Abstract
Increased tubular epithelial cell proliferation is a prerequisite for cyst formation and expansion in polycystic kidney disease (PKD). Rapamycin is a potent antiproliferative agent. The aim of the present study was to determine the effect of rapamycin on tubular cell proliferation, cyst formation, and renal failure in the Han:SPRD rat model of PKD. Heterozygous (Cy/+) and littermate control (+/+) male rats were weaned at 3 wk of age and then treated with rapamycin 0.2 mg/kg per d intraperitoneally or vehicle (ethanol) for 5 wk. Vehicle-treated Cy/+ rats had a more than doubling of kidney size compared with +/+ rats. Rapamycin reduced the kidney enlargement by 65%. Rapamycin significantly reduced the cyst volume density in Cy/+ rats by >40%. Blood urea nitrogen was 59% increased in vehicle-treated Cy/+ rats compared with +/+ rats. Rapamycin reduced the blood urea nitrogen to normal in Cy/+ rats. The number of proliferating cell nuclear antigen (PCNA)-positive cells per noncystic tubule was eightfold increased in vehicle-treated Cy/+ compared with +/+ rats. Rapamycin significantly reduced the number of PCNA-positive cells in noncystic tubules of Cy/+ rats. In addition, the number of PCNA-positive cells per cyst in Cy/+ rats was significantly reduced by rapamycin. In summary, in a rat model of PKD, rapamycin treatment (1) decreases proliferation in cystic and noncystic tubules, (2) markedly inhibits renal enlargement and cystogenesis, and (3) prevents the loss of kidney function.
Autosomal dominant polycystic kidney disease (ADPKD) is the most common life-threatening hereditary disease in the United States. ADPKD accounts for approximately 5 to 10% of end-stage renal failure that requires dialysis and renal transplantation in the United States (1). There is no effective treatment for ADPKD.
The heterozygous Han:SPRD rat exhibits many of the features of ADPKD in humans, including (1) autosomal dominant inheritance; (2) relatively slow progression to end-stage renal failure with uremia, hypertension, and anemia; (3) bilateral renal involvement; (4) increased proliferation of renal cyst cells; and (5) more aggressive disease in males (2). The Han:SPRD rat is a suitable and well-documented animal model of PKD even though the defect is based on a mutation of a gene locus that is not a homologue of either the PKD1 or the PKD2 locus (3–6).
Human and experimental data provide strong evidence that abnormal proliferation in tubular epithelial cells plays a crucial role in cyst development and/or growth in PKD (7). Genetic manipulations that induce the proliferation of tubular epithelial cells in mice cause cysts to form in the kidney (8,9). Rapamycin is a Food and Drug Administration–approved immunosuppressive and powerful antiproliferative drug (10). In view of the importance of tubular cell proliferation in cyst formation and the antiproliferative effects of rapamycin, we developed the hypothesis that rapamycin would reduce cyst formation and disease progression in PKD via inhibition of tubular cell proliferation.
Materials and Methods
Animals
The study was conducted in heterozygous (Cy/+) and normal littermate control (+/+) Han:SPRD rats. All of the normal rats and Cy/+ rats studied were male. The Cy/+ Han:SPRD rat develops clinically detectable PKD by 8 wk of age as evidenced by a doubling of kidney size and kidney failure compared with +/+ control rats (2,4). A colony of Han:SPRD rats was established in our animal care facility from a litter that was obtained from the Polycystic Kidney Program at the University of Kansas Medical Center. The study protocol was approved by the University of Colorado Health Sciences Center Animal Care and Use Committee. Rats had free access to tap water and standard rat diet.
Experimental Protocol
Male Cy/+ and +/+ rats were weaned at 3 wk of age and then treated with rapamycin 0.2 mg/kg per d intraperitoneally or vehicle (ethanol) for 5 wk. Rapamycin was obtained from LC Laboratories (Woburn, MA), and a 1 mg/ml stock solution in 100% ethanol was kept at 4°C. At the end of the eighth week of age, rats were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt), and kidneys were removed and weighed. The left kidney was fixed in 4% paraformaldehyde in PBS for 120 min and then put into 70% ethanol and embedded in paraffin for histologic examinations.
Cyst Volume Density
Hematoxylin-eosin–stained sections were used to determine the cyst volume density. This was performed by a reviewer who was blinded to the identity of the treatment modality, using point counting stereology (11). Areas of the cortex at 90, 180, and 270 degrees from the hilum of each section were selected to guard against field selection variation.
Immunohistochemistry
Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) staining was performed using an anti-PCNA antibody (Santa Cruz Biotechnology, Santa Cruz, CA; 1:50). The sections were incubated with alkaline-phosphatase–labeled polymer (DAKO EnVision System, Cat# K4016; DAKO, Carpinteria, CA) and visualized with the substrate chromogen fast red. Negative control sections showed no staining.
Tubular Cell Proliferation
The number of PCNA-positive cells per tubule was counted using a Nikon Eclipse E400 microscope equipped with a digital camera connected to Spot Advanced imaging software (Version 3.5) by an observer who was blinded to the treatment modality. Noncystic tubules were defined as tubules that were <50 μm in diameter. At least 10 noncystic tubules or cysts in the cortex and medulla per sample were randomly selected and counted.
To avoid confusion between noncystic tubules and small cysts as well as potential changes in tubular cells lining massive cysts, PCNA-positive tubular cells were counted in “medium-sized cysts” of approximately 250 μm in diameter. At least 10 cysts per sample in the cortex were randomly selected and counted.
Chemistry
Serum urea nitrogen levels were measured using a Beckman autoanalyzer (Beckman Instruments, Fullerton, CA).
Statistical Analyses
Nonnormally distributed data were analyzed by the nonparametric unpaired Mann-Whitney test. Multiple group comparisons were performed using a one-way ANOVA with posttest according to Newman-Keuls. P < 0.05 was considered statistically significant. Values are expressed as means ± SEM.
Results
Effect of Rapamycin on Body Weight, Two-Kidney/Total Body Weight Ratio, Cyst Volume Density, and Blood Urea Nitrogen
The body weight was 248 g in vehicle-treated +/+ rats (n = 8), 193 g in rapamycin-treated +/+ rats (P < 0.01 versus vehicle-treated +/+; n = 6), 260 g in vehicle-treated Cy/+ rats (n = 9), and 200 g in rapamycin-treated Cy/+ rats (P < 0.01 versus vehicle-treated Cy/+; n = 5). Thus rapamycin-treated +/+ and Cy/+ rats had a 22% decrease in body mass.
Despite the loss in body weight, all of the rats seemed healthy during the study. Two rats of a total of 16 rats that were treated with rapamycin died. These rats were not noted to be sick before death. None of a total of 17 vehicle-treated rats died.
Representative kidney sections of vehicle-treated +/+, vehicle treated Cy/+, and rapamycin-treated Cy/+ rats, stained with hematoxylin-eosin, at the same magnification are demonstrated in Figure 1, A through C. The two-kidney/total body weight ratio was determined to correct for the lower body mass caused by the rapamycin. Two-kidney/total body weight ratio (%) was 0.9 ± 0.04 in vehicle-treated +/+ rats (n = 8), 0.9 ± 0.03 in rapamycin-treated +/+ rats (n = 6), 2.0 ± 0.1 in vehicle-treated Cy/+ rats (P < 0.001 versus +/+; n = 11), and 1.3 ± 0.1 in rapamycin-treated Cy/+ rats (P < 0.001 versus vehicle-treated Cy/+ rats; n = 9; Figure 1D). Cy/+ rats had a more than doubling of kidney size compared with +/+ controls. Rapamycin reduced the kidney enlargement by 65%.
Effects of rapamycin (Rapa) on the development of PKD in Han:SPRD rats. (A through C) Representative kidney sections, stained with hematoxylin-eosin, at the same magnification of vehicle-treated control (+/+; A), vehicle-treated heterozygous (Cy/+; B), and rapamycin-treated Cy/+ rats (C). (D) The two-kidney/total body weight ratio was unaffected by rapamycin in +/+ rats. Cy/+ rats had a more than doubling of kidney size compared with +/+ controls. Rapamycin reduced the kidney enlargement in Cy/+ rats by 65%. *P < 0.001 versus +/+; **P < 0.001 versus vehicle-treated Cy/+. (E) The cyst volume density was hugely increased in vehicle-treated Cy/+ rats. Rapamycin reduced the cyst volume density by >40%. *P < 0.001 versus +/+, **P < 0.01 versus vehicle treated Cy/+. (F) Blood urea nitrogen (BUN) was unaffected by rapamycin in +/+ rats. Cy/+ rats with polycystic kidney disease have kidney failure as indicated by a close to doubling in BUN. Rapamycin completely prevented the increase in BUN in the Cy/+ rats. *P < 0.01 versus vehicle-treated and rapamycin-treated +/+ rats; **P < 0.01 versus vehicle-treated Cy/+ rats, NS versus vehicle-treated and rapamycin-treated +/+ rats.
Cyst volume density (CVD; %) was 0.6 ± 0.1 in vehicle-treated +/+ rats (n = 7), 46 ± 4 in vehicle-treated Cy/+ rats (P < 0.001 versus +/+; n = 8), and 25 ± 4 in rapamycin-treated Cy/+ rats (P < 0.01 versus vehicle-treated Cy/+; n = 5; Figure 1E). Thus, rapamycin reduced the CVD by >40%.
Blood urea nitrogen was 22 ± 4 in vehicle-treated +/+ rats (n = 5), 21 ± 2 in rapamycin-treated +/+ rats (n = 5), 35 ± 2 in vehicle-treated Cy/+ rats (P < 0.01 versus vehicle-or rapamycin-treated +/+; n = 6), and 23 ± 1 in rapamycin-treated Cy/+ rats (P < 0.01 versus vehicle-treated Cy/+, NS versus rapamycin-treated +/+; n = 5; Figure 1F). Thus, rapamycin completely prevented the increase in blood urea nitrogen in the Cy/+ rats.
Tubular Cell Proliferation
The number of PCNA-positive cells per tubule in noncystic tubules in the cortex and medulla was 0.08 ± 0.02 in vehicle-treated +/+ rats, 0.17 ± 0.04 in rapamycin-treated +/+ rats, 0.65 ± 0.1 in vehicle-treated Cy/+ rats (P < 0.05 versus vehicle or rapamycin-treated +/+), and 0.2 ± 0.1 in rapamycin-treated Cy/+ rats (P < 0.05 versus vehicle treated, NS versus +/+; Figure 2A). Representative pictures are shown in Figure 2, B through D. The number of PCNA-positive cells per cyst in the cortex was 2.3 ± 0.5 in vehicle-treated Cy/+ rats and 0.4 ± 0.3 in rapamycin-treated Cy/+ rats (P < 0.05 versus vehicle-treated rats; Figure 3A). Representative pictures are shown in Figure 3, B and C.
Tubular cell proliferation in noncystic tubules. (A) The number of proliferating cell nuclear antigen (PCNA)-positive cells per tubule in noncystic tubules in the cortex and medulla was increased in vehicle-treated Cy/+ rats compared with vehicle-treated and rapamycin-treated +/+ rats. Rapamycin-treated Cy/+ rats had significantly fewer PCNA-positive cells per tubule compared with vehicle-treated Cy/+ rats. *P < 0.05 versus +/+ rats; **P < 0.05 versus vehicle treated Cy/+ rats, NS versus +/+. (B) Vehicle-treated +/+ rats had relatively few PCNA-positive cells per tubule (arrows) compared with vehicle-treated Cy/+ rats (C; arrows). (D) Rapamycin decreased the number of PCNA-positive cells per tubule in Cy/+ rats (arrows).
Tubular cell proliferation in cysts. (A) The number of PCNA-positive cells in tubular epithelial cells lining the cysts was significantly decreased by rapamycin (Rapa). *P < 0.05 versus vehicle-treated Cy/+ rats. (B) Representative picture of the PCNA staining (arrows) in cysts of vehicle-treated Cy/+ rats. (C) Representative picture of the PCNA staining (arrows) in cysts of rapamycin-treated Cy/+ rats.
Discussion
The bacterially-derived drug rapamycin, also known as sirolimus, specifically inhibits the mammalian target of rapamycin. The mammalian target of rapamycin is a serine threonine kinase that plays a central role in the regulation of cell proliferation, growth, differentiation, migration, and survival (12). Rapamycin is known to cause reduced cell growth, a reduced rate of cell-cycle progression, and reduced cell proliferation (13). Thus, rapamycin and its analogs CCI-779 and RAD001 have demonstrated promising anticancer activity and relatively mild side effects in phase I and II clinical studies (12). In addition, rapamycin inhibits proliferation of hematopoietic cells and is used to inhibit kidney transplant rejection. Because proliferation of tubular cells is one of the mechanisms of cyst enlargement in PKD, we examined the effect of rapamycin. In this regard, an EGF receptor tyrosine kinase inhibitor attenuated the cystic disease but not the renal failure in Han:SPRD rats (14). Another antiproliferative agent, paclitaxel, had no effect on PKD and increased mortality in Han:SPRD rats (15), whereas it had a remarkable effect on kidney size, renal function, and survival in congenital PKD (16). The immunosuppressive drug methylprednisolone retards interstitial fibrosis and the progression of PKD in the Han:SPRD rat (17).
A consistent finding in the present study was that rapamycin decreased tubular cell proliferation in noncystic as well as cystic tubules. Although the proliferation index has been found to be consistently highest in cystic tubular epithelium, noncystic tubules from mice with polycystic kidneys (18) and Han:SPRD rats (6) have higher proliferation rates than tubules from age-matched controls. In the present study, noncystic tubules in the cortex and medulla from Cy/+ rats with PKD had an eightfold increase in PCNA-positive cells compared with +/+ controls without PKD. These studies suggest that tubular cell proliferation precedes cyst formation in the Han:SPRD rat (6). Thus, rapamycin seems to decrease cyst formation, in part, by decreasing tubular cell proliferation in noncystic tubules.
Another finding of the present study was that rapamycin modestly decreased the body weight in both Cy/+ and control rats despite no apparent difference in food intake. Other studies in rats (19) and mice (20) have described weight loss caused by rapamycin. Long-term treatment with rapamycin has not been reported to cause weight loss in adults or children (21,22). The effect of lower doses of rapamycin and treatment for a longer period of time are the subject of ongoing animal studies.
The rats were treated from a young age just after being weaned from their mothers. The rapamycin seemed to have no major side effects as the rats seemed healthy. Rapamycin has an excellent safety profile in both children and adults (21,22). In fact, transplant patients with nephrotoxic effects of calcineurin antagonists are often changed to rapamycin (23).
ADPKD is a significant cause of renal failure in adults. There are currently no effective treatments. Rapamycin is a Food and Drug Administration–approved immunosuppressive drug to inhibit kidney transplant rejection (10). The ability of rapamycin to inhibit markedly cyst formation and prevent renal failure in rats demonstrates its potential to delay the onset of renal failure and subsequent dialysis in ADPKD patients. However, the usefulness of rapamycin and its analogs remains to be proved in other animal models of PKD and in randomized clinical trials in human ADPKD. In view of the absence of effective therapies in ADPKD and the safety of rapamycin as evidenced by long-term use in adults and children to prevent transplant rejection, future studies in other animal models of PKD and even future clinical studies are an exciting prospect.
Acknowledgments
This work was supported by National Institutes of Health Grants DK56851 and DK34039 (section 3) to C.L.E. and DK52599 and DK34039 to R.W.S.
- © 2005 American Society of Nephrology
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