Sirolimus attenuates disease progression in an orthologous mouse model of human autosomal dominant polycystic kidney disease
2010; Elsevier BV; Volume: 78; Issue: 8 Linguagem: Inglês
10.1038/ki.2010.250
ISSN1523-1755
AutoresIram Zafar, Kameswaran Ravichandran, Franck A. Belibi, R. Brian Doctor, Charles L. Edelstein,
Tópico(s)Renal Diseases and Glomerulopathies
ResumoIn autosomal dominant polycystic kidney disease (ADPKD), abnormal proliferation of tubular cells drives cyst development and growth. Sirolimus, an inhibitor of the protein kinase mammalian target of rapamycin (mTOR) and a potent anti-proliferative agent, decreases cyst growth in several genetically distinct rodent models of polycystic kidney disease (PKD). We determined here the effect of sirolimus on renal cyst growth in Pkd2WS25/− mice; an ortholog of human ADPKD involving mutation of the Pkd2 gene. In Pkd2WS25/− mice treated with sirolimus, both the two kidney/total body weight (2K/TBW) ratio and the cyst volume density (CVD) were significantly decreased by over half compared with untreated mice suffering with PKD. However, there was no effect on the increased blood urea nitrogen (BUN) levels as an index of kidney function. There are two distinct complexes containing mTOR depending on its binding partners: mTORC1 and mTORC2. Western blot analysis of whole kidney lysates and immunohistochemistry of the cysts found that phospho-S6 ribosomal protein, a marker of mTORC1 activity, was increased in Pkd2WS25/− mice and its phosphorylation was decreased by sirolimus treatment. Phospho-Akt at serine 473, a marker associated with mTORC2 activity, was not different between Pkd2WS25/− mice and normal littermate controls. Hence, our study found that inhibition of mTORC1 by sirolimus correlated with decreased renal cyst growth in this model of human ADPKD but had no effect on the decline in renal function. In autosomal dominant polycystic kidney disease (ADPKD), abnormal proliferation of tubular cells drives cyst development and growth. Sirolimus, an inhibitor of the protein kinase mammalian target of rapamycin (mTOR) and a potent anti-proliferative agent, decreases cyst growth in several genetically distinct rodent models of polycystic kidney disease (PKD). We determined here the effect of sirolimus on renal cyst growth in Pkd2WS25/− mice; an ortholog of human ADPKD involving mutation of the Pkd2 gene. In Pkd2WS25/− mice treated with sirolimus, both the two kidney/total body weight (2K/TBW) ratio and the cyst volume density (CVD) were significantly decreased by over half compared with untreated mice suffering with PKD. However, there was no effect on the increased blood urea nitrogen (BUN) levels as an index of kidney function. There are two distinct complexes containing mTOR depending on its binding partners: mTORC1 and mTORC2. Western blot analysis of whole kidney lysates and immunohistochemistry of the cysts found that phospho-S6 ribosomal protein, a marker of mTORC1 activity, was increased in Pkd2WS25/− mice and its phosphorylation was decreased by sirolimus treatment. Phospho-Akt at serine 473, a marker associated with mTORC2 activity, was not different between Pkd2WS25/− mice and normal littermate controls. Hence, our study found that inhibition of mTORC1 by sirolimus correlated with decreased renal cyst growth in this model of human ADPKD but had no effect on the decline in renal function. We have previously shown in the Han:SPRD rat model of polycystic kidney disease (PKD) that sirolimus treatment decreases proliferation in cystic and non-cystic tubules, markedly inhibits renal enlargement and cystogenesis and prevents the loss of kidney function.1.Tao Y. Kim J. Schrier R.W. et al.Rapamycin markedly slows disease progression in a rat model of polycystic kidney disease (PKD).J Am Soc Nephrol. 2005; 16: 46-51Crossref PubMed Scopus (302) Google Scholar Subsequently two other studies have shown that mammalian target of rapamycin (mTOR) inhibition with sirolimus2.Wahl P.R. Serra A.L. Le Hir M. et al.Inhibition of mTOR with sirolimus slows disease progression in Han:SPRD rats with autosomal dominant polycystic kidney disease (ADPKD).Nephrol Dial Transplant. 2006; 21: 598-604Crossref PubMed Scopus (222) Google Scholar or everolimus3.Wu M. Wahl P.R. Le Hir M. et al.Everolimus retards cyst growth and preserves kidney function in a rodent model for polycystic kidney disease.Kidney Blood Press Res. 2007; 30: 253-259Crossref PubMed Scopus (89) Google Scholar reduces cyst formation and renal failure in the Han:SPRD rat. It has also recently been shown that sirolimus decreases cyst formation and renal failure in the orpk-rescue mouse (defective cilia protein polaris) and the bpk mouse (overexpressing myelin and lymphocyte protein) models of PKD.4.Shillingford J.M. Murcia N.S. Larson C.H. et al.The mTOR pathway is regulated by polycystin-1, and its inhibition reverses renal cystogenesis in polycystic kidney disease.Proc Natl Acad Sci USA. 2006; 103: 5466-5471Crossref PubMed Scopus (586) Google Scholar Autosomal dominant polycystic kidney disease (ADPKD) in humans is caused by a mutation in the Pkd1 or Pkd2 gene. The Han:SPRD rat, orpk, and bpk mouse do not have primary abnormalities of the Pkd1 or Pkd2 genes as the cause of the PKD. In this study, we tested the hypothesis that sirolimus therapy would decrease PKD caused by a mutation in the Pkd2 gene, in the Pkd2WS25/− mouse model. These mice were engineered to have one of the Pkd2 alleles knocked out and the other Pkd2 allele capable of undergoing high rates of recombination with one of the recombination products resulting in the loss of a functional gene. This study in Pkd2WS25/− mice is important as not all therapies that ameliorate murine cystic disease in diverse models may be effective in disease caused by Pkd gene mutations and the Pkd2WS25/− mice model closely resembles human ADPKD. The mTOR exists in association with two different complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). The mTORC1 is a complex of mTOR and Raptor (regulatory associated protein of mTOR) whereas mTORC2 is a complex of mTOR and Rictor (sirolimus-independent companion of mTOR). The mTORC1 complex regulates cell growth (size), proliferation, apoptosis, and autophagy. Activation of mTORC1 has been shown in PKD in rodents3.Wu M. Wahl P.R. Le Hir M. et al.Everolimus retards cyst growth and preserves kidney function in a rodent model for polycystic kidney disease.Kidney Blood Press Res. 2007; 30: 253-259Crossref PubMed Scopus (89) Google Scholar, 4.Shillingford J.M. Murcia N.S. Larson C.H. et al.The mTOR pathway is regulated by polycystin-1, and its inhibition reverses renal cystogenesis in polycystic kidney disease.Proc Natl Acad Sci USA. 2006; 103: 5466-5471Crossref PubMed Scopus (586) Google Scholar, 5.Weimbs T. Regulation of mTOR by polycystin-1: is polycystic kidney disease a case of futile repair?.Cell Cycle. 2006; 5: 2425-2429Crossref PubMed Scopus (46) Google Scholar and in humans.6.Fischer D.C. Jacoby U. Pape L. et al.Activation of the AKT/mTOR pathway in autosomal recessive polycystic kidney disease (ARPKD).Nephrol Dial Transplant. 2009; 24: 1819-1827Crossref PubMed Scopus (59) Google Scholar The effect of PKD and sirolimus on mTORC2 is not known. The mTORC2 increases proliferation, inhibits apoptosis, regulates the actin cytoskeleton, and can phosphorylate Akt at serine 473.7.Boletta A. Emerging evidence of a link between the polycystins and the mTOR pathways.Pathogenetics. 2009; 2: 6Crossref PubMed Google Scholar As proliferation and apoptosis8.Edelstein C.L. What is the role of tubular epithelial cell apoptosis in polycystic kidney disease (PKD)?.Cell Cycle. 2005; 4: e141-e145Crossref Scopus (40) Google Scholar, 9.Tao Y. Kim J. Faubel S. et al.Caspase inhibition reduces tubular apoptosis and proliferation and slows disease progression in polycystic kidney disease (PKD).Proc Natl Acad Sci USA. 2005; 102: 6954-6959Crossref PubMed Scopus (89) Google Scholar cytoskeleton abnormalities10.Witzgall R. Polycystic kidney disease–a tale of calcium channels and the actin cytoskeleton.Anna Anat. 2001; 183: 391-392Crossref PubMed Scopus (2) Google Scholar, 11.Boca M. D'Amato L. Distefano G. et al.Polycystin-1 induces cell migration by regulating phosphatidylinositol 3-kinase-dependent cytoskeletal rearrangements and GSK3beta-dependent cell cell mechanical adhesion.Mol Biol Cell. 2007; 18: 4050-4061Crossref PubMed Scopus (78) Google Scholarand activation of Akt3.Wu M. Wahl P.R. Le Hir M. et al.Everolimus retards cyst growth and preserves kidney function in a rodent model for polycystic kidney disease.Kidney Blood Press Res. 2007; 30: 253-259Crossref PubMed Scopus (89) Google Scholar are features of PKD, the degree of mTORC2 activation was determined. In this study, we tested the hypotheses that there would be increased mTORC1 and mTORC2 activation in Pkd2WS25/− mouse kidneys and that the mTORC1 inhibitor, sirolimus would decrease PKD in Pkd2WS25/− mice. Sirolimus had no effect on the body weight (Table 1). This is in contrast to previous reports showing that short-term (5 week) 0.2 mg/kg/d sirolimus treatment in Han:SPRD rats and wild-type male rats resulted in a 22% loss of body weight.1.Tao Y. Kim J. Schrier R.W. et al.Rapamycin markedly slows disease progression in a rat model of polycystic kidney disease (PKD).J Am Soc Nephrol. 2005; 16: 46-51Crossref PubMed Scopus (302) Google Scholar In this study, mice received a higher dose of sirolimus (that is, 0.5 mg/kg/d) for a longer period of time (that is, 8–12 weeks) but did not have weight loss.Table 1Effects of sirolimus in Pkd2WS25/− mice+/+ vehicle (n=24)+/+ sirolimus (n=8)Pkd2WS25/− vehicle (N=10)Pkd2WS25/− sirolimus (N=10)Body weight (g)22.8±3.123.4±0.727.4±127.6±1Kidney weight (g)0.31±0.010.27±0.020.60±0.11*P<0.01 vs +/+ vehicle and +/+ sirolimus0.44±0.04**P<0.05 vs Pkd2WS25/− vehicle, NS vs +/+ vehicle and +/+ sirolimus.2K/TBW (%)1.24±0.021.15±0.052.20±0.4*P<0.01 vs +/+ vehicle and +/+ sirolimus1.63±0.1**P<0.05 vs Pkd2WS25/− vehicle, NS vs +/+ vehicle and +/+ sirolimus.CVD (%)0.5±0.20.5±0.238.8 ±3.7†P<0.001 +/+ vehicle and +/+ sirolimus20.4±5.1P<0.01 vs Pkd2WS25/− vehicleBUN (mg/dl)16.7±0.917.5±0.933.8±4.2*P<0.01 vs +/+ vehicle and +/+ sirolimus29.2±1.5‡NS vs Pkd2WS25/− vehicle. The P-value for the interaction between sirolimus and genotype was NS for kidney weight, NS for 2K/TBW (%), 0.009 for CVD (%) and NS for BUN.Abbreviations: BUN, blood urea nitrogen; CVD, cystic volume density; NS, not significant; 2K/TBW, two kidney/total body weight.* P<0.01 vs +/+ vehicle and +/+ sirolimus** P<0.05 vs Pkd2WS25/− vehicle, NS vs +/+ vehicle and +/+ sirolimus.† P<0.001 +/+ vehicle and +/+ sirolimus†† P<0.01 vs Pkd2WS25/− vehicle‡ NS vs Pkd2WS25/− vehicle. The P-value for the interaction between sirolimus and genotype was NS for kidney weight, NS for 2K/TBW (%), 0.009 for CVD (%) and NS for BUN. Open table in a new tab Abbreviations: BUN, blood urea nitrogen; CVD, cystic volume density; NS, not significant; 2K/TBW, two kidney/total body weight. Representative kidney sections of vehicle-treated Pkd2WS25/− mice and sirolimus-treated Pkd2WS25/− mice, stained with hematoxylin-eosin, at the same magnification are shown in Figure 1a and b. These representative sections show that the percentage of the kidney that is occupied by cysts is markedly reduced in the kidney obtained from the sirolimus-treated Pkd2WS25/− mice. The two kidney/total body weight ratio (2K/TBW), which corrects for differences in body weight, was nearly double in Pkd2WS25/− mice compared with vehicle-treated +/+ mice. Sirolimus resulted in a 61% decrease in 2K/TBW (Table 1). The increase in kidney weights in the Pkd2WS25/− mice was directly paralleled by increases in the measured cyst volume density (CVD) in the kidneys. Mean CVD percentage was 39% in Pkd2WS25/− mice treated with vehicle and 20% in Pkd2WS25/− mice treated with sirolimus. Sirolimus resulted in a 51% decrease in CVD (Table 1). The blood urea nitrogen (BUN) level was increased in vehicle-treated Pkd2WS25/− mice and sirolimus-treated Pkd2WS25/− mice compared with vehicle-treated +/+ mice and sirolimus-treated +/+ mice. BUN concentration was not different between vehicle-treated Pkd2WS25/− mice and sirolimus-treated Pkd2WS25/− mice (Table 1). Female Pkd2WS25/− mice have less severe PKD than that that in males.12.Doctor R.B. Serkova N. Hasebroock K. et al.Distinct patterns of kidney and liver cyst growth in Pkd2WS25/− mice.Nephrol Dial Transplant. 2010Google Scholar Only two of the ten Pkd2WS25/− vehicle and two of the ten Pkd2WS25/−sirolimus-treated mice were female. The two female Pkd2WS25/− mice treated with sirolimus had a decrease in 2K/TBW (%) and CVD compared with female littermate controls. In the Pkd2WS25/− model, liver cyst formation increases significantly after 16 weeks of age.12.Doctor R.B. Serkova N. Hasebroock K. et al.Distinct patterns of kidney and liver cyst growth in Pkd2WS25/− mice.Nephrol Dial Transplant. 2010Google Scholar This study ended at 16 weeks of age. At the end of the study, liver weight was identical (1.6 g) in Pkd2WS25/− treated with vehicle and Pkd2WS25/− treated with sirolimus. To evaluate renal fibrosis, quantitation of collagen deposition by Masson's trichrome staining was carried out. As shown in Figure 2, sirolimus reduced collagen staining. The renal failure in PKD has been linked to fibrosis. However, despite the reduction in collagen staining by sirolimus, there was no effect on kidney function by sirolimus. The number of terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive apoptotic cells per cyst was 0.15±0.06 in Pkd2WS25/− mice treated with the vehicle and 0.26±0.09 in Pkd2WS25/− mice treated with sirolimus (P=not significant vs vehicle-treated, N=4) (Figure 3). Representative pictures of TUNEL staining are shown in Figure 3. Caspase-3 is the major mediator of apoptosis. In support of the data that there is no significant difference in apoptosis with sirolimus treatment, caspase-3 activity was not significantly affected by sirolimus. Caspase-3 activity (nmol/min/mg) was 5.4±2.3 in Pkd2WS25/− mice treated with the vehicle and 2.9±1.7 in Pkd2WS25/− mice treated with sirolimus (P=not significant vs vehicle-treated, N=5). Activation of mTORC1, but not mTORC2, results in the phosphorylation of S6 ribosomal protein. On immunoblotting there was an increase in phospho-S6 ribosomal protein in vehicle-treated Pkd2WS25/− mouse kidneys compared with kidneys from normal littermate controls (+/+) (Figure 4). The increase in phospho-S6 ribosomal protein in Pkd2WS25/− kidneys was inhibited by sirolimus (Figure 4). Total S6 ribosomal protein, used as a control, was not significantly different in vehicle-treated controls (+/+), vehicle-treated Pkd2WS25/−, vehicle-treated Pkd2WS25/−, and sirolimus-treated Pkd2WS25/− kidneys (Figure 4). To identify the localization of phospho-S6 ribosomal protein, immunohistochemical staining was performed. On immunohistochemical staining, there was increased phospho-S6 ribosomal protein in both noncystic and cystic tubules of Pkd2WS25/− mice. The increase in phospho-S6 ribosomal protein was decreased by sirolimus. Representative pictures of phospho-S6 ribosomal protein staining are shown in Figure 5. On immunoblotting of whole kidney lysates, Rictor protein was not significantly different between normal littermate controls (+/+) and Pkd2WS25/− mice kidneys (Figure 6a). Interestingly, sirolimus treatment of Pkd2WS25/− resulted in a significant decrease in the abundance of Rictor (Figure 6b). Phospho-AKT (serine 473) is directly phosphorylated by mTORC2 and phospho-AKT is a marker of mTORC2 activation.13.Wullschleger S. Loewith R. Hall M.N. TOR signaling in growth and metabolism.Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4362) Google Scholar, 14.Sarbassov D.D. Ali S.M. Sengupta S. et al.Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB.Molecular Cell. 2006; 22: 159-168Abstract Full Text Full Text PDF PubMed Scopus (2057) Google Scholar, 15.Toschi A. Lee E. Xu L. et al.Regulation of mTORC1 and mTORC2 complex assembly by phosphatidic acid: competition with rapamycin.MolCell Biol. 2009; 29: 1411-1420Crossref PubMed Scopus (242) Google Scholar Phospho-AKT (serine 473) was not different in whole kidney lysates between normal littermate controls and Pkd2WS25/− kidneys (Figure 7a). Furthermore, sirolimus had no inhibitory effect on levels of phospho-AKT (serine 473) (Figure 7b). Two mutations have been induced in the mouse homolog Pkd2 gene: an unstable allele (WS25; also known as Pkd2WS25/− mice) that can undergo homologous recombination-based somatic rearrangement to form a null allele; and a true null mutation (WS183; also known as Pkd2−/− mice).16.Wu G. Markowitz G.S. Li L. et al.Cardiac defects and renal failure in mice with targeted mutations in Pkd2.Nat Genet. 2000; 24: 75-78Crossref PubMed Scopus (276) Google Scholar Adult Pkd2+/− mice have intermediate survival in the absence of cystic disease or renal failure, providing the first indication of a deleterious effect of haploinsufficiency of Pkd2 on long-term survival. As in human ADPKD, formation of kidney cysts in adult Pkd2WS25/− mice is associated with renal failure and early death. The vasopressin V2 receptor antagonist OPC31260 decreases renal CVD and BUN in Pkd2WS25/− mice.17.Torres V.E. Wang X. Qian Q. et al.Effective treatment of an orthologous model of autosomal dominant polycystic kidney disease.Nat Med. 2004; 10: 363-364Crossref PubMed Scopus (357) Google Scholar Treatment of Pkd2WS25/− mice between 4 and 8 months of age with the VEGFR inhibitor, SU-5416, markedly reduced liver weight and CVD of the liver.18.Amura C.R. Brodsky K.S. Groff R. et al.VEGF receptor inhibition blocks liver cyst growth in pkd2(WS25/−) mice.Am J Physiol Cell Physiol. 2007; 293: C419-C428Crossref PubMed Scopus (51) Google Scholar These adult Pkd2WS25/− mice were used in this study. Study of mice with targeted mutations of the PKD gene have advanced our understanding of ADPKD as these mouse models recapitulate the complex human ADPKD phenotype.19.Somlo S. Markowitz G.S. The pathogenesis of autosomal dominant polycystic kidney disease: an update.Curr Opin Nephrol Hypertens. 2000; 9: 385-394Crossref PubMed Scopus (25) Google Scholar Human and experimental data provide strong evidence that abnormal proliferation in tubular epithelial cells has a crucial role in cyst development and/or growth in PKD.20.Wilson P.D. Polycystic kidney disease.N Engl J Med. 2004; 350: 151-164Crossref PubMed Scopus (582) Google Scholar Genetic manipulations that induce the proliferation of tubular epithelial cells in mice cause cysts to form in the kidney.21.Trudel M. D′Agati V. Costantini F. C-myc as an inducer of polycystic kidney disease in transgenic mice.Kidney Int. 1991; 39: 665-671Abstract Full Text PDF PubMed Scopus (180) Google Scholar, 22.Schaffner D.L. Barrios R. Massey C. et al.Targeting of the rasT24 oncogene to the proximal convoluted tubules in transgenic mice results in hyperplasia and polycystic kidneys.Am J Pathol. 1993; 142: 1051-1060PubMed Google Scholar The therapeutic effect of sirolimus in PKD is related to its anti-proliferative effect. Although the proliferation index is consistently highest in cystic tubular epithelium, non-cystic tubules from mice suffering with PKD23.Trudel M. Barisoni L. Lanoix J. et al.Polycystic kidney disease in SBM transgenic mice: role of c-myc in disease induction and progression.Am J Pathol. 1998; 152: 219-229PubMed Google Scholar and Han:SPRD rats1.Tao Y. Kim J. Schrier R.W. et al.Rapamycin markedly slows disease progression in a rat model of polycystic kidney disease (PKD).J Am Soc Nephrol. 2005; 16: 46-51Crossref PubMed Scopus (302) Google Scholar, 24.Ramasubbu K. Gretz N. Bachmann S. Increased epithelial cell proliferation and abnormal extracellular matrix in rat polycystic kidney disease.J Am Soc Nephrol. 1998; 9: 937-945PubMed Google Scholar have higher proliferation rates than tubules from age-matched controls. These studies suggest that tubular cell proliferation precedes cyst formation in the Han:SPRD rat.24.Ramasubbu K. Gretz N. Bachmann S. Increased epithelial cell proliferation and abnormal extracellular matrix in rat polycystic kidney disease.J Am Soc Nephrol. 1998; 9: 937-945PubMed Google Scholar In Pkd2WS25/− mice, increased cell proliferation is an early event preceding cyst formation.25.Chang M.Y. Parker E. Ibrahim S. et al.Haploinsufficiency of Pkd2 is associated with increased tubular cell proliferation and interstitial fibrosis in two murine Pkd2 models.Nephrol Dial Transplant. 2006; 21: 2078-2084Crossref PubMed Scopus (65) Google Scholar In this study non cystic tubules from Pkd2WS25/− mice had higher expression of phospho-S6 protein than non cystic tubules from wild-type mice (Figure 4) suggesting that activation of the mTORC1 signaling pathway is an early event preceding cyst formation. In this study, sirolimus therapy was started at 4–8 weeks of age, a time when there is already significant proliferation and kidney cyst formation in Pkd2WS25/− mice.16.Wu G. Markowitz G.S. Li L. et al.Cardiac defects and renal failure in mice with targeted mutations in Pkd2.Nat Genet. 2000; 24: 75-78Crossref PubMed Scopus (276) Google Scholar, 25.Chang M.Y. Parker E. Ibrahim S. et al.Haploinsufficiency of Pkd2 is associated with increased tubular cell proliferation and interstitial fibrosis in two murine Pkd2 models.Nephrol Dial Transplant. 2006; 21: 2078-2084Crossref PubMed Scopus (65) Google Scholar Thus, it is likely that initiation of therapy earlier would have had a better therapeutic effect. On the other hand sirolimus therapy initiated after significant proliferation and cyst formation, as in this study, still had a therapeutic effect. Thus, this study has implications for the treatment of patients, which in many cases will be initiated after significant cyst formation. The Pkd2WS25/− mice had a 2K/TBW ratio that was nearly double that of normal littermate control mice. The Pkd2WS25/− mice had chronic kidney disease as indicated by a BUN concentration that was double that of normal littermate control mice. Sirolimus significantly decreased kidney and cyst size but had no effect on the chronic kidney disease as assessed by the BUN. Although an inverse relationship between increasing cyst volume and decreasing kidney function is known,26.Chapman A.B. Guay-Woodford L.M. Grantham J.J. Glockner J.F. Wetzel L.H. Brummer M.E. O′Neill W.C. Robbin M.L. Bennett W.M. Klahr S. Hirschman G.H. Kimmel P.L. Thompson P.A. Miller J.P. et al.Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease, cohort: Renal structure in early autosomal-dominant polycystic kidney disease (ADPKD): The Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease (CRISP) cohort.Kidney Int. 2003; 64: 1035-1045Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar it is shown that the 51% decrease in CVD caused by sirolimus was not enough to affect renal function in Pkd2WS25/− mice. Therapeutic blood levels of sirolimus in humans are 5–15 ng/ml. The mean trough sirolimus levels in '++' and 'ws25' mice was above therapeutic levels. The high blood levels raise the possibility that sirolimus nephrotoxicity may have contributed to the lack of an effect of sirolimus on BUN. However, sirolimus has minimal functional and histopathological effects in normal rat and mouse kidney. In Sprague–Dawley rats, sirolimus (10 mg/kg/d), a dose three times higher than its effective immunosuppressive dose, had no functional or histological effects on the kidney.27.DiJoseph J.F. Sharma R.N. Chang J.Y. The effect of rapamycin on kidney function in the Sprague-Dawley rat.Transplantation. 1992; 53: 507-513Crossref PubMed Scopus (95) Google Scholar In mice, sirolimus (up to 100 mg/kg), a dose 50 times higher than its therapeutic dose, reduced body weight after 7 days but had minimal effect on kidney function and histology.28.Di Joseph J.F. Sehgal S.N. Functional and histopathologic effects of rapamycin on mouse kidney.Immunopharmacol Immunotoxicol. 1993; 15: 45-56Crossref PubMed Scopus (13) Google Scholar Thus, as opposed to cyclosporin, we do not think that there was nephrotoxicity due to sirolimus. In addition, unlike Han:SPRD rats treated with sirolimus (0.2 mg/kg/d) for 5 weeks1.Tao Y. Kim J. Schrier R.W. et al.Rapamycin markedly slows disease progression in a rat model of polycystic kidney disease (PKD).J Am Soc Nephrol. 2005; 16: 46-51Crossref PubMed Scopus (302) Google Scholar or 1 year29.Zafar I. Belibi F.A. He Z. et al.Long-term rapamycin therapy in the Han:SPRD rat model of polycystic kidney disease (PKD).Nephrol Dial Transplant. 2009; 24: 2349-2353Crossref PubMed Scopus (63) Google Scholar that 22% loss in body weight, the Pkd2WS25/− mice or normal littermate control mice treated with 0.5 mg/kg/d did not experience weight loss suggesting that weight loss was not a side effect of sirolimus therapy in mice. Next we sought to determine why sirolimus did not have a better therapeutic effect. It is possible that a higher dose of sirolimus or earlier initiation of therapy may have had a better therapeutic effect. As kidney size and CVD were not normalized by sirolimus, we investigated the mTORC2 pathway that is known to be proliferative through phosphorylation of phospho-Akt (serine 473).7.Boletta A. Emerging evidence of a link between the polycystins and the mTOR pathways.Pathogenetics. 2009; 2: 6Crossref PubMed Google Scholar, 13.Wullschleger S. Loewith R. Hall M.N. TOR signaling in growth and metabolism.Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4362) Google Scholar Rictor and phospho-Akt (serine 473), the functional readout of the mTOR–Rictor mTORC2 complex activity, were not different between normal littermate control and Pkd2WS25/− kidneys at 16 weeks of age. However, Rictor in whole kidney lysates was decreased by sirolimus in Pkd2WS25/− mice. A possible reason for the difference between Rictor and phospho-Akt (serine 473) expression is that Rictor expression in whole kidney lysates does not accurately reflect mTORC2 assembly or the effect of sirolimus on mTORC2 assembly.14.Sarbassov D.D. Ali S.M. Sengupta S. et al.Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB.Molecular Cell. 2006; 22: 159-168Abstract Full Text Full Text PDF PubMed Scopus (2057) Google Scholar, 15.Toschi A. Lee E. Xu L. et al.Regulation of mTORC1 and mTORC2 complex assembly by phosphatidic acid: competition with rapamycin.MolCell Biol. 2009; 29: 1411-1420Crossref PubMed Scopus (242) Google Scholar Rather, the Rictor-mTOR complex on immunopreciptates of cells, not whole cell lysates, reflects mTORC2 assembly and Akt (serine 473) activity.14.Sarbassov D.D. Ali S.M. Sengupta S. et al.Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB.Molecular Cell. 2006; 22: 159-168Abstract Full Text Full Text PDF PubMed Scopus (2057) Google Scholar, 15.Toschi A. Lee E. Xu L. et al.Regulation of mTORC1 and mTORC2 complex assembly by phosphatidic acid: competition with rapamycin.MolCell Biol. 2009; 29: 1411-1420Crossref PubMed Scopus (242) Google Scholar Long term or high-dose sirolimus therapy14.Sarbassov D.D. Ali S.M. Sengupta S. et al.Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB.Molecular Cell. 2006; 22: 159-168Abstract Full Text Full Text PDF PubMed Scopus (2057) Google Scholar, 30.Zeng Z. Sarbassov dD. Samudio I.J. et al.Rapamycin derivatives reduce mTORC2 signaling and inhibit AKT activation in AML.Blood. 2007; 109: 3509-3512Crossref PubMed Scopus (290) Google Scholar is able to inhibit mTORC2 assembly, as seen on immunoprecipitation, and resultant phosphorylation of Akt (serine 473). In this study, we show activation of mTORC1 (increased ribosomal S6 protein), but not mTORC2, in PKD caused by a mutation of the Pkd2 gene. Having shown that there is increased mTORC1 signaling in Pkd2WS25/− kidneys and that sirolimus significantly decreases kidney size and CVD in Pkd2WS25/− mice, we speculated on how the mutation in the Pkd2 gene as seen in Pkd2WS25/− mice, may affect mTORC1 signaling. PC-2, the protein product of the Pkd2 gene binds to polycystin-1 (PC-1), a large transmembrane receptor.31.Qian F. Germino F.J. Cai Y. et al.PKD1 interacts with PKD2 through a probable coiled-coil domain.Nat Genet. 1997; 16: 179-183Crossref PubMed Scopus (534) Google Scholar PC-1 and PC-2 are proposed to constitute a flow-sensitive ion channel complex in the primary cilium. It has recently been discovered that the PC-1/PC-2 ratio regulates pressure sensing.32.Sharif-Naeini R. Folgering J.H. Bichet D. et al.Polycystin-1 and -2 dosage regulates pressure sensing.Cell. 2009; 139: 587-596Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar The PC-1 and PC-2 proteins associate in a complex to prevent cyst formation.7.Boletta A. Emerging evidence of a link between the polycystins and the mTOR pathways.Pathogenetics. 2009; 2: 6Crossref PubMed Google Scholar For example, PC-1 induces the formation of a complex with tuberin and the ser/thr kinase of mTOR resulting in inhibition of mTORC1 activity.5.Weimbs T. Regulation of mTOR by polycystin-1: is polycystic kidney disease a case of futile repair?.Cell Cycle. 2006; 5: 2425-2429Crossref PubMed Scopus (46) Google Scholar Most recently, direct evidence that PC-1 inhibits mTORC1 in a tuberin-dependent manner by regulation of ERK-dependent phosphorylation was shown.33.Distefano G. Boca M. Rowe I. et al.Polycystin-1 regulates extracellular signal-regulated kinase-dependent phosphorylation of tuberin to control cell size through mTOR and its downstream effectors S6K and 4EBP1.Mol Cell Biol. 2009; 29: 2359-2371Crossref PubMed Scopus (143) Google Scholar Thus, the PC-1/TSC-1-TSC-2 complex inhibits mTORC1.34.Huang J. Dibble C.C. Matsuzaki M. et al.The TSC1-TSC2 complex is required for proper activation of mTOR complex 2.Mol Cell Biol. 2008; 28: 4104-4115Crossref PubMed Scopus (364) Google Scholar In contrast, the TSC-1—TSC-2 complex activates mTORC2 in a manner that is independent of the effec
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