdehyde
(MDA), and reduced gluthatione (GSH) as markers of lipid per-
oxidation.
Free radical formation seems to play an important role in the
pathogenesis of shockwave-induced renal tubular injury; there-
fore, our aim was to investigate the potential protective effect
of pyrolidium dithiocarbamate (PDTC) on shockwave-induced
oxidative stress in rat kidney.
MATERIALS AND METHODS
Animals
Forty-eight adult male Sprague-Dawley rats (weight
250–300 g) were acquired from the Experimental Animal Lab-
oratory of Istanbul University, Cerrahpasa Medical Faculty vi-
varium, and maintained in a 14-hour light/10-hour dark cycle
with free access to food and water.
Experimental conditions
Rats were first divided into three equal groups. Rats in the
SWL  PDTC group were injected with PDTC (Sigma-Aldrich
Chemical Corp., St. Louis, MO) (100 mg/kg/day given in-
traperitoneally) and were exposed to 1500 shock waves at 15
kV in a lithotriptor (Stonelith-V5 lithotriptor; PCK, Ankara,
Turkey). In the SWL group, rats were exposed to 1500 shock-
waves at 15 kV. In the control group, the rats received anes-
thesia and contrast medium, but were not exposed to SWL.
Each of the three groups were subdivided into two equal groups
according to the experimental sampling period: 7 and 35 days af-
ter SWL. Finally, we named the groups: group 1: SWL  PDTC
7; group 2: SWL 7; group 3: control 7; group 4: SWL  PDTC
35; group 5: SWL 35; and group 6: control 35.
The animals were placed in cages 7 days before beginning
the experiment to acclimate them to their surroundings. During
TUGCU ET AL.
the experimental period, all groups had free access to regular
rat chow. PDTC was given starting 1 day before and continu-
ing for 5 days after SWL (for a total of 6 days).
The rats were anesthetized (intraperitoneal sodium pento-
barbital, 50 mg/kg body weight), they were fixed in a supine
position on the platform of the lithotriptor, and fixed at the tho-
rax and hip to allow direct entry of the shockwaves through the
abdominal wall into the left kidney. All the rats were injected
intravenously with 0.7 mL of nonionic contrast medium (io-
promide; Ultravist® Injection 370 mg/50 mL; Bayer Health
Care Pharmaceuticals Inc., Wayne, Germany) and the left kid-
ney was treated after it was located using fluoroscopy.
At the end of the experimental period (days 7 and 35) the
rats were killed via intraperitoneal injection with sodium pen-
tobarbital, 50 mg/kg body weight. The kidneys were quickly
removed and decapsulated, the renal cortex was carefully
separated from the medulla, and then was homogenized as de-
scribed previously.20 Small samples were fixed in formalde-
hyde for histopathologic and immunohistochemical examina-
tion.
Histopathalogic examination
The tissues were prepared for histopathologic examination
by light microscopy (Nikon, Tokyo, Japan). The kidney sec-
tions were analyzed semi-quantitatively by a pathologist who
was blinded to the group being examined, using the technique
of Houghton and associates.21
Immunohistochemical evaluation
For immunohistochemical evaluation, the specimens were
processed as for light microscopy and sections were incubated
at 60°C overnight and then dewaxed in xylene for 30 minutes.
After soaking in decreasing concentrations of ethanol, the
sections were washed with distilled water and phosphate-
buffered saline for 10 minutes. The sections were treated with
2% trypsin in 50 mM Tris buffer (pH 7.5) at 37°C for 15 min-
utes, and then washed with phosphate-buffered saline. The
sections were marked with a Dako pen (Dako, Glostrup, Den-
mark) and incubated in a solution of 3% hydrogen peroxide
for 15 minutes to inhibit endogenous peroxidase activity. Then
the sections were incubated with NF-B/p65 (Rel A) Ab-1
(Neomarkers R-B-1638-R7), and iNOS Ab-1 (Neomarkers
R-B-1605-R7) antibodies. They were then stained using the
Ultravision HRP-AEC staining protocol (Lab Vision, Fre-
mont, CA).
Prepared sections from each group were examined by light
microscopy. Sections of rat lung were used as controls for im-
munohistochemical staining specificity according to data pro-
vided by the company that produced the antibodies.
The cases were evaluated for diffuseness and staining. With
regard to the diffuseness of staining, the sections were graded
as follows: 0  no staining; 1  staining 25%; 2  staining
of 25% to 50%; 3  staining of 50% to 75%; and 4  staining
75%. With regard to staining intensity, the sections were
graded as follows: 0  no staining; 1  weak but detectable
staining above the control level; 2  distinct staining; and 3 
intense staining. Immunohistochemical values were obtained by
adding diffuseness and staining intensity scores.
PROTECTIVE EFFECTS OF PDTC IN SWL-INDUCED RENAL INJURY 561

TABLE 1. SERUM, URINE, AND TISSUE LEVELS IN THE STUDY GROUPS

Day 7 Day 35

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

SWL  PDTC 7 SWL 7 Control 7 SWL  PDTC 35 SWL 35 Control 35

MDA 59.2  9.6a,c 133.4  12.4b,c,d 48.6  8.6 53.1  8.7 90.5  16.3b,c,d,e 51.1  9.4
(nmol/g wet tissue)
NOx(NO2NO3) 58.2  9.3a,c 95.6  12.4b,c,d 47.3  9.1 52.3  8.1 82.8  6.5b,c,d,e 49.4  7.5
(nmol/g wet tissue)
GSH 1.3  0.2a,c 0.9  0.3a,e,d 1.7  0.2 1.5  0.3 1.1  0.2a,c,d 1.6  0.2
(nmol/g wet tissue)
aP  0.05.
bP  0.01.
cSignificant compared to control group.
dSignificant compared to SWL  PDTC group.
eSignificant compared to day 7.

Biochemical determinations the Mann-Whitney U test. Probability values 0.05 were con-
All tissues were washed twice with cold saline solution and sidered significant.
immediately stored at –80°C for the measurement of MDA,
GSH, and NO concentrations. The tissues were homogenized
in four volumes of ice-cold buffer containing 20 mM Tris and RESULTS
10 mM EDTA (pH 7.4).
Total nitrite was quantified by the Griess reaction after in- Biochemical variables in urine, serum, and tissue
cubation of the supernatant with E. coli nitrate reductase to con-
There were significantly lower MDA and NO levels (P 
vert NO3 to NO2.
0.01) and higher GSH concentrations (P  0.05) in kidney cor-
Thiobarbituric acid reacts with lipoperoxidation aldehydes
tices in the SWL  PDTC group compared with the other
such as MDA, and this is the most common method used
groups (SWL day 7 and SWL day 35) (Table 1). There was a
to assess lipid peroxidation in biological samples. The proce-
significant difference in MDA and NO concentrations of the
dure was modified from the one described by Buege and
SWL groups (P  0.01). The mean tissue MDA level on day
Aust.22
7 was 133.4  12.4 nmol/g wet tissue. On day 35, it was de-
GSH concentration was determined using the spectropho-
creased to 90.5  16.3 nmol/g wet tissue. The mean tissue
tometric method of Ellman, which is based on the develop-
NO concentration on day 7 was 95.6  12.4 nmol/g wet tissue.
ment of a yellow color when DTNB (5,5dithiobis-2-ni-
On day 35, it had decreased to 82.8  6.5 nmol/g wet tissue.
trobenzoic acid) is added to compounds containing sulfhydryl
(Table 1).
groups.20
There was significant difference between controls and the
SWL  PDTC groups in concentrations of MDA, NO, and
Statistical analysis
GSH for day 7. MDA and NO concentrations were higher and
Statistical analyses of histopathologic and immunohisto- GSH concentrations were lower (P  0.05) in kidney cortices
chemical evaluation of the groups were carried out with chi- in control group compared with the SWL  PDTC group for
square testing, and biochemical values were compared using day 7 (Table 1). There was no significant difference between

TABLE 2. HISTOPATHOLOGIC EFFECTS OF SWL AND PDTC

Day 7 Day 35

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

SWL  PDTC 7 SWL 7 Control 7 SWL  PDTC 35 SWL 35 Control 35

Glomerular bleeding   – –  –
Tubular dilatation   – –  –
Tubular atrophy   – –  –
Tubular red blood cells   – –  –
Interstitial necrosis   –   –
Interstitial fibrosis –  – –  –
Venous dilatation   – –  –
Neutrophilic infiltration   – –  –

Histopathologic findings: – none,  isolated,  frequent,  highly frequent.

 562
controls and the SWL  PDTC group for MDA, NO, and GSH
concentrations for day 35 (P  0.05) (Table 1).
Histologic examination
The microscopic examination revealed a similar pattern of
histologic damage in the SWL groups and the SWL  PDTC
groups. Histologic damage was more apparent in day 7 groups
compared with day 35 groups (SWL 7 and SWL  PDTC
7 compared to SWL  PDTC 35 and SWL  PDTC 35)
(Table 2).
Subcapsular and intraparenchymal hemorrhages were more
severe at the corticomedullary borders and perivascular areas
in the SWL group compared to the SWL  PDTC group on
day 7. Also, changes in the tubules including blood and protein
A B
TUGCU ET AL.
in the tubule lumen, swollen tubules, and mononuclear inflam-
matory cells infiltrating into the interstitium were observed in
the SWL group, but only rarely in the SWL  PDTC group,
for day 7. There were also dilated tubules and ruptured arter-
ies and veins in SWL day 7 group.
Some of these pathologic findings seemed to be recovered
by day 35, but glomerular, tubular, and interstitial necrosis,
tubular atrophy, and hydropic degeneration were more appar-
ent in the SWL day 35 group than the SWL day 7 and SWL 
PDTC groups (days 7 and 35) (Table 2) (Fig. 1a, b, and c).
Immunohistochemical studies
In the immunohistochemical evaluation, there was more in-
tensely positive iNOS and p65 staining in the renal cortex and
FIG. 1. (A) As shown here, there was no hydropic degener-
ation or fibrosis in the SWL  PDTC 35 group (hematoxylin
and eosin 400). (B) As shown here, there was mild hydropic
degeneration and limited fibrosis in the SWL 7 group (hema-
toxylin and eosin 400). (C) As shown here, interstitial ne-
crosis, hydropic degeneration, and fibrosis were more apparent
in the SWL 35 group (hematoxylin and eosin 400).
PROTECTIVE EFFECTS OF PDTC IN SWL-INDUCED RENAL INJURY
within the proximal tubules in the SWL groups (SWL 7 and
SWL 35) compared with the other groups. It appeared that SWL
causes more damage to the tubules than other structures (Table
3 and Fig. 2a, b, and c).
Immunohistochemical scores of group SWL 7 were higher
than those of group SWL 35, but there was no significant dif-
ference between these two groups.
There was poorly or mildly positive staining of iNOS and p65
in the SWL  PDTC groups (days 7 and 35) and control groups
compared with the SWL groups at all time intervals (Table 3).
iNOS and p65 expression in group SWL  PDTC 35 was more
weakly positive than in group SWL  PDTC 7. Recovery to-
ward the end of the experimental period was apparent.
DISCUSSION
The mechanisms behind SWL-induced renal damage have
not been elucidated in detail. Significant free radical produc-
tion after SWL has been documented in early investigations.6,23
PDTC is known to be a free radical scavenger, and free rad-
ical formation is thought to be an important mechanism in
shockwave-induced renal injury.4–6 Reactive oxygen species
are also important in other types of renal injury (e.g., post-isch-
emic and toxic acute renal failure).24
By using fluorescent dyes, the generation of free radicals by
shockwave-induced cavitations has been demonstrated in sus-
pended cells.6
Ozgüner and associates25 found that the novel free radical
scavenger caffeic acid phenethyl ester (CAPE) provided sig-
nificant protection against SWL-induced free radical damage.
Following shockwave exposure there was a significant rise in
MDA, N-acetyl-beta-glucosaminidase (NAG), and uric acid
and white cell counts. They found that CAPE reduced the rise
in MDA, NAG, uric acid, and white cell counts.
Serel and colleagues9 demonstrated that melatonin signifi-
cantly reduced shockwave-induced renal impairment resulting
in increased MDA levels and urine NAG activity.
In in vitro experiments, Strohmaier and co-workers26 used
cultivated Madin-Darby Canine Kidney (MDCK) cells to dem-
onstrate that the nephroprotective antibiotic fosfomycin and the
calcium channel blocker verapamil could limit shockwave-in-
duced cell damage.27,28 Li and associates demonstrated that
TABLE 3. HISTOPATHOLOGIC EFFECTS
Total Day 7
immunohistochemical
staining score Group 1 Group 2
iNOS/p65 SWL  PDTC 7 SWL 7
0 — — 6/6
1 — — 6/4
2 5/4 — 4/6
3 6/6 — —
4 5/6 — —
5 — 5/4
6 — 6/7
7 — 5/5
563
PDTC attenuated SWL-induced MDCK cellular injury by in-
hibiting NF-B activation.1
Similar to the findings of Li’s group, our results demonstrate
that PDTC significantly reduces shockwave- induced renal im-
pairment resulting in increased MDA and NO levels in renal
tissue when compared with controls. The SWL  PDTC groups
had significantly higher GSH concentrations in kidney cortical
tissue than the groups given SWL only.
Reactive oxygen intermediates (ROI) have been shown to be
involved in NF-B activation in many studies.14,29 Animal stud-
ies have demonstrated that endotoxemia,30 blood loss,31,32 and
hyperoxia,33,34 all result in increased NF-B activation.
NO plays an important role in a variety of physiologic and
pathophysiologic processes in the kidney.34,35 NO can interact
with reactive oxygen species to form peroxynitrite, which in-
duces protein damage by formation of nitrotyrosine.
The contribution of NO to tissue injury can be due to a di-
rect effect mediated by the nitric oxide molecule itself, 36 or
an indirect effect mediated by reactive nitrogen species pro-
duced by the interaction of NO with superoxide anions or
oxygen.36,37 For example, iNOS-mediated NO production
was significantly elevated during renal ischemia/reperfusion
injury.34,35,38,39
The expression of iNOS is mainly controlled by activation
of its transcriptional factors, including NF-B. Zhang and as-
sociates observed that at pathophysiologic concentrations, ho-
mocysteine was able to activate NF-B, causing enhanced
iNOS expression in macrophages.40
In our study, we showed immunohistochemically that iNOS
and p65 expression were significantly reduced in rats given
SWL plus an NF-B inhibitor (PDTC), compared with SWL
only.
On light microscopy, acute morphologic changes such as
glomerular bleeding, tubular dilatation, atrophy, and partial ne-
crosis occurred immediately after SWL throughout the kidney.
In the long-term groups, the hematomas progressed to intersti-
tial fibrosis with segmental retraction of the renal cortices.
There was no fibrosis seen in the SWL  PDTC groups for day
7 and day 35.
RT-PCR and Western blotting analyses are functional as-
says used to measure the actual activity of iNOS. Western
blotting provides more accurate measurements of iNOS and
p65 activity. Thus, one limitation of this study is that we did
not use Western blotting analyses. However, there are a num-
OF SWL AND PDTC
Day 35
Group 3 Group 4 Group 5 Group 6
Control 7 SWL  PDTC 35 SWL 35 Control 35
— — 6/5
4/4 — 5/7
10/8 — 5/4
2/4 — —
— 3/4 —
— — 5/3 —
— — 8/9 —
— — — —
 564
A B
ber of studies in the literature in which the authors made use
of immunohistochemical grading of iNOS to evaluate NO ac-
tivity.29 This method was also used in another recent study.25
Monitoring urinary excretion of enzymes, particularly NAG,
is considered a relatively simple, cheap, fast, and noninvasive
method to detect and follow-up renal tubular function under
various conditions. The determination of urinary NAG con-
centrations provides a sensitive and reliable indicator of renal
damage, such as that occurring through injury or dysfunction.
In this study we examined the kidneys to measure kidney dam-
age directly, and thus we did not need to measure urinary NAG
excretion. But urinary NAG excretion can be used to assess kid-
ney damage, especially in human models.41
TUGCU ET AL.
FIG. 2. (A) As shown here, there was intense p65 staining in
the SWL 35 group (immunohistochemical staining 400). (B)
As shown here, there is poor p65 staining in the SWL  PDTC
35 group (immunohistochemical staining 400). (C) As shown
here, the p65 staining is quite poor in the control 35 group (im-
munohistochemical staining 400).
CONCLUSIONS
We propose that the NF-B inhibitor PDTC acts in the kid-
ney as a potent scavenger of free radicals, and prevents some
of the side effects of SWL. These results also reinforce the sig-
nificant role of free radicals in the pathogenesis of nephropa-
thy induced by SWL. In particular, NF-B inhibitors such as
PDTC are among the most potent and specific antioxidants for
hydroxyl radicals.
Inhibiting the NF-B pathway by using PDTC could decrease
iNOS expression and thus reduce cellular damage. More study
is required to clarify the precise mechanisms of its action, and to
ascertain whether it could be used to treat human nephropathy.
PROTECTIVE EFFECTS OF PDTC IN SWL-INDUCED RENAL INJURY
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566 TUGCU ET AL.

Address reprint requests to: ABBREVIATIONS USED

Vulcan Tugcu, M.D.
Department of Urology CAPE  caffeic acid phenethyl ester; GSH  reduced
Bakirkoy Research and Training Hospital gluthatione; iNOS  inducible nitric oxide synthase; MDA 
Tevfik Sağlam Cad. No. 11 malondialdehyde; MDCK  Madin-Darby Canine Kidney
Zuhuratababa (cell); NF-B  nuclear factor kappa-B; NAG  N-acetyl-
34740 Istanbul, Turkey beta-glucosaminidase; NO  nitric oxide; PDTC  pyrolidium
dithiocarbamate; ROI  reactive oxygen intermediates;
E-mail: volkantugcu@yahoo.com SWL  extracorporeal shockwave lithotripsy.
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