sirolimus and Polycystic-Kidney-Diseases

Nutrients such as glucose, amino acids and lipids are fundamental sources for the maintenance of essential cellular processes and homeostasis in all organisms. The nutrient-sensing kinases mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) are expressed in many cell types and have key roles in the control of cell growth, proliferation, differentiation, metabolism and survival, ultimately contributing to the physiological development and functions of various organs, including the kidney. Dysregulation of these kinases leads to many human health problems, including cancer, neurodegenerative diseases, metabolic disorders and kidney diseases. In the kidney, physiological levels of mTOR and AMPK activity are required to support kidney cell growth and differentiation and to maintain kidney cell integrity and normal nephron function, including transport of electrolytes, water and glucose. mTOR forms two functional multi-protein kinase complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Hyperactivation of mTORC1 leads to podocyte and tubular cell dysfunction and vulnerability to injury, thereby contributing to the development of chronic kidney diseases, including diabetic kidney disease, obesity-related kidney disease and polycystic kidney disease. Emerging evidence suggests that targeting mTOR and/or AMPK could be an effective therapeutic approach to controlling or preventing these diseases.

Topics: AMP-Activated Protein Kinases; Humans; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Multiprotein Complexes; Nutrients; Polycystic Kidney Diseases; Renal Insufficiency, Chronic; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The mTOR pathway plays an important role in a number of common renal diseases, including acute kidney injury (AKI), diabetic nephropathy (DN), and polycystic kidney diseases (PKD). The activity of mTOR complex 1 (mTORC1) is necessary for renal regeneration and repair after AKI, and inhibition of mTORC1 by rapamycin has been shown to delay recovery from ischemic AKI in animal studies, and to prolong delayed graft function in humans who have received a kidney transplant. For this reason, administration of rapamycin should be delayed or discontinued in patients with AKI until full recovery of renal function has occurred. On the other hand, inappropriately high mTORC1 activity contributes to the progression of the metabolic syndrome, the development of type 2 diabetes, and the pathogenesis of DN. In addition, chronic hyperactivity of mTORC1, and possibly also mTORC2, contributes to cyst formation and enlargement in a number of forms of PKD. Inhibition of mTOR, using either rapamycin (which inhibits predominantly mTORC1) or "catalytic" inhibitors (which effectively inhibit both mTORC1 and mTORC2), provide exciting possibilities for novel forms of treatment of DN and PKD. In this second part of the review, we will examine the role of mTOR in the pathophysiology of DN and PKD, as well as the potential utility of currently available and newly developed inhibitors of mTOR to slow the progression of DN and/or PKD.

Topics: Acute Kidney Injury; Diabetic Nephropathies; Humans; Kidney; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Polycystic Kidney Diseases; Proteins; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors

Polycystic kidney diseases (PKDs) comprise a large group of genetic disorders characterized by formation of cysts in the kidneys and other organs, ultimately leading to end-stage renal disease. Although PKDs can be caused by mutations in different genes, they converge on a set of common molecular mechanisms involved in cystogenesis and ciliary dysfunction, and can be qualified as ciliopathies. Recent advances in understanding the mechanisms regulating disease progression have led to the development of new therapies that are being tested in both preclinical and clinical trials. In this article, we briefly review a network of molecular pathways of cystogenesis that are regulated by ciliary functions. We discuss the mTOR pathway in depth, highlighting recent progress in understanding its role in PKD and the current results of clinical trials.

Topics: Animals; Cilia; Clinical Trials as Topic; Humans; Kidney; Mutation; Polycystic Kidney Diseases; Protein Kinase D2; Protein Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; TRPP Cation Channels

Sirolimus is a member of a novel class of immunosuppressant drug that potently suppresses T cell proliferation and expansion by inhibition of the Target of Rapamycin Complex 1 (TORC1) protein kinase. Sirolimus also has anti-proliferative effects on intrinsic cells of the kidney, and increasing evidence suggests that it may have a therapeutic role in non-transplant renal diseases. In the normal kidney, sirolimus is considered to be non-nephrotoxic. In the diseased kidney, sirolimus may be beneficial or detrimental, depending on the type of renal injury. In polycystic kidney disease, TORC1 activation mediates renal tubular epithelial cell (TEC) proliferation and cyst growth in animals, and Phase III clinical trials are underway to determine the effect of sirolimus in attenuating disease progression in humans. In contrast, in acute kidney injury, sirolimus transiently impairs proximal TEC regeneration and delays renal recovery. In animal models of lupus nephritis and diabetic kidney disease, sirolimus prevents disease progression. However, the efficacy of sirolimus in human glomerulonephritis as well as in diabetic chronic kidney disease remains unclear, as it paradoxically exacerbates renal dysfunction when the baseline glomerular filtration rate is low (< 40 ml/min/1.73 m(2)) and there is heavy proteinuria (> 300 mg/day). This may, in part, be due to inhibition of compensatory glomerular capillary repair through the suppression of endothelial cell proliferation and angiogenic growth factor production by podocytes. Therefore, at present, polycystic kidney disease is the most promising therapeutic application for sirolimus in non-transplant renal diseases, and further studies are needed to clarify its role in other situations.

Topics: Animals; Disease Progression; Glomerular Filtration Rate; Humans; Kidney Diseases; Polycystic Kidney Diseases; Sirolimus

Polycystic kidney disease (PKD) is the most common genetic cause of chronic renal failure. Mouse models of PKD, especially those with mutations in genes that are orthologous to human disease genes, have provided insights into the pathogenesis of cyst formation and advanced the preclinical testing of new drugs.. PKD is a ciliopathy that arises from abnormalities in the primary cilium, a sensory organelle present on the surface of most cells. The primary cilium is required for the maintenance of planar cell polarity, which regulates tubular diameter. Acute kidney injury stimulates cell proliferation and promotes cyst formation in a mouse model of PKD. Studies of signaling pathways that are perturbed in PKD have identified new potential therapeutic targets. Drugs that have shown beneficial effects in orthologous animal models of PKD include tolvaptan, octreotide, src inhibitors, CFTR inhibitors, pioglitazone, etanercept, and triptolide.. Abnormalities in the primary cilium perturb signaling pathways that regulate renal epithelial cell growth and differentiation and lead to the formation of kidney cysts. Acute kidney injury promotes cyst formation and may underlie the variability in disease progression that is observed in affected individuals. Several promising new therapeutic agents that have been validated in orthologous animal models have entered clinical trials in humans.

Topics: Animals; Antidiuretic Hormone Receptor Antagonists; Cell Polarity; Cilia; Humans; Kinesins; Octreotide; Polycystic Kidney Diseases; Purines; Roscovitine; Sirolimus; TRPP Cation Channels

Polycystic kidney disease is an inherited multisystem disorder. It causes progressive loss of kidney function, flank pain, urinary tract infection, arterial hypertension and vascular abnormalities. Until the present time the treatment of polycystic kidney disease has been symptomatic. New approaches based on cell culture of cyst wall epithelia and on the discovery of polycystins 1 and 2 have lead to novel treatment protocols to attack the origin of the disease. These protocols involve vasopressin antagonists, rapamycin and somatostatin at the present time.

Topics: Animals; Humans; Polycystic Kidney Diseases; Sirolimus; Somatostatin; Vasopressins

Topics: Animals; Calcium-Binding Proteins; Caroli Disease; Cholangiopancreatography, Magnetic Resonance; Cysts; Disease Models, Animal; Elasticity Imaging Techniques; Glucosidases; Humans; Immunosuppressive Agents; Intracellular Signaling Peptides and Proteins; Liver Cirrhosis; Liver Diseases; Magnetic Resonance Imaging; Membrane Proteins; Molecular Biology; Molecular Chaperones; Octreotide; Peptides, Cyclic; Polycystic Kidney Diseases; Prevalence; RNA-Binding Proteins; Sirolimus; Somatostatin; Tomography, X-Ray Computed

Sirolimus is a potent immunosuppressant drug with a novel mechanism of action. It inhibits the mammalian target of rapamycin (mTOR) and blocks the cell cycle of various cell types, including T- and B-lymphocytes. Sirolimus is widely used as a maintenance immunosuppressive agent in organ transplantation. Also, a potentially benefit of this valuable drug in some immunologic and malignant diseases is currently under scrutiny.Classical side effects: hematological (anaemia, leucopenia, thrombocytopenia), hypercholesterolemia, arthralgias, extremity oedema and impaired wound healing have been frequently associated with the use of sirolimus. Additionally with its increased use, transplant professionals are encountering a variety of previously unreported and potentially more severe side effects.Here, we review the most recent data on sirolimus unexpected side effects (with an emphasis on pulmonary and renal toxicity), its use in renal transplantation and its new potential therapeutic indications (chronic glomerulopathies, polycystic kidney disease, different types of cancer). A brief description of the current knowledge of sirolimus therapeutic drug monitoring, methods of analysis, pharmacokinetics and drug interactions with calcineurin inhibitors is also included.

Topics: Angioedema; Animals; Humans; Immunosuppressive Agents; Kidney Diseases; Kidney Transplantation; Pneumonia; Polycystic Kidney Diseases; Sirolimus; Uveitis

Although rapamycin is a very effective drug for rodents with polycystic kidney disease (PKD), it is not encouraging in the clinical trials due to the suboptimal dosages compelled by the off-target side effects. We here report the generation, characterization, specificity, functionality, pharmacokinetic, pharmacodynamic and toxicology profiles of novel polycystic kidney-specific-targeting nanoparticles (NPs). We formulated folate-conjugated PLGA-PEG NPs, which can be loaded with multiple drugs, including rapamycin (an mTOR inhibitor) and antioxidant 4-hydroxy-TEMPO (a nephroprotective agent). The NPs increased the efficacy, potency and tolerability of rapamycin resulting in an increased survival rate and improved kidney function by decreasing side effects and reducing biodistribution to other organs in PKD mice. The daily administration of rapamycin-alone (1 mg/kg/day) could now be achieved with a weekly injection of NPs containing rapamycin (379 μg/kg/week). This polycystic kidney-targeting nanotechnology, for the first time, integrated advances in the use of 1) nanoparticles as a delivery cargo, 2) folate for targeting, 3) near-infrared Cy5-fluorophore for in vitro and in vivo live imaging, 4) rapamycin as a pharmacological therapy, and 5) TEMPO as a combinational therapy. The slow sustained-release of rapamycin by polycystic kidney-targeting NPs demonstrates a new era of nanomedicine in treatment for chronic kidney diseases at clinically relevant doses.

Topics: Animals; Folic Acid; Mice; Nanomedicine; Nanoparticles; Polycystic Kidney Diseases; Sirolimus; Tissue Distribution; TOR Serine-Threonine Kinases

A hallmark of polycystic kidney diseases (PKDs) is aberrant proliferation, which leads to the formation and growth of renal cysts. Proliferation is mediated by cyclin-dependent kinases (Cdks), and the administration of roscovitine (a pan-Cdk inhibitor) attenuates renal cystic disease in juvenile cystic kidney (jck) mice. Cdk2 is a key regulator of cell proliferation, but its specific role in PKD remains unknown. The aim of this study was to test the hypothesis that Cdk2 deficiency reduces renal cyst growth in PKD. Three studies were undertaken: (i) a time course (days 28, 56, and 84) of cyclin and Cdk activity was examined in jck mice and compared with wild-type mice; (ii) the progression was compared in jck mice with or without Cdk2 ablation from birth; and (iii) the effect of sirolimus (an antiproliferative agent) on Cdk2 activity in jck mice was investigated. Renal disease in jck mice was characterized by diffuse tubular cyst growth, interstitial inflammation and fibrosis, and renal impairment, peaking on day 84. Renal cell proliferation peaked during earlier stages of disease (days 28-56), whereas the expression of Cdk2-cyclin partners (A and E) and Cdk1 and 2 activity, was maximal in the later stages of disease (days 56-84). Cdk2 ablation did not attenuate renal disease progression and was associated with persistent Cdk1 activity. In contrast, the postnatal treatment of jck mice with sirolimus reduced both Cdk2 and Cdk1 activity and reduced renal cyst growth. In conclusion, (i) the kinetics of Cdk2 and Cdk2-cyclin partners did not correlate with proliferation in jck mice; and (ii) the absence of Cdk2 did not alter renal cyst growth, most likely due to compensation by Cdk1. Taken together, these data suggest that Cdk2 is dispensable for the proliferation of cystic epithelial cells and progression of PKD.

Topics: Animals; Cell Proliferation; Cyclin-Dependent Kinase 2; Disease Models, Animal; Disease Progression; Female; Kidney; Male; Mice; Polycystic Kidney Diseases; Sirolimus

Primary cilia are sensory organelles that regulate cell cycle and signaling pathways. In addition to its association with cancer, dysfunction of primary cilia is responsible for the pathogenesis of polycystic kidney disease (PKD) and other ciliopathies. Because the association between cilia formation or length and cell cycle or division is poorly understood, we here evaluated their correlation in this study. Using Spectral Karyotyping (SKY) technique, we showed that PKD and the cancer/tumorigenic epithelial cells PC3, DU145, and NL20-TA were associated with abnormal ploidy. We also showed that PKD and the cancer epithelia were highly proliferative. Importantly, the cancer epithelial cells had a reduction in the presence and/or length of primary cilia relative to the normal kidney (NK) cells. We then used rapamycin to restore the expression and length of primary cilia in these cells. Our subsequent analyses indicated that both the presence and length of primary cilia were inversely correlated with cell proliferation. Collectively, our data suggest that restoring the presence and/or length of primary cilia may serve as a novel approach to inhibit cancer cell proliferation.

Topics: Antibiotics, Antineoplastic; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cilia; Epithelial Cells; Humans; Polycystic Kidney Diseases; Sirolimus

Topics: Animals; Drug Delivery Systems; Folic Acid; Kidney; Mice; Polycystic Kidney Diseases; Polycystic Kidney, Autosomal Dominant; Sirolimus

Previous studies report a cross-talk between the polycystic kidney disease (PKD) and tuberous sclerosis complex (TSC) genes. mTOR signalling is upregulated in PKD and rapamycin slows cyst expansion, whereas renal inactivation of the Tsc genes causes cysts. Here we identify a new interplay between the PKD and TSC genes, with important implications for the pathophysiology of both diseases. Kidney-specific inactivation of either Pkd1 or Tsc1 using an identical Cre (KspCre) results in aggressive or very mild PKD, respectively. Unexpectedly, we find that mTORC1 negatively regulates the biogenesis of polycystin-1 (PC-1) and trafficking of the PC-1/2 complex to cilia. Genetic interaction studies reveal an important role for PC-1 downregulation by mTORC1 in the cystogenesis of Tsc1 mutants. Our data potentially explain the severe renal manifestations of the TSC/PKD contiguous gene syndrome and open new perspectives for the use of mTOR inhibitors in autosomal dominant PKD caused by hypomorphic or missense PKD1 mutations.

Topics: Animals; Cilia; Cysts; Down-Regulation; Gene Expression Regulation; Gene Silencing; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Polycystic Kidney Diseases; Sirolimus; TOR Serine-Threonine Kinases; TRPP Cation Channels; Tuberous Sclerosis; Up-Regulation

Topics: Animals; Ciliopathies; Disease Models, Animal; Elafin; Epithelial Cells; Germ-Line Mutation; Humans; Kidney; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Phosphoric Monoester Hydrolases; Polycystic Kidney Diseases; Proto-Oncogene Proteins c-akt; Sequence Deletion; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The disease-modifying effects of target of rapamycin complex 1 (TORC1) inhibitors during different stages of polycystic kidney disease (PKD) are not well defined. In this study, male Lewis Polycystic Kidney Disease (LPK) rats (a genetic ortholog of human NPHP9, phenotypically characterised by diffuse distal nephron cystic growth) and Lewis controls received either vehicle (V) or sirolimus (S, 0.2 mg/kg by intraperitoneal injection 5 days per week) during the early (postnatal weeks 3 to 10) or late stages of disease (weeks 10 to 20). In early-stage disease, sirolimus reduced kidney enlargement (by 63%), slowed the rate of increase in total kidney volume (TKV) in serial MRI by 78.2% (LPK+V: 132.3±59.7 vs. LPK+S: 28.8±12.0% per week) but only partly reduced the percentage renal cyst area (by 19%) and did not affect the decline in endogenous creatinine clearance (CrCl) in LPK rats. In late-stage disease, sirolimus reduced kidney enlargement (by 22%) and the rate of increase in TKV by 71.8% (LPK+V: 13.1±6.6 vs. LPK+S: 3.7±3.7% per week) but the percentage renal cyst area was unaltered, and the CrCl only marginally better. Sirolimus reduced renal TORC1 activation but not TORC2, NF-κB DNA binding activity, CCL2 or TNFα expression, and abnormalities in cilia ultrastructure, hypertension and cardiac disease were also not improved. Thus, the relative treatment efficacy of TORC1 inhibition on kidney enlargement was consistent at all disease stages, but the absolute effect was determined by the timing of drug initiation. Furthermore, cystic microarchitecture, renal function and cardiac disease remain abnormal with TORC1 inhibition, indicating that additional approaches to normalise cellular dedifferentiation, inflammation and hypertension are required to completely arrest the progression of PKDs.

Topics: Animals; Cardiovascular Diseases; Chemokine CCL2; Cilia; Creatinine; Disease Models, Animal; Gene Expression; Kidney; Male; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Multiprotein Complexes; Myocardium; NF-kappa B; Polycystic Kidney Diseases; Rats; Rats, Inbred Lew; Severity of Illness Index; Sirolimus; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha

Polycystic kidney disease (PKD) is a life-threatening disease that leads to a grotesque enlargement of the kidney and significant loss of function. Several imaging studies with MRI have demonstrated that cyst size in polycystic kidneys can determine disease severity and progression. In the present study, we found that, although kidney volume and cyst volume decreased with drug treatment, renal function did not improve with treatment. Here, we applied dynamic contrast-enhanced MRI to study PKD in a Glis3 (GLI-similar 3)-deficient mouse model. Cysts from this model have a wide range of sizes and develop at an early age. To capture this crucial stage and assess cysts in detail, we imaged during early development (3-17 weeks) and applied high spatiotemporal resolution MRI (125 × 125 × 125 cubic microns every 7.7 s). A drug treatment with rapamycin (also known as sirolimus) was applied to determine whether disease progression could be halted. The effect and synergy (interaction) of aging and treatment were evaluated using an analysis of variance (ANOVA). Structural measurements, including kidney volume, cyst volume and cyst-to-kidney volume ratio, changed significantly with age. Drug treatment significantly decreased these metrics. Functional measurements of time-to-peak (TTP) mean and TTP variance were determined. TTP mean did not change with age, whereas TTP variance increased with age. Treatment with rapamycin generally did not affect these functional metrics. Synergistic effects of treatment and age were not found for any measurements. Together, the size and volume ratio of cysts decreased with drug treatment, whereas renal function remained the same. The quantification of renal structure and function with MRI can comprehensively assess the pathophysiology of PKD and response to treatment.

Topics: Animals; DNA-Binding Proteins; Image Interpretation, Computer-Assisted; Imaging, Three-Dimensional; Immunosuppressive Agents; Magnetic Resonance Imaging; Mice; Mice, Inbred C57BL; Mice, Knockout; Polycystic Kidney Diseases; Repressor Proteins; Reproducibility of Results; Sensitivity and Specificity; Sirolimus; Trans-Activators; Treatment Outcome

Topics: Angioedema; Cyclosporine; Everolimus; Humans; Immunosuppressive Agents; Kidney Transplantation; Mycophenolic Acid; Polycystic Kidney Diseases; Sirolimus

Both experimental and clinical studies have suggested that any potential treatment of polycystic kidney disease (PKD) should start early and last for a long time to be effective, with unavoidable side reactions and considerable costs. The aim of the present study was to test how low doses of rapamycin (RAPA; 0.15 mg/kg ip for 4 days/wk), tolvaptan (TOLV; 0.005% in diet), or AEZ-131 (AEZ; a novel ERK inhibitor, 30 mg/kg for 3 days/wk by gavage), alone and in association, affect the progression of polycystic renal disease in PCK rats. Rats were treated for 8 wk starting at 4-6 wk of age. The efficacy of low doses of such drugs in inhibiting their respective targets was confirmed by immunoblot experiments. Compared with rats in the control (CON) group, RAPA treatment caused a significant reduction in cyst volume density (CVD; -19% vs. the CON group) and was numerically similar to that in TOLV-treated rats (-18%, not significiant), whereas AEZ treatment was not effective. RAPA + TOLV treatment resulted in a significantly lower CVD (-49% vs. the CON group) and was associated with a striking decrease in cAMP response element-binding protein phosphorylation, and similar data were detected in RAPA + AEZ-treated rats (-42%), whereas TOLV + AEZ treatment had virtually no effect. RAPA administration significantly lessened body weight gain, whereas TOLV administration resulted a mild increase in diuresis and a significant increase in cAMP urinary excretion. Histological data of tubular proliferation were in full agreement with CVD data. In conclusion, this study demonstrates that the association of low doses of RAPA, TOLV, and AEZ slows the progression of PKD with limited side effects, suggesting the use of combined therapies also in clinical trials.

Topics: Animals; Benzazepines; Cyclic AMP; Disease Models, Animal; Disease Progression; Drug Therapy, Combination; Enzyme Inhibitors; Kidney; Male; MAP Kinase Signaling System; Polycystic Kidney Diseases; Rats; Rats, Inbred Strains; Rats, Mutant Strains; Rats, Sprague-Dawley; Sirolimus; Tolvaptan; TOR Serine-Threonine Kinases; Treatment Outcome

Autosomal dominant polycystic kidney disease (ADPKD) is the most common life-threatening hereditary disease in the USA. In human ADPKD studies, sirolimus, a mammalian target of rapamycin complex 1 (mTORC1) inhibitor, had little therapeutic effect. While sirolimus robustly inhibits mTORC1, it has a minimal effect on mTOR complex 2 (mTORC2). Polycystic kidneys of Pkd2WS25/- mice, an orthologous model of human ADPKD caused by a mutation in the Pkd2 gene, had an early increase in pS6 (marker of mTORC1) and pAktSer(473) (marker of mTORC2). To investigate the effect of combined mTORC1 and 2 inhibition, Pkd2WS25/- mice were treated with an mTOR anti-sense oligonucleotide (ASO) that blocks mTOR expression thus inhibiting both mTORC1 and 2. The mTOR ASO resulted in a significant decrease in mTOR protein, pS6 and pAktSer(473). Pkd2WS25/- mice treated with the ASO had a normalization of kidney weights and kidney function and a marked decrease in cyst volume. The mTOR ASO resulted in a significant decrease in proliferation and apoptosis of tubular epithelial cells. To demonstrate the role of mTORC2 on cyst growth, Rictor, the functional component of mTORC2, was silenced in Madin-Darby canine kidney cell cysts grown in 3D cultures. Silencing Rictor significantly decreased cyst volume and expression of pAktSer(473). The decreased cyst size in the Rictor silenced cells was reversed by introduction of a constitutively active Akt1. In vitro, combined mTORC1 and 2 inhibition reduced cyst growth more than inhibition of mTORC1 or 2 alone. In conclusion, combined mTORC1 and 2 inhibition has therapeutic potential in ADPKD.

Topics: Animals; Carrier Proteins; Cell Proliferation; Disease Models, Animal; Dogs; Gene Expression Regulation; Genetic Therapy; Humans; Kidney; Madin Darby Canine Kidney Cells; Mice; Mice, Inbred C57BL; Oligonucleotides, Antisense; Polycystic Kidney Diseases; Rapamycin-Insensitive Companion of mTOR Protein; Sirolimus; TOR Serine-Threonine Kinases; TRPP Cation Channels

Inhibition of the mammalian target of rapamycin (mTOR) shows beneficial effects in animal models of polycystic kidney disease (PKD); however, two clinical trials in patients with autosomal dominant PKD failed to demonstrate a short-term benefit in either the early or progressive stages of disease. The stage of disease during treatment and the dose of mTOR inhibitors may account for these differing results. Here, we studied the effects of a conventional low dose and a higher dose of sirolimus (blood levels of 3 ng/ml and 30-60 ng/ml, respectively) on mTOR activity and renal cystic disease in two Pkd1-mutant mouse models at different stages of the disease. When initiated at early but not late stages of disease, high-dose treatment strongly reduced mTOR signaling in renal tissues, inhibited cystogenesis, accelerated cyst regression, and abrogated fibrosis and the infiltration of immune cells. In contrast, low-dose treatment did not significantly reduce renal cystic disease. Levels of p-S6Rp(Ser240/244), which marks mTOR activity, varied between kidneys; severity of the renal cystic phenotype correlated with the level of mTOR activity. Taken together, these data suggest that long-term treatment with conventional doses of sirolimus is insufficient to inhibit mTOR activity in renal cystic tissue. Mechanisms to increase bioavailability or to target mTOR inhibitors more specifically to kidneys, alone or in combination with other compounds, may improve the potential for these therapies in PKD.

Topics: Animals; Dose-Response Relationship, Drug; Female; Immunosuppressive Agents; Male; Mice; Mice, Inbred C57BL; Polycystic Kidney Diseases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Activation of the mTOR pathway has been implicated in the mediation of the progression of polycystic kidney disease (PKD). Whereas targeted inhibition of mTOR has been proven to be effective in various animal models of autosomal dominant PKD, its efficacy in autosomal recessive PKD (ARPKD) remains to be elucidated. We examined the effects of sirolimus in PCK rats, an orthologous animal model of human ARPKD.. Weaned PCK rats (n = 85) and SD-control rats (n = 72) received drinking water without and with sirolimus (corresponding to a daily intake of 2 mg/kg body weight) for 4, 8 and 12 weeks, respectively. The renal and hepatic functions were monitored throughout the treatment periods. Kidneys and livers were harvested and investigated with respect to progression of fibrosis, and number and size of cysts using the QWin image analysis programme. Expression of Akt, mTOR and its downstream target pS6K were assessed by immunohistochemistry.. Five out of 43 sirolimus-treated PCK rats, but none of the controls, died during the study. Sirolimus treatment resulted in slightly reduced weight gain. In PCK rats, grossly enlarged kidney and livers as well as hepatic fibrosis together with enlarged bile ducts were readily detectable. Whereas activation of Akt/mTOR signalling was hardly detectable in the kidneys of SD rats, strong signals were seen in the kidneys of PCK rats. Despite a significantly reduced relative kidney weight after 12 weeks of treatment (P < 0.05), neither fibrosis and cyst area nor renal function improved during treatment. Sirolimus-treated PCK rats showed only a minor inhibition of renal mTOR-specific phosphorylation of S6K. Male PCK rats on sirolimus presented with increased concentrations of bile acids and bilirubin compared with controls (each P < 0.05 at 12 weeks). Similar, albeit non-significant, effects were noted in female PCK rats.. Sirolimus failed to attenuate progression of kidney and liver disease in PCK rats. The lack of a protective effect might be due to intrinsic or acquired rapamycin resistance in this animal model of ARPKD.

Topics: Animals; Blotting, Western; Disease Progression; Female; Humans; Immunoenzyme Techniques; Immunosuppressive Agents; Liver Diseases; Male; Polycystic Kidney Diseases; Rats; Sirolimus; TOR Serine-Threonine Kinases

Aberrant activation of the mammalian target of rapamycin (mTOR) pathway occurs in polycystic kidney disease (PKD). mTOR inhibitors, such as rapamycin, are highly effective in several rodent models of PKD, but these models result from mutations in genes other than Pkd1 and Pkd2, which are the primary genes responsible for human autosomal dominant PKD. To address this limitation, we tested the efficacy of rapamycin in a mouse model that results from conditional inactivation of Pkd1. Mosaic deletion of Pkd1 resulted in PKD and replicated characteristic features of human PKD including aberrant mTOR activation, epithelial proliferation and apoptosis, and progressive fibrosis. Treatment with rapamycin was highly effective: It reduced cyst growth, preserved renal function, inhibited epithelial cell proliferation, increased apoptosis of cyst-lining cells, and inhibited fibrosis. These data provide in vivo evidence that rapamycin is effective in a human-orthologous mouse model of PKD.

Topics: Animals; Apoptosis; Blood Urea Nitrogen; Cell Division; Disease Models, Animal; Fibrosis; Gene Expression; Humans; Immunosuppressive Agents; Intermediate Filament Proteins; Intracellular Signaling Peptides and Proteins; Kidney Tubules, Collecting; Kidney Tubules, Distal; Mice; Mosaicism; Nerve Tissue Proteins; Nestin; Phenotype; Polycystic Kidney Diseases; Protein Serine-Threonine Kinases; Sirolimus; TOR Serine-Threonine Kinases; TRPP Cation Channels

A 38-year-old Afro-Caribbean woman, who was pre-dialysis with polycystic kidney disease, received a live-donor kidney transplant from her 55-year-old mother. This study documents her imunosuppression therapy including resolution of an oral Kaposi's sarcoma and explores the many underlying problems with converting to an mTOR inhibitor.

Topics: Adult; Female; Graft Rejection; Humans; Immunocompromised Host; Immunosuppressive Agents; Kidney Transplantation; Mouth Neoplasms; Polycystic Kidney Diseases; Sarcoma, Kaposi; Sirolimus

Short-term studies have demonstrated that rapamycin or everolimus treatment decreases cyst formation and improves renal function in animal models of polycystic kidney disease (PKD). Autosomal dominant polycystic kidney disease (ADPKD) patients would likely require life-long treatment with rapamycin.. Male Han:SPRD rats with PKD (Cy/+) were treated with rapamycin (0.2 mg/kg/day IP) or vehicle from 1 to 12 months of age. Mean trough levels of rapamycin (ng/mL) were 6.6 +/- 0.1 at 8 weeks of age. Twelve-month-old littermates (+/+) were used as normal controls.. Twelve-month-old male Cy/+ rats treated with the vehicle had a more than doubling of kidney volume, severe chronic renal failure, severe hypertension and increased heart weight compared to normal littermate controls (+/+). After rapamycin treatment, 12-month-old Cy/+ rats had markedly improved kidney volume, renal function, blood pressure and heart weight not statistically different from controls. Rapamycin reduced the cyst volume density (CVD) by 72%. Mammalian target of rapamycin (mTOR) activation in the heart, as evidenced by a marked increase in the phospho-S6 protein that was inhibited by rapamycin, was demonstrated in 12-month-old Cy/+ rats.. In conclusion, long-term rapamycin treatment in Cy/+ rats results in a normalization of kidney volume, renal function, blood pressure and heart weight. The novel finding that rapamycin decreases hypertension, heart enlargement and mTOR signalling in the heart in PKD rats is reported. The only side effect of rapamycin treatment was an 11% decrease in body weight.

Topics: Animals; Blood Pressure; Body Weight; Disease Models, Animal; Heart; Hypertension; Immunoblotting; Immunosuppressive Agents; Kidney Function Tests; Male; Organ Size; Polycystic Kidney Diseases; Rats; Rats, Sprague-Dawley; Sirolimus; Time Factors

The efficacy of mammalian target of rapamycin (mTOR) inhibitors is currently tested in patients affected by autosomal dominant polycystic kidney disease. Treatment with mTOR inhibitors has been associated with numerous side effects. However, the renal-specific effect of mTOR inhibitor treatment cessation in polycystic kidney disease is currently unknown. Therefore, we compared pulse and continuous everolimus treatment in Han:SPRD rats. Four-week-old male heterozygous polycystic and wild-type rats were administered everolimus or vehicle by gavage feeding for 5 wk, followed by 7 wk without treatment, or continuously for 12 wk. Cessation of everolimus did not result in the appearance of renal cysts up to 7 wk postwithdrawal despite the reemergence of S6 kinase activity coupled with an overall increase in cell proliferation. Pulse everolimus treatment resulted in striking noncystic renal parenchymal enlargement and glomerular hypertrophy that was not associated with compromised kidney function. Both treatment regimens ameliorated kidney function, preserved the glomerular-tubular connection, and reduced proteinuria. Pulse treatment at an early age delays cyst development but leads to striking glomerular and parenchymal hypertrophy. Our data might have an impact when long-term treatment using mTOR inhibitors in patients with autosomal dominant polycystic kidney disease is being considered.

Topics: Administration, Oral; Animals; Drug Administration Schedule; Emulsions; Everolimus; Hypertrophy; Immunosuppressive Agents; Kidney; Kidney Glomerulus; Male; Polycystic Kidney Diseases; Protein Kinases; Proteinuria; Pulse Therapy, Drug; Rats; Sirolimus; TOR Serine-Threonine Kinases

A pilot study was performed on adult polycystic kidney disease (PCKD) patients to examine the effects of the anti-proliferative mammalian target of rapamycin inhibitor sirolimus on the growth of renal cysts. Eight consecutive PCKD patients were given sirolimus (1 mg/d PO) for 6 consecutive months, in addition to an angiotensin receptor blocker (ARB), namely telmisartan. Another 8 PCKD patients served as a control group given only telmisartan. All PCKD patients had a serum creatinine value <2 mg/dL with a negative urine culture before enrollment. All patients were diagnosed by renal magnetic resonance imaging (MRI) to measure renal volumes. After a 6-month follow-up, patients were rescanned to remeasure the MRI volumes. Renal function was stable in 5/8 subjects in the sirolimus group, improved in 2 cases, and worsened in 1 with an increase of serum creatinine to >2 mg/dL resulting in his withdrawal after 5 months of follow-up. In contrast, the serum creatinine value was stable in 3 control group subjects, worsen in 3, and improved in 2. Four patients in the sirolimus group experienced infectious complications, namely, urinary tract infections (UTI) in 2 which were treated with antibiotics, and monilial pharyngitis in 2, who were treated and cured with a topical antifungal. In the control group, only 2 developed and were treated for UTIs. Hematologic tests were normal in all patients. There was an insignificant rise in kidney volume as measured by MRI in the sirolimus group (2845 vs 3221 mL after 6 months; P = NS) compared with a significant increase in the control group (2667 vs 3590 mL after 6 months; P < .05). We concluded that sirolimus, in addition to an ARB, might be beneficial for PCKD patients who present early in their illness.

Topics: Adult; Angiotensin-Converting Enzyme Inhibitors; Benzimidazoles; Benzoates; Blood Pressure; Creatinine; Female; Glomerular Filtration Rate; Humans; Immunosuppressive Agents; Kidney; Magnetic Resonance Imaging; Male; Middle Aged; Pilot Projects; Polycystic Kidney Diseases; Sirolimus; Telmisartan

We aimed to explore the effect of Mycophenolate mofetil (MMF) on loss of renal function and cyst progression compared to rapamycin in Han: SPRD rats. We also sought to assess whether the effect of combination therapy of MMF plus rapamycin was better than that of monotherapy.. Sixty heterozygous (Cy/+) and littermate control (+/+) male Han: SPRD rats were weaned at 4 weeks of age, then divided into four groups randomly to receive different treatments by intragastric administration for 2 months: vehicle-treated group as control, MMF-treated group (20mg/kg/day), rapamycin-treated group (2mg/kg/day), and MMF+Rapa- treated group (MMF 20mg/kg/day plus Rapamycin 2mg/kg/day). RESULLS: After 2 months of treatment, rapamycin caused a 22 % decrease in body weight in comparison to the control group, whereas MMF had no significant effect on weight gain. The steady increase of BUN in Cy/+ rats was reduced by 15% in MMF-treated Cy/+ rats. However, rapamycin and combination therapy reduced BUN by 42% and 43%, respectively. CCr was 0.93+/-0.11ml/min in vehicle-treated Cy/+ rats, 1.67+/-0.23 ml/min in MMF-treated Cy/+ rats (P<0.05), 1.72+/-0.44 ml/min and 1.83+/-0.21 ml/min in rapamycin- and MMF+Rapa-treated Cy/+ rats, respectively (.P<0.01). Cyst volume density was 57.1 % in vehicle-treated Cy/+ rats, 45.2% in MMF-treated Cy/+ rats (P<0.05), 32.9% and 37.7% in rapamycin- and MMF+Rapa-treated Cy/+ rats, respectively (P<0.01). MMF markedly ameliorated interstitial inflammation and fibrosis. Rapamycin showed a similar effect on interstitial inflammation and fibrosis, but to a lesser degree.. MMF is more tolerable than rapamycin and can retard deterioration of renal function in Han: SPRD rats, though its effect is weaker than that of rapamycin. Combination therapy does not exert more favorable effect than monotherapy.

Topics: Animals; Disease Models, Animal; Drug Therapy, Combination; Immunosuppressive Agents; Male; Mycophenolic Acid; Polycystic Kidney Diseases; Rats; Rats, Sprague-Dawley; Sirolimus; Time Factors

The ciliopathies are a class of rare human genetic disease whose aetioligies lie in defective primary cilia. Typical ciliopathies include Bardet-Biedl syndrome (BBS), nephronophthisis (NPHP), Jeune, Joubert, oro-facial-digital (OFD1) and Meckel (MKS) syndromes. All ciliopathies have the common denominator of renal disease, often including tubular cysts. In this study, we have modelled a range of ciliopathies in zebrafish and shown in all cases that knocking down these genes causes cystic lesions in the kidney. We have identified two drugs, rapamycin and roscovitine, which ameliorate the renal phenotype, both morphologically and functionally. This is the first study in which zebrafish has been used to identify potential therapeutic modalities for ciliopathic renal disease, and the results pave the way for further investigations in mammalian models.

Topics: Animals; Antineoplastic Agents; Bardet-Biedl Syndrome; Cilia; Disease Models, Animal; Immunosuppressive Agents; Kidney; Polycystic Kidney Diseases; Purines; Recovery of Function; Roscovitine; Sirolimus; Zebrafish

Remodelling of matrix and tubular basement membranes (TBM) is a characteristic of polycystic kidney disease. We hypothesized that matrix and TBM degradation by metalloproteinases (MMPs) could promote cyst formation. We therefore investigated the renal expression of MMPs in the Han:SPRD rat model of autosomal dominant polycystic kidney disease (ADPKD) and examined the effect of sirolimus treatment on MMPs.. 5-week-old male heterozygous (Cy/+) and wild-type normal (+/+) rats were treated with sirolimus (2 mg/kg/day) through drinking water for 3 months.. The mRNA and protein levels of MMP-2 and MMP-14 were markedly increased in the kidneys of heterozygous Cy/+ animals compared to wild-type +/+ as shown by RT-PCR and Western blot analyses for MMP-2 and MMP-14, and by zymography for MMP-2. Strong MMP-2 expression was detected by immunoperoxidase staining in cystic epithelial cells that also displayed an altered, thickened TBM. Tissue inhibitor of metalloproteinases-2 (TIMP-2) expression was not changed in Cy/+ kidneys. Sirolimus treatment leads to decreased protein expression of MMP-2 and MMP-14 in Cy/+, whereas MMP-2 and MMP-14 mRNA levels and TIMP-2 protein levels were not affected by sirolimus.. In summary, in kidneys of the Han:SPRD rat model of ADPKD, there is a marked upregulation of MMP-2 and MMP-14. Sirolimus treatment was associated with a marked improvement of MMP-2 and MMP-14 overexpression, and this correlated also with less matrix and TBM alterations and milder cystic disease.

Topics: Animals; Basement Membrane; Disease Models, Animal; Extracellular Matrix; Immunosuppressive Agents; Kidney; Male; Matrix Metalloproteinase 14; Matrix Metalloproteinase 2; Oligonucleotide Array Sequence Analysis; Polycystic Kidney Diseases; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sirolimus; Tissue Inhibitor of Metalloproteinase-2

Patients with Birt-Hogg-Dubé (BHD) syndrome harbor germline mutations in the BHD tumor suppressor gene that are associated with an increased risk for kidney cancer. BHD encodes folliculin, a protein that may interact with the energy- and nutrient-sensing 5'-AMP-activated protein kinase-mammalian target of rapamycin (AMPK-mTOR) signaling pathways.. We used recombineering methods to generate mice with a conditional BHD allele and introduced the cadherin 16 (KSP)-Cre transgene to target BHD inactivation to the kidney. Kidney cell proliferation was measured by BrdU incorporation and phospho-histone H3 staining. Kidney weight data were analyzed with Wilcoxon's rank-sum, Student's t, and Welch's t tests. Hematoxylin and eosin staining and immunoblot analysis and immunohistochemistry of cell cycle and signaling proteins were performed on mouse kidney cells and tissues. BHD knockout mice and kidney cells isolated from BHD knockout and control mice were treated with the mTOR inhibitor rapamycin. Mouse survival was evaluated by Kaplan-Meier analyses. All statistical tests were two-sided.. BHD knockout mice developed enlarged polycystic kidneys and died from renal failure by 3 weeks of age. Targeted BHD knockout led to the activation of Raf-extracellular signal-regulated protein kinase (Erk)1/2 and Akt-mTOR pathways in the kidneys and increased expression of cell cycle proteins and cell proliferation. Rapamycin-treated BHD knockout mice had smaller kidneys than buffer-treated BHD knockout mice had (n = 4-6 mice per group, relative kidney/body weight ratios, mean = 4.64% vs 12.2%, difference = 7.6%, 95% confidence interval = 5.2% to 10.0%; P < .001) and longer median survival time (n = 4-5 mice per group, 41.5 vs 23 days; P = .0065 ).. Homozygous loss of BHD may initiate renal tumorigenesis in the mouse. The conditional BHD knockout mouse may be a useful research model for dissecting multistep kidney carcinogenesis, and rapamycin may be considered as a potential treatment for Birt-Hogg-Dubé syndrome.

Topics: Animals; Antibiotics, Antineoplastic; Blotting, Southern; Cell Proliferation; Disease Models, Animal; Estrone; Fluorescent Antibody Technique; Gene Silencing; Genotype; Germ-Line Mutation; Immunoblotting; Immunohistochemistry; Kaplan-Meier Estimate; Kidney; Kidney Neoplasms; Mice; Mice, Knockout; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Polycystic Kidney Diseases; Protein Kinases; Proto-Oncogene Proteins; Random Allocation; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sirolimus; Syndrome; TOR Serine-Threonine Kinases; Tumor Suppressor Proteins

The use of proliferative signal inhibitors (PSIs) in immunosuppression-related malignancies opens new roads for increasing the survival and quality of life in patients with solid organ transplantation. A 56-year-old female recipient of a living donor renal allograft (1990), who was immunosuppressed with cyclosporine (CsA; Neoral), azathioprine, and steroids, did initially well with acceptable renal function. During the last 5 years she required local therapy due to posterior vaginal lip human papillomavirus (HPV) lesions. In 2000, she discontinued azathioprine and the CsA doses were reduced to 100 mg daily. The local lesion showed a good response to reduced immunosuppression. In February 2005, the lesion reappeared and a biopsy showed malignancy. Local surgery was performed and CsA was replaced by everolimus (EVL; Certican). Two months after treatment initiation, the patient developed cough, dyspnea, and low-grade fever. Chest X-ray showed a lesion at the base of the left lung compatible with pneumonitis. After fiberbronchoscopy a diagnosis of bronchiolitis obliterans organizing pneumonia (BOOP) was obtained. She was treated with increased doses of oral steroids. EVL was never discontinued. The radiological lesion disappeared and the malignancy is currently in remission. In summary, a case of gynecological cancer in a renal transplant recipient was treated by surgical removal. After 1 year of immunosuppression with EVL, no recurrence has been observed. The adverse event (BOOP) was probably related to the PSI treatment and was controlled with an increased dose of steroids without discontinuing EVL.

Topics: Adrenal Cortex Hormones; Azathioprine; Cryptogenic Organizing Pneumonia; Cyclosporine; Everolimus; Female; Humans; Kidney Transplantation; Methylprednisolone; Middle Aged; Papillomavirus Infections; Polycystic Kidney Diseases; Postoperative Complications; Sirolimus; Vaginal Neoplasms

Rapamycin inhibits cyst growth in polycystic kidney disease by targeting the mammalian target of rapamycin (mTOR). To determine if this is a class effect of the mTOR inhibitors, we examined the effect of everolimus, the analogue of rapamycin, on disease progression in the Han:SPRD rat model of polycystic kidney disease.. Four-week-old male heterozygous cystic (Cy/+) and wild-type normal (+/+) Han:SPRD rats were administered everolimus or vehicle (3 mg/kg/day) by gavage for 5 weeks. Kidney function and whole-blood trough levels of everolimus were monitored. After treatment kidney weight and cyst volume density were assessed. Tubule epithelial cell proliferation was assessed by BrdU staining.. Everolimus trough levels between 5 and 7 microg/l were sufficient to significantly reduce kidney and cyst volume density by approximately 50 and 40%, respectively. The steady decrease of kidney function in Cy/+ rats was reduced by 30% compared with vehicle-treated Cy/+ rats. Everolimus treatment markedly reduced the number of 5-bromo-2-deoxyuridine-labeled nuclei in cyst epithelia. Body weight gain and kidney function were impaired in everolimus-treated wild-type rats.. Moderate dosage of everolimus inhibits cystogenesis in Han:SPRD rats. The inhibitory effect of everolimus appears to represent a class effect of mTOR inhibitors.

Topics: Animals; Cysts; Disease Models, Animal; Everolimus; Kidney Function Tests; Male; Polycystic Kidney Diseases; Rats; Rats, Mutant Strains; Sirolimus

Autosomal-dominant polycystic kidney disease (ADPKD) is a common genetic disorder that frequently leads to renal failure. Mutations in polycystin-1 (PC1) underlie most cases of ADPKD, but the function of PC1 has remained poorly understood. No preventive treatment for this disease is available. Here, we show that the cytoplasmic tail of PC1 interacts with tuberin, and the mTOR pathway is inappropriately activated in cyst-lining epithelial cells in human ADPKD patients and mouse models. Rapamycin, an inhibitor of mTOR, is highly effective in reducing renal cystogenesis in two independent mouse models of PKD. Treatment of human ADPKD transplant-recipient patients with rapamycin results in a significant reduction in native polycystic kidney size. These results indicate that PC1 has an important function in the regulation of the mTOR pathway and that this pathway provides a target for medical therapy of ADPKD.

Topics: Animals; Apoptosis; Humans; Kidney; Mice; Polycystic Kidney Diseases; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases; TRPP Cation Channels

Topics: ABO Blood-Group System; Adult; Creatinine; Cyclosporine; Histocompatibility Testing; Humans; Immunosuppressive Agents; Kidney Transplantation; Male; Polycystic Kidney Diseases; Sirolimus; Treatment Outcome

Sirolimus is currently used to prevent rejection of solid organ transplant, and sirolimus-eluting stents have shown promise for the prevention of coronary artery restenosis. Thrombocytopenia is a well-known adverse effect of sirolimus limiting its use. Herein we report on a patient in whom sirolimus caused a platelet-independent hemostasis defect. The patient was a 52-year-old woman who underwent renal transplant with consequent normal kidney function. The immunosuppressive regimen included basiliximab, steroids, and cyclosporine induction later shifted to sirolimus and mycophenolate due to biopsy findings of tubular necrosis on day 6 posttransplantation. At discharge the serum creatinine was 0.7 mg/dL. Four months after transplantation the patient was admitted to our hospital because of fever (37.5 degrees C to 38 degrees C), anorexia, and asthenia. Blood analysis showed: creatinine 1.7 mg/dL, Hb 9.6 g/dL, WBC 6 x 10(3)/microL, PLT 123 x 10(3)/microL, liver function tests normal, LDH 720 mU/mL, fibrinogen 628 mg/dL, d-dimer 0.42 ng/mL, FDP > 40 ng/mL, INR 1.10, PT 87%, aPTT 40 seconds. Cultures and tests for infection were negative. Serum sirolimus level was 25.9 ng/mL. The following day the serum creatinine rose to 2.3 mg/dL and diuresis fell to 20 mL/h. Multiple bleeding times (Ivy test) performed before the renal biopsy were repeatedly over 30 minutes (normal 3 to 5 minutes), despite normal platelet count and platelet function studies. There was no spontaneous aggregation and in vitro aggregation was normal (collagen, ADP, adrenalin, and ristocetin induced). Coagulation studies showed a defect in fibrin formation and a reduction of fibrinolysis. Suspension of sirolimus treatment was followed by remission of fever, improvement of renal function (serum creatinine 1.2 mg/dL), and normalization of bleeding time.

Topics: Blood Platelets; Drug Therapy, Combination; Female; Hemostasis; Humans; Immunosuppressive Agents; Kidney Transplantation; Middle Aged; Polycystic Kidney Diseases; Sirolimus