Attenuation of cisplatin nephrotoxicity by streptozotocin-induced diabetes

FUNDAMENTALANDAPPLIEDTOXICOLOGY

(1989)

12,530-539

Attenuation of Cisplatin Nephrotoxicity Streptozotocin-Induced Diabetes’ LAURIE

A. SCOTT, ELIO MADAN,

AND MONICA

by

A. VALENTOVI?

Departments ofPharmacology and Pathology, Marshall University School of Medicine. Huntington, West Virginia 25755-9310

Received May 25, 1988; accepted September 13.1988 Attenuation of Cisplatin Nephrotoxicity by Streptozotocin-Induced Diabetes. SCOTT, L. A., E., AND VALENTOVIC, M. A. (1989). Fundarn. Appl. Toxicol. 12,530-539. The therapeutic use of cisplatin is associated with acute renal failure. The purpose of this study was to determine (a) if streptozotocin (STZ) was toxic to renal proximal tubules and (b) the nephrotoxicity of cisplatin in STZ-diabetic rats. Male Sprague-Dawley rats were injected with STZ (55 mg/kg, ip) to induce a diabetic state. BUN and renal cortical slice uptake ofpaminohippurate (PAH) and tetraethylammonium (TEA) were not altered, relative to normoglycemic rats, 3, 16, and 28 days following STZ treatment. These results indicate that STZ is not toxic to renal proximal tubules. Cisplatin nephrotoxicity studies were then conducted in STZdiabetic and normoglycemic rats. Cisplatin nephrotoxicity was also evaluated in diabetic rats pretreated for 8 days with insulin. Diabetic and normoglycemic rats were administered 5 mg/kg cisplatin or water (ip). Increased kidney weight, BUN levels, glucosuria, and proteinuria were measured in normoglycemic rats 4 days after cisplatin administration. Renal cortical TEA and lactate-stimulated PAH uptake (p i 0.05) wexe diminished in the normoglycemic rats 4 days after cisplatin injection. No change in kidney weight, BUN levels, or renal cortical slice accumulation of PAH and TEA was observed in diabetic rats treated with cisplatin. However, cisplatin administration to diabetic rats pretreated with insulin resulted in increased mortality, proteinuria, glucosuria and elevated kidney weight. These results indicate that the diabetic state attenuates cisplatin nephrotoxicity. Additionally, these results indicate that diabetes attenuation of cisplatin nephrotoxicity is dependent on the severity of the diabetic state. 8 1989 society ofToxicology. MADAN,

Streptozotocin (STZ)-induced diabetes has been reported to protect against gentamicin nephrotoxicity (Teixeira et al., 1982). Teixeira and co-workers showed that gentamicin administration to diabetic rats failed to induce histological alterations or lysozymuria or to diminish creatinine clearance. Studies by Vaamonde and associates ( 1984) have also shown that protection from gentamicin-in’ Presented in part at the 71st Annual Federation of American Societies for Experimental Biology Meeting, March 29-April3, 1987, Washington, DC, and the 29th National Student Research Forum, April 6-8,1988, Galveston, TX. * To whom correspondence should be addressed. 0272-0590/89 $3.00 Copyrieht 0 1989 by the Society ofToxicology. All ri@ts of reproduction in any form mered.

530

duced nephrotoxicity is independent of the duration of diabetes since lysozymuria and diminished creatinine clearance are not produced by gentamicin in rats diabetic for 5 days or 5 months. The purpose of this investigation is to examine (a) if STZ is toxic to renal proximal tubules and (b) if the diabetic state attenuates the nephrotoxicity of cis-diamminedichloroplatinum (cisplatin). The ability of diabetes to alter cisplatin nephrotoxicity was investigated for several different reasons. First, these studies would determine if diabetes modulates the nephrotoxicity of compounds that are not structurally related to the aminogly-

CISPLATIN

NEPHROTOXICITY

coside antibiotics. Second, this investigation would determine if diabetes could modulate the renal damage induced by other compounds that are toxic to renal proximal tubules. Cisplatin is an antineoplastic agent that induces proximal tubular damage. The nephrotoxicity of cisplatin is characterized by a rise in blood urea nitrogen (BUN) levels, increased kidney weight (Ward and Fauvie, 1976), diminished renal blood flow, and decreased glomerular filtration rate (Dentin0 et al., 1978). Renal toxicity in patients treated with cisplatin is localized in the distal and collecting tubules (Gonzalez-Vitale et al., 1977). On the other hand, the primary site in rats for cisplatin-induced renal toxicity is the !$ segment of the proximal tubule (Dobyan et al., 1980). A single injection of cisplatin results in cellular swelling, loss of brush border, sloughing of tubular epithelial cells, and necrosis of the S3 segment (Dobyan et al., 1980). The studies described here examined if the diabetic state modulates cisplatin nephrotoxicity. MATERIALS

AND

METHODS

Animals and induction of diabetes. Male SpragueDawley rats (175-340 g) were purchased from Hilltop Lab Animals, Inc. (Scottdale, PA) and housed at a constant ambient temperature (21-23°C) and light cycle (0600-l 800 hr). Animals were permitted a minimum 5day quarantine period prior to initiation of any experimental procedures. Blood was obtained from the tail to quantitate baseline BUN and glucose levels. Rats were individually housed in stainless-steel metabolism cages and permitted free accessto food (powdered Purina Rodent Chow) and water during a 24hr acclimation period. On completion of the 24hr period, food was withheld from 0900 to 1200 hr to collect urine free of food contamination. The urine was analyzed for protein, glucose, ketones, and pH using Multistix or Combistix (Ames Division, Miles Laboratories). Rats were then permitted free access to food for 21 hr. Baseline values for urine output and food and water intake were measured at the end of the 24-hr period and denoted Control Day values. Rats were injected (ip) the following day at 0900 hr with 55 m&kg STZ (12/group) or citrate buffer, pH 4.6 (8/ group). Food intake and water and urine output were measured 1,3, 7,2 1, and 28 days after STZ injection as

IN DIABETES

531

described above. Protein, ketones, glucose, and pH were analyzed on urine wllected from 0900 to 1200 hr. Kidney weight, renal cortical organic ion transport, serum glucose, and BUN levels were quantitated 3, 16, and 28 days after STZ ((i/group) or vehicle (4/group) treatment. Cisplatin treatment of STZ-diabetic and normoglycemic rats. Animals were divided into four groups (>4/ group): normoglycemiovehicle (NV), normoglycemiccisplatin (NC), diabetic-vehicle (DV), and diabeticcisplatin (DC). Blood was obtained from the tail of all animals to quantitate baseline serum glucose and BUN levels. Rats were individually housed in stainless-steel metabolism cages and permitted a 24hr acclimation period. Baseline values for body weight, urine output, and food and water intake were monitored and denoted Control Day values. Urine was analyzed using Multistix or Combistix for protein, glucose, and pH. Normoglycemic and 14-day diabetic rats were injected on Day 1 at 0900 hr with 5 mp/kg cisplatin (4 ml/kg) or water (ip). Body weight, urinalysis, and food and water intake were measured 1,2, and 4 days after cisplatin or vehicle treatment. Vehicle-injected groups were pair-fed with respective treated animals to eliminate variability in data due to decreased food intake. Serum glucose, BUN levels, kidney weight, and renal cortical uptake of paminohippurate (PAH) and tetraethylammonium (TEA) were quantitated 2 days after cisplatin or vehicle treatment. Impact of insulin treatment on cisplatin nephrotoxicity. Diabetes was confirmed 9 days aher STZ injection by serum glucose levels exceeding 200 mg/dl. Rats were divided into the following groups (N = 4-8): normoglycemic-vehicle (NSV), normoglycemic-cisplatin (NSC), diabetic-vehicle (DSV), diabetic-cisplatin (DSC), diabetic-insulin-vehicle (DIV), and diabetic-insulin-cisplatin (DIC). Animals in the DIC and DIV group were initially injected with 5 U/kg NPH insulin (SC)twice daily beginning 10 days after STZ injection. Urinary glucose was monitored with Combistix (Ames Laboratories Inc) prior to each injection. Insulin dosage was adjusted daily to maintain urine glucose levels less than 250 mg/dl in the DIC and DIV groups. Beginning 16 days after STZ injection, rats were individually housed in metabolism cages.Body weight, urinalysis, and food and water intake were measured 17 days after STZ injection and denoted Control Day values. Cisplatin (5 mglkg) was injected (ip) at 0900 hr into 18-day diabetic or normoglycemic rats. Urinalysis and food and water intake were measured at 24hr intervals in all animals. Vehicle-treated rats were pair-fed with respective cisplatin-treated groups. Body weight, kidney weight, organic ion transport, serum glucose, and BUN levels were measured 4 days after cisplatin administration. Organic ion transport. Rats were anesthetized with diethyl ether and laparotomized and blood was drawn from the dorsal aorta. The left kidney was quickly excised, decapsulated, and placed in ice-cold Krebs-Ringer phosphate buffer (pH 7.4) containing 1 mM calcium. Renal

532

SCOTT, MADAN,

AND VALENTOVIC

TABLE 1 SERUM

BUN Day

AND

GLUCOSE

LEVELS

AFTER

0

STZ

INJECTION’ After

Group

BUN bg/W

Glucose bx.k-ll)

Day

Control Diabetic Control Diabetic Control Diabetic

26 + 1.1 28 f 0.5 20+ 1.5 21 kO.8 26+2.1 27+ 1.5

124k9.0 139 + 9.0 89 + 9.4 104+ 11.6 106 + 5.4 101 f 11.2

3 3 16 16 28 28

STZ

treatment

BUN b-Ml) 24+ 25 + 19 + 32 + 19 f 28 +

1.2 1.2 0.6 1.9*~? 0.2 1.3t

Glucose (mid@ 110 + 9.6 385 + 50.6’ 163 f 6.7* 736 k 46.8* 127 f 8.4 703+32.1*

’ Serum glucose and BUN levels were measured O-28 days after STZ injection. Values for age-matched normoglycemic rats are denoted control values. * Significantly different (p c 0.05) from respective Day 0. t Significantly different (p < 0.05) corresponding control group.

cortical slice accumulation of PAH and TEA was quantitated by the method of Rankin (1982). Briefly, renal cortical slices were prepared freehand, 70- 100 mg of tissue was incubated for 90 min at 25’C with constant shaking in an oxygen atmosphere with PAH or TEA. PAH accumulation was quantitated in the presence of 0 or 10 mM lactate. Renal cortical slice accumulation was expressed as slice to media (S/M) ratios, where Sequals radioactiv-

TABLE 2 DAILY

Foot

AND

AI=~ER Group

Control

WATER

CI~PLATIN day

Day 1 Food

NC DC

24.2 kO.3 43.4k2.8

43.5 + 3.5 40.0+ 1.8 176.0 + 27.3 195.0 + 21.9

O-2

DAYS

hY2

intake(g) 3.4 + 0.49 26.4 f 2.9*

Water NV NC DV DC

INTAKE INJECIION~

intake

5.0f 1.0’ 32.6 k 3.0

RESULTS

(ml)

19.0 I!z 4.78 18.5 f 1.5* 131.5+ 19.6' 127.0* 22.29

ity (dpm) per gram oftissue, and Mrepresents radioactivity (dpm) per milliliter of medium. Blood obtained from the dorsal aorta was centrifuged and serum samples were frozen at -2o’C until assayed. Serum glucose was quantitated using a spectrophotometric glucose oxidase (Sigma) kit and BUN was assayed spectrophotometrically using a urease (Sigma) assay. Histological examination. The right kidney was excised, weighed, cut in half lengthwise, and fixed in 10% neutral buffered formalin solution. Fixed tissues were embedded in pa&in, sectioned at 6-8 @cm,and stained with hematoxylin and eosin (HE) reagent before histologic examination. Statistical analysis. Values are reported as means + SE. Differences between normoglycemic and diabetic groups were quantitated using Student’s t test. Diff‘erences between groups were determined using an analysis of variance (ANOVA) followed by a Newman-Keuls test with a 95% confidence interval.

20.0 + 4.2* 20.5 f 1.7' 138.0 f 17.4* 167.0f 23.5*

DValues for 24-hr food and water intake are reported O-2 days after cisplatin injection. Data are expressed as means + SE. NV, normoglycemic vehicle; NC, normoglycemic-cisplatin; DV, diabetic-vehicle; DC, diabetio cisplatin. *Significantly different (p < 0.05) from respective Control Day values.

Impact of diabetes. A single injection of STZ induced a permanent diabetic state as indicated by polyuria, polyphagia, and hyperglycemia. Urine output was increased beginning 24 hr after STZ injection and continued to be elevated in all diabetic animals. Beginning 3 days after STZ administration, serum glucose levels were elevated more than twoto sixfold (Table 1) above normoglycemic

CISPLATIN

NEPHROTOXICITY

533

IN DIABETES

TABLE 3 BODY WEIGHT, KIDNEY WEIGHT, SERUM GLUCOSE, AND BUN LEVELS 2 DAYS AFTER CISPLATIN INJECTION Group Day

NV

NC

DV

DC

MY weight 64 ControP 2

357 If: 10.2 323 z!z7.8

365 k 13.9 330 * 9.3*

295 + lO.S§ 285 A 14.25

266 + 4.45 255 k 7.25

0.44 f O.Ol§

0.50 k 0.02?$

66.4 + 2.6s 42.8 f 4.O+j

70.9 + 0.69 52.0 + 2.4**$

758 f 18.48 579 + 50.6*-g

765 2 19.5s 730 * 47.5*g

Kidney weight (g/100 g Body w-t) 2

0.36 f 0.01

0.39 + 0.01 BUN (w/W

Control 2

37.2 + 1.5 23.8 + 1.5*

39.9 ? 2.6 53.9 + 1.2*q Glucose (mg/dl)

Control 2

142 f 6.0 177 f 6.5*

142 f 6.3 177 f 3.9*

’ Values were measured 2 days after cisplatin administration in the following groups: NV, normoglycemic-vehicle; NC, normoglycemic-cisplatin; DV, diabetic-vehicle; DC, diabetic-cisplatin. Data are expressed as means f SE. b Control Day measurements denote Baseline values prior to cisplatin administration. * Statistically different (p < 0.05) from respective Control Day values. t Different (p -z 0.05) from respective vehicle group. 8 DV or DC different (p -z 0.05) from age-matched NV group.

rats. The diabetic state was also associated with a significant (p < 0.05) elevation, relative to the normoglycemic group, in serum BUN levels (Table 1) 16 and 28 days after ST2 administration. Kidney weights were also increased by STZ administration. Ridney weights 16 and 28 days after STZ injection were 0.61 + 0.02 and 0.62 + 0.09 g/100 g body wt in the diabetic rats (p < 0.05) and 0.39 + 0.01 and 0.42 f 0.02 g/100 g body wt in the normoglycemic rats, respectively. To examine the nephrotoxicity of cisplatin in diabetic rats, it was necessary to first determine the impact of STZ on renal function. Renal cortical slice accumulation of PAH or TEA was not statistically different between diabetic and citrate buffer controls 3-28 days after STZ injection. These results indicate that STZ (55 mg/kg) is not toxic to proximal tubules.

Cisplatin nephrotoxicity ajer 48 hr. Food intake in the normoglycemic rats (Table 2) was decreased by cisplatin at 24 and 48 hr, whereas food intake was decreased only at 24 hr in the diabetic animals. Variability in data due to altered food intake was prevented by pair feeding of respective vehicle-treated groups. A loss in body weight was associated with cisplatin treatment in the NC group (Table 3), whereas body weight was unaffected in diabetic rats. Total daily urine output was diminished 24 hr after cisplatin injection in the DC group (Fig. 1). The decreased urine output can be attributed to diminished food and water intake (Table 2), since comparable changes occurred in the DV rats. Serum BUN levels were increased 35% in the NC group 48 hr after cisplatin injection (Table 3). Diabetes attenuated cisplatin

534

SCOTT, MADAN, OEJIEm NV tic

g

125.

iw h5

loo-

DV

DC

75-

B

50.

s

25. OJCONTROL

DAY 1

DAY2

FIG. 1. Total daily urine output O-2 days after cisplatin administration. NV, normoglycemic-vehicle; NC, normoglycemic-cisplatin; DV, diabetic-vehicle; DC, diabetic&p&in. Values prior to cisplatin injection are denoted Control Day. *Statistically different (p < 0.05) from respective Control Day values.

nephrotoxicity since serum BUN levels were not altered after 48 hr. Renal slice uptake of TEA (Fig. 2) was diminished at 48 hr in the normoglycemic rats (p < 0.05). However, cisplatin administration did not alter PAH and TEA uptake in diabetic rats. Kidney weights were increased at 48 hr by cisplatin in the diabetic rats. Cisplatin increased kidney weight in the normoglycemic rats; however, these values were not statistically significant. Impact of insulin pretreatment on cisplatin nephrotoxicity. Diabetes is associated with decreased body weight, polyuria, polydipsia, and polyphagia. Total daily urine output (Fig. 3) and food and water intake (Table 4) were partially normalized in insulin-treated diabetic rats. These results indicate that insulin pretreatment partially regulated the diabetic state. These studies were also conducted to examine cisplatin nephrotoxicity after 4 days since maximal damage produced by cisplatin is manifested after 3-5 days. Marked decreases in food intake (Table 4) were observed in normoglycemic rats. Cisplatin administration diminished food intake 100% on Days 3 and 4 in the NSC group. Food intake was decreased in the DSC and DIC rats 39% (p < 0.05) and 35% (nonsignificant), re-

AND VALENTOVIC

spectively, of Control Day values. The diminished food intake was associated with reduced water consumption in all groups (Table 4). Urine volume (Fig. 3) and composition were altered by cisplatin treatment. Total daily urine output was diminished in all animals. The diminished volume should be attributed to decreased food and water intake since comparable changes were observed between treated and pair-fed controls. Urine content was greatly altered by cisplatin administration. The earliest detection of proteinuria and glucosuria occurred in the NSC group (Table 5). Glucosuria appeared by Day 2 in the DIC group, whereas protein was not detected until Day 4 in the DSC and DIG rats. Cisplatin administration most markedly elevated serum BUN levels (Table 6) in the normoglycemic rats, No significant increase in BUN levels was detected in the DIC or DSC groups. Cisplatin increased kidney weights in the DIC and NSC rats on Day 4 (Table 6). Since kidney weights were not elevated in the DSC rats, these results indicate cisplatin is nephrotoxic in normoglycemic and insulin-treated diabetic rats. omlm NV NC

DV

DC

15 52 2 ffi

10

$f/l 5

0

PAH

PAH

TEA

LA&ATE 2. Renal cortical slice accumulation of PAH and TEA was measured2 daysaftercisplatinadministration in 16-day diabetic or age-matched normoglycemic rats. NV, normoglycemic-vehicle; NC, normoglycemic-cisplatin; DV, diabetic-vehicle; DC, diabetic-cisplatin. tSignificant difference (p < 0.05) from respective vehicletreated rats. Cisplatin decreased renal cortical slice accumulation of TEA in the NC group. FIG.

CISPLATIN

NBPHROTOXICITY

IN DIABETES

535

OmEsYm~m NSV

NSC

CONTROL

DIV

DIG

DSV

DAY 1

DSC

DAY 3

DAY 4

RG. 3. Total 24-hr urine output was measured O-4 days following cisplatin injection. NSV, normoglycemic-vehicle; NSC, normoglycemiocisplatin treated; DIV, diabetic-insulin-treated-vehicle; DIC, diabetic-insulin-treated-cisplatin; DSV, diabetic-vehicle; DSC, diabetic-cisplatin. DIC and DIV groups were treated with insulin for 8 days prior to cisplatin or vehicle injection. Values are reported as means rt_SE, N > 4. Total daily urine output prior to cisplatin treatment is denoted Control Day. *Statistically different from respective Control Day values. tDifferences (p c 0.05) Between cisplatin- and respective vehicletreated group.

Cisplatin administration to insulin-treated diabetic rats was associated with increased mortality. Survival rates following cisplatin administration were 100% in diabetic rats and 75% in insulin-treated diabetic rats. Mortality cannot be attributed to an insulin overdose since insulin treatment was terminated on cisplatin injection. Insulin administration was discontinued after treatment with cisplatin since food intake decreased and urinary glucose tests were negative in the DIV rats. Renal cortical slice accumulation of TEA (Fig. 4) was diminished in the normoglycemic rats 4 days after cisplatin administration (a < 0.05). Cisplatin also decreased lactate-stimulated PAH uptake in the NSC group. No change in renal cortical slice uptake of PAH or TEA was measured in the DSC or DIC rats. Histological examination of renal tissue showed proximal tubular damage in normoglycemic rats treated with cisplatin. Cisplatin produced dilation of tubules, vacuolated cells, and tubular epithelial cell necrosis. Histological examination of the kidneys from the

DSC and DIC rats indicated the presence of tubule dilation. Cisplatin administration, however, did not produce tubular epithelial cell necrosis in the DIC or DSC rats. DISCUSSION The results of these studies indicate that STZ modulates cisplatin nephrotoxicity. Cisplatin had no effect on serum BUN levels or kidney weight in the diabetic rats, while normoglycemic animals had marked elevations in BUN levels (Table 6) and kidney weight. Cisplatin administration also produced vacuolated cells, dilation of proximal tubules, and acute tubular necrosis in the normoglycemic rat. However, the extent of tubular damage and the presence of vacuolated cells were less severe in the diabetic group. Renal organic ion accumulation was monitored 2 and 4 days after cisplatin injection to examine tubule function during maximal degeneration. Previous work by Ward and Fauvie (1976) has shown that cisplatin produces maximal elevations in serum creati-

536

SCOTT, MADAN,

AND VALENTOVIC

TABLE 4 DAILY FOOD AND WATER INTAKE O-4 DAYS AFTER CISPLATIN TREATMENT After cisplatin treatment Group

Control day

Day 1

Day 3

Day 4

Ok0 23.0 k 3.2* 12.8 + 7.4*.t

Ok0 25.2 + 3.0* 20.1 + 10.4

6+ 1* 14+2* 85 f 9* 104 f 8* 38 + 14t 39 k 5*q

10*5* 10 f 1* 104 1- 20* 103*4* 84?22 57 + **q

Food intake (g) NSC DSC DIC

20.2 f 1.5 41.0* 1.4 30.8 + 4.2t

4.1 + 1.0* 21.8 + 5.5* 14.9 + 4.9 Water intake (ml)

NSC NSV DSC DSV DIC DIV

33+ 1 37+1 185 IL 10 206 + 15 67 + 197 84 +- 20t

12 + 1’ 15f2* 97 + 22* 1212 16* 48 f 6*st 37 + 5*q

u Daily food and water itnake was measured O-4 days after cisplatin administration. Values are expressed as means f SE. NSV, normoglycemic-vehicle; NSC, normoglycemic cisplatin; DSV, diabetic-vehicle; DSC, diabetic-cisplatin; DIV, diabetioinsuhn-vehicle; DIC, diabetic-insulin-cisplatin. DIC and DIV groups were treated for 8 days with insulin prior to cisplatin or vehicle treatment. * Different (p < 0.05) from respective Control Day values. t Control Day values for DIC or DIV are different (p < 0.05) from DSV. Insulin pretreatment (DIC, DIV) partially normalized the diabetic condition as indicated by the decreased food and water intake.

nine, increased BUN levels, and degeneration of proximal tubules within 3-5 days. Renal cortical accumulation of PAH and TEA was measured since it is a very sensitive parameter for detection of proximal tubular damage (Mazze et al., 1973; Kluwe, 1981). Attenuation of cisplatin nephrotoxicity by STZ-induced diabetes is further indicated by the inability of cisplatin to significantly lower PAH and TEA accumulation. This was in contrast to the marked decrease in renal cortical TEA and lactate-stimulated PAH uptake in the cisplatin-treated normoglycemic group. The results of this study also demonstrated that insulin treatment partially reversed the protective effect of diabetes. Cisplatin induced an earlier onset of glucosuria and a greater severity of proteinuria in the insulintreated diabetic rats. Additionally, increased kidney weight and lethality occurred after cisplatin administration in insulin-treated dia-

betic rats, whereas no change in kidney weight or survival rates occurred in the DSC group. The mechanism for diabetes-induced attenuation of cisplatin nephrotoxicity is not known. Modulation of cisplatin nephrotoxicity is probably mediated by some physiologic or cellular parameter inherently unique to the diabetic state. Additionally, the factor(s) attenuating cisplatin toxicity is dependent on the severity of the diabetic state, since partial correction of the diabetic condition reversed the protective effect of diabetes. Cisplatin nephrotoxicity may be dependent on proximal tubular retention of the toxin. Therefore, diminished accumulation of cisplatin is one plausible mechanism for attenuation of cisplatin nephrotoxicity. Cisplatin accumulates heterogeneously between the cortex and medulla, with the highest levels of platinum located in the corticomedullary region (Choie et al., 1981). Cisplatin ac-

CISPLATIN

NEPHROTOXICITV

TABLE 5 URINALYSIS IN NORM~GLYCEMIC AND DIABETIC RATS TREATED WITH CISPLATI@ After cisplatin treatment Group

Day 0

Day 1

Day 2

Day 4

Proteinb NSV NSC DSV DSC DIV DIG

++

++

+

++

+

++

+++

++++

+ +

+ -I0 ++

+ ++

+ +

+ ++

+ ++

+ ++’

Glucosec NSV NSC DSV DSC DIV DIC

0 0 ++++ ++++ +++ +++

0 0 ++++ ++++ ++ +++

0 0 ++ ++++ 0 ++++

0 +++ ++ ++++ 0 ++++

r?The presence of protein and glucose was determined using Combistix or Multistix. NSV, normoglycemicvehicle; NSC, normoglycemic-cisplatin; NSV, diabeticvehicle; DSC, diabetic-cisplatin; DIV, diabetic-insulinvehicle; DIG, diabetic-insulin-cisplatin. b Semiquantitative values for protein represent in maJ dlz (+) 30, (++) 100, (+++) 300, (++++) >2000. ’ Semiquantitative values for urinary glucose represent (m&h): (+) 250, (++) 500, (+++) 1000, (++++) 32000.

cumulates in the S3 segment due to active transport of the toxin from the peritubular side of the cell. The !$ segment, which is located in the corticomedullary region, is also the primary site of proximal tubular damage. Consequently, a decreased accumulation of cisplatin would be a likely mechanism for diabetes modulation of cisplatin nephrotoxicity. A decreased level of cisplatin would be expetted if diabetes produces a defect in proximal tubular transport processes on the peritubular side. However, no differences in renal cortical slice uptake of PAH or TEA were measured between normoglycemic and 3- to 28&y diabetic rats.

537

IN DIABETES

Another possible mechanism for diabetesmediated protection of cisplatin nephrotoxicity is enhanced renal excretion. Total urine output in the STZdiabetic rat is over 1O-fold higher than that in the normoglycemic group (Fig. 1). The increased urine output is the result of a glucose-induced osmotic effect. The enhanced diuresis associated with diabetes may promote enhanced excretion of cisplatin and therefore reduce nephrotoxicity. However, further studies are needed to determine if diabetes enhances renal cisplatin excretion. A considerable amount of work has been performed by other investigators examining diabetes-mediated reduction of gentamicin nephrotoxicity. Gentamicin is an aminoglycoside antibiotic that is structurally unrelated to cisplatin. However, similarities may exist in the mechanisms involved in diabetes-mediated reduction of cisplatin and gentamicin nephrotoxicity. Modulation of gentamicin nephrotoxicity is attributed to diminished accumulation of the antibiotic by the diabetic kidney (Elliott et al., 1985). The mechanisms responsible for the diminished accumulation are not entirely known but studies have been

NSV

NSC

DlV

DIC

DSV

DSC

241

PAH

PAH LAC+TATE

TEA

PIG. 4. Renal cortical slice accumulation of PAH and TEA was measured 4 days alter cisnlatin ink&ion. Values represent means + SE. NSV, normoglycemiovehicle; NSC, normoglycemic-cisplatin; DIV, diabetic-insucle; DIC, diabetic-insulin treated-cis~~ti~$$e~~ abetic-vehicle; DSC, diabetic-cisplatin. +r&rence ip 1: 0.05) between cisplatin- and respective vehicle-treated group.

538

SCOTT, MADAN,

AND VALENTOVIC

TABLE 6 BODY WEIGHT,

KIDNEY

WEIGHT,

SERUM GLUCOSE,

AND BUN

LEVELS 4 DAYS AITER CISPLATIN

INJECTIONS

Group Day

NSV

NSC

DSV WY

Control

4

367 + 9’ 294 + 8*

342 f 7 265 ?I 2*

weight

DSC

DIV

DIC

257-+8 262+ 11

323zk5 295 -t 9*

286 Y!I12 289 + 40

0.57 + 0.03

0.44 + 0.02

0.55 iI 0.127

30.2 k 2.2 33.6 + 8.4

33.8 f 2.7 19.0 + 1.7*

35.2 + 2.7 26.5 t 3.0*

608 f 48 783 + 82*

636 k 40 305 2 49*

678 + 62 476 + 14*

(63

295 k 12 252 + 12*

Kidney weight (g/ 100 g Body wt) 4

0.38 -t 0.01

0.53 + 0.01t

0.53 f 0.01 BUN (mg/dl)

Control 4

21.6k0.9 17.0 + o.s*

21.8 + 1.7 28 1.8 f 34.6*x?

29.5 + 1.3 27.5 + 2.0 Glucose (mg/dl)

Control 4

139 f 12.4 142 -+ 8.9

134 + 12.4 420 f 57**t

676 f 47 482 + 57*

’ Body weight, serum glucose, BUN, and kidney weight were measured O-4 days after cisplatin administration. NSV, normoglycemiovehicle; NSC, normoglycemic-cisplatin; DSV, diabetic-vehicle; DSC, diabetic-cisplatin; DIV, diabetic-insulin-vehicle; DIG, diabetic-insulin-cisplatin. All values are expressed as means k SE. * Significant difference (p < 0.05) from respective Control Day values. 7 Significantly different (p -z 0.05) from respectively pair-fed vehicle group.

conducted to examine the contribution of glucosuric diuresis to diabetes-mediated reduction of gentamicin nephrotoxicity. Interestingly, isosorbide-induced diuresis of normoglycemic rats (Cronin et al., 1983) failed to protect against the proximal tubular damage of gentamicin. These results would indicate that diabetes does not reduce gentamicin nephrotoxicity through enhanced excretion of the toxin. A third possible mechanism for diabetesmediated modulation of cisplatin nephrotoxicity is increased renal cellular regeneration. Kidney weight is increased in diabetic patients (Morgensen and Anderson, 1973) as well as experimental animal models of diabetes (Jensen et al., 198 1). The elevated organ weight is due to increased renal growth, which occurs at a very early stage in the development of diabetes. Studies by other investi-

gators have shown an increased content of protein and RNA (Cartes et al., 1980) in the kidney of diabetic animals. Further studies have demonstrated that the content and the actual rate of renal RNA synthesis (Cortes et al., 1987) are accelerated in STZ-diabetic rats. The increased rate of cellular growth in the diabetic kidney is a very plausible mechanism for diabetes-mediated reduction of cisplatin nephrotoxicity. Diabetic animals may recover more rapidly after cisplatin treatment simply because of an inherent capacity to rapidly regenerate new proximal epithe1ia.l cells. Further studies are needed to investigate this hypothesis. These studies have shown that STZ-induced diabetes modulates cisplatin nephrotoxicity. Additionally, insulin treatment partially reverses the protective effect of diabetes. These results indicate that diabetes mediates

CISPLATIN

NEPHROTOXICITY

a reversible change in the kidney which attenuates cisplatin nephrotoxicity. Further studies are needed to investigate the mechanisms involved in diabetes-mediated reduction of cisplatin nephrotoxicity.

ACKNOWLEDGMENTS

REFERENCES D. D., L~NGNE~KER,

D. S., AND DEL CAMPO,

A. A. ( 198 1). Acute and chronic cisplatin nephropathy in rats. Lab. Invest. 44,397-402. TORTES,

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The authors are grateful for the donation of cisplatin by Bristol-Myers Pharmaceutical Company. The expert assistance of Cindy Elliott and Darla Kennedy, as well as the cooperation of the Pathology Laboratory at the Veterans Administration Hospital (Huntington, WV), is gratetitlly appreciated. This research was supported by NIH RR-05870.

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