sirolimus and Diabetic-Nephropathies

Mechanistic/Mammalian target of rapamycin (mTOR) orchestrates cellular homeostasis by controlling cell growth, proliferation, metabolism, and survival by integrating various growth factors, nutrients and amino acids. Eccentric synchronization of mTOR has been incriminated in various diseases/disorders like cancer, neurodegenerative disorders, and diabetes mellitus and its complications. Recent reports also highlight the role of mTOR in diabetes and its associated complications. This review tries to fathom the role of mTOR signaling in diabetes mellitus and its complications- diabetic cardiomyopathy, diabetic nephropathy, and diabetic retinopathy and highlights mTOR as a putative target for the development of novel anti-diabetic drug candidates.

Topics: Diabetes Mellitus; Diabetic Cardiomyopathies; Diabetic Nephropathies; Humans; 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

The immunosuppressive drug rapamycin has helped to identify a large signaling network around the target of rapamycin (TOR) protein that integrates information on nutrient availability and growth factors to control protein synthesis and cell size. Studies using rapamycin in animal models of kidney disease indicate that mTOR deregulation has a role in glomerular disease, polycystic kidney disease, and renal cancer. The role of mTOR activation in podocytes is context dependent, and indirect evidence suggests that mTOR may have a role in chronic podocyte loss. Several lines of evidence show that cyst formation in polycystic kidney disease (PKD) involves mTOR activation and its upstream regulator TSC. Polycystin 1 regulates mTOR activity through different pathways, and TSC intersects with the primary cilium, a crucial cell organelle in the pathogenesis of PKD. Data from hamartoma syndromes provide clear evidence that mutation of members of the mTOR network results in renal cancers. The detailed analysis of renal cell carcinomas has revealed a positive feedback loop involving VHL and mTOR. Rapamycin and its derivatives have been approved for the treatment of advanced renal cancer and are being investigated for the treatment of PKD. Discrepancies exist between the effects of rapamycin in animal models and the clinical experience with patients, precluding the widespread use of mTOR inhibitors in kidney disease. The details of mTOR signaling in the kidney need to be clarified to hopefully develop targeted treatments for renal disease in the future.

Topics: Animals; Autophagy; Cilia; Clinical Trials as Topic; Diabetic Nephropathies; Humans; Immunosuppressive Agents; Kidney Diseases; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Nephrons; Podocytes; Proteins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; TRPP Cation Channels

Many reviews have been written on protein kinase B/Akt focusing on its history dating back from the isolation of the Akt8 transforming murine leukemia virus by Staal in 1977, to the co-discovery of the Akt1 gene by the three groups in 1991 (reviewed in 7). There are currently over 22,000 publications in the PubMed database with "Akt" as a keyword - these publications describe a wealth of diverse data on the physiological functions of Akt isoforms. Many of these publications describe roles of Akt ranging from its requirement for cellular processes such as glucose uptake, cell survival and angiogenesis to roles in diseases such as cancer and ischaemia (22). This review will focus on the evidence for Akt signaling in different kidney cells during diabetes, or diabetic nephropathy (DN).

Topics: Diabetic Nephropathies; Humans; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Kidney transplantation is the best replacement therapy of type 2 diabetic patients and recently similar graft and patient survival between diabetic and nondiabetic recipients has been reported. However, standard immunosuppressive protocols are lacking. We present our experience with sirolimus-based immunosuppression in a population of 24 type 2 diabetic patients who underwent a kidney transplantation.. From January 2001 to December 2006, 396 kidney transplantations were performed. Twenty-four patients had type 2 diabetes mellitus as a cause of end-stage renal disease. They were randomized in two groups: thirteen patients (group A) received an immunosuppressive treatment with sirolimus, low-dose tacrolimus and steroids, while 11 patients (group B) received sirolimus, mycophenolate mofetil and steroids.. Clinical characteristics were similar between the two groups. A slightly better kidney functionality was observed in group B patients. There were neither acute rejection episodes nor severe infectious complications in both groups. One patient in each group underwent a foot amputation. Graft and patient survival was 100% for both groups at a median follow-up of 29 months.. Sirolimus-based immunosuppression is safe and efficacious in type 2 diabetic patients who underwent a kidney transplantation, allowing a better glucose metabolism control.

Topics: Diabetes Mellitus, Type 2; Diabetic Nephropathies; Humans; Immunosuppression Therapy; Immunosuppressive Agents; Kidney Transplantation; Middle Aged; Retrospective Studies; Sirolimus

Sirolimus (RAPA) and corticosteroids (CS) both inhibit wound healing. To evaluate the possibility that RAPA and CS have additive effects on wound healing, we evaluated the effects of corticosteroid avoidance (CSAV) on wound healing complications in patients treated with RAPA.. One hundred nine patients treated with a CSAV regimen (no pretransplantation or posttransplantation CS) were compared with a historical control group (n = 72) that received cyclosporine (CsA), mycophenolate mofetil (MMF), and CS. The CSAV group received low-dose CsA, MMF, RAPA, and thymoglobulin induction. Complications were classified as follows: wound healing complications (WHC) or infectious wound complications (IWC). WHC included lymphocele, hernia, dehiscence, diastasis, and skin edge separation. IWC included wound abscess and empiric antibiotic therapy for wound erythema.. The CSAV group was largely CS-free: 11% of patients received CS for rejection, 12% of patients received CS for recurrent disease, and 85% of patients are currently off CS. The CSAV group had a significantly lower incidence of WHC (13.7% vs 28%; P = .03) and lymphoceles (5.5% vs 16%; P = .02) than the control group. There was no difference in the incidence of IWC between the 2 groups. Patients who received CSAV were 18% less likely (P = .57) to develop any type of complication, 41% less likely (P = .20) to develop a WHC, and 71% less likely (P = .018) to develop a lymphocele.. CSAV in a RAPA-based regimen results in a marked reduction in WHC and lymphoceles. Therefore, CSAV provides a promising approach for addressing WHC associated with RAPA therapy.

Topics: Adrenal Cortex Hormones; Cyclosporine; Diabetic Nephropathies; Drug Administration Schedule; Drug Therapy, Combination; Female; Humans; Immunosuppressive Agents; Lymphocele; Male; Middle Aged; Mycophenolic Acid; Sirolimus; Wound Healing

Topics: Acute Disease; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Drug Therapy, Combination; Graft Rejection; Humans; Immunosuppressive Agents; Kidney Failure, Chronic; Kidney Transplantation; Pancreas Transplantation; Sirolimus; Survival Rate; Treatment Outcome

Topics: Adult; Cholesterol; Creatinine; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Drug Therapy, Combination; Follow-Up Studies; Humans; Immunosuppressive Agents; Kidney Failure, Chronic; Pancreas Transplantation; Pilot Projects; Prospective Studies; Sirolimus; Tacrolimus; Time Factors

Diabetic nephropathy (DN) is the most frequent cause of end-stage renal failure. Zinc oxide nanoparticles (ZnO-NPs) are promising antidiabetic agents. Our aim was to evaluate the prospective efficacy of ZnO-NPs in treating DN in streptozotocin-induced diabetic rats. Rats were randomly dispersed into three sets: control group, DN group and DN + ZnO-NPs group. ZnO-NPs were given at a dose of 10 mg/kg/day by oral gavage for 4 weeks. Urine and blood samples were processed for biochemical analyses. Kidney samples were managed for light and electron microscopy studies. Immune histochemical staining of P53, aquaporin11 (AQP11) and mechanistic target of rapamycin (mTOR) were performed. Gene analyses of nephrin, podocin, beclin-1, LC3 and p62 were done. Administration of ZnO-NPs ameliorated the functional and histopathological alterations of the kidney in a rat model of diabetic nephropathy. ZnO-NPs retained the constancy of the glomerular filtration barrier and restored almost normal renal structure. This was confirmed by upregulation of mRNA expression of podocyte markers (nephrin and podocin) and AQP11 immune histochemical expression in the renal tubules. The beneficial outcomes of ZnO-NPs might be attributed to activation of autophagy through inhibiting mTOR signaling pathway. ZnO-NPs enhanced beclin-1 and LC3 mRNA expressions and reduced p62 mRNA expression. ZnO-NPs also exerted anti-apoptotic potential (evidenced by the decrease in p53 immune expression), anti-inflammatory and anti-oxidant effect [endorsed by suppression of serum cyclooxygenase-2 (COX-2) enzyme activity, tissue nuclear factor kappa beta (NF-κB) level and blood hypoxia-inducible factors (HIF-1α) level]. These results may point the way to an effective therapy of DN.Abbreviations: AQP11 Aquaporin11; BUN: Blood urea nitrogen; COX-2: Cyclooxygenase-2; DAB: 3, 3'-diaminobenzidine; DM: Diabetes mellitus; DN: Diabetic nephropathy; ELISA: Enzyme-linked immunosorbent assay; H&E: Hematoxylin & eosin; HIF-1α: Hypoxia-inducible factors; iNOS: inducible nitric oxide synthase; LC3: Microtubule-associated protein 1 light chain 3; mTOR: Mechanistic target of rapamycin; NF-κB: Nuclear factor kappa beta; NPs: Nanoparticles; PAS: Periodic acid Schiff; PCR: Polymerase chain reaction; PGE2: Prostaglandin E2; ROS: Reactive oxygen species; STZ: Streptozotocin; X ± SEM: Mean ± standard error of means; Zn: Zinc; ZnO-NPs: Zinc oxide nanoparticles.

Topics: Animals; Beclin-1; Cyclooxygenase 2; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Hypoxia; Nanoparticles; NF-kappa B; Prospective Studies; Rats; RNA, Messenger; Sirolimus; Streptozocin; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53; Zinc Oxide

Autophagy has been demonstrated to have a beneficial effect on diabetic nephropathy (DN). Rapamycin, an inhibitor of mTOR, was shown to stimulate β-cell autophagy. However, its effects on preventing or ameliorating DN is unclear, and its effects are worth studying. As fasting is now an attractive protective strategy, we aim to compare its effect to rapamycin effects on pancreatic and renal cells. Twenty-eight adult male Wistar Albino rats were randomly divided into four groups, using streptozotocin (STZ) to induce diabetes mellitus (DM). Autophagy was induced by two ways; rapamycin or fasting. The extent of autophagy and apoptosis were investigated by measuring the level of LC3B and p53 proteins, respectively, in pancreatic and kidney tissues using Western blotting (WB) technique and imaging the renal cells under transmission electron microscope. The efflux transporter P-glycoprotein was quantified by WB as well. Rapamycin-induced autophagy occurred concurrently with apoptosis. On the other hand, fasting supported P-glycoprotein recovery and renal cell survival together with disabling β-cells apoptosis. In conclusion, this study provides a potential link between rapamycin or fasting for the cross-regulation of apoptosis and autophagy in the setting of cell stress as DN. Unlike rapamycin, fasting enhanced the active expression of ABCB1 efflux protein, providing insights on the potential ameliorative effects of fasting in DN that require further elucidation.

Topics: Animals; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Autophagosomes; Autophagy; Biomarkers; Cell Survival; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Models, Animal; Disease Susceptibility; Fasting; Kidney Function Tests; Liver Function Tests; Oxidative Stress; Rats; Sirolimus

Drug-induced nephrotic syndrome (NS) can be resolved by eliminating the causative agents. However, patients with metastatic cancer have not been previously reported to achieve complete recovery from anticancer drug-induced NS after discontinuation of treatment, because many patients die of cancer progression before NS is restored.. A 67-year-old man presented with edema of both lower extremities. He received pazopanib therapy for recurrent metastatic renal cell carcinoma (mRCC) for 17 months. Laboratory examinations revealed 7484.58 mg/day of 24-h urine protein, 434 mg/dL of serum cholesterol, and 2.9 g/dL of serum albumin. He was diagnosed with NS, and pazopanib treatment was discontinued. Four months later, he completely recovered from NS. He was then treated with temsirolimus and nivolumab sequentially for > 26 months. Pazopanib was re-introduced following disease progression, and demonstrated antitumor effects for 7 months without NS recurrence.. Pazopanib-induced NS can occur late in patients with mRCC, and its subsequent discontinuation can enable patients to completely recover from its adverse effects. Moreover, pazopanib treatment may be re-introduced without the recurrence of NS.

Topics: Aged; Amlodipine; Angiogenesis Inhibitors; Antihypertensive Agents; Antineoplastic Agents; Carcinoma, Renal Cell; Combined Modality Therapy; Diabetic Nephropathies; Dihydropyridines; Drug Substitution; Edema; Everolimus; Humans; Hypertension; Indazoles; Kidney Failure, Chronic; Lung Neoplasms; Male; Nephrotic Syndrome; Nivolumab; Pancreatic Neoplasms; Pancreaticoduodenectomy; Pneumonectomy; Protein Kinase Inhibitors; Pyrimidines; Sirolimus; Sulfonamides; Sunitinib

Huangkui capsule (HKC), an anti-inflammatory Chinese modern patent medicine, has been now widely applied to the clinical therapy of diabetic nephropathy (DN). However, the overall therapeutic mechanisms in vivo are still unclear. Renal tubular epithelial-to-mesenchymal transition (EMT) is one of the major pathogenesis of renal interstitial fibrosis in DN. Recently, the physiological roles of NLRP3 inflammasome activation and toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB signaling are closely linked to EMT. But, it remains elusive whether HKC regulates renal tubular EMT in vivo through targeting NLRP3 inflammasome activation and TLR4/NF-κB signaling in the kidneys.. This study thereby aimed to clarify the therapeutic effects of HKC on renal tubular EMT in DN and its underlying mechanisms in vivo, compared to rapamycin (RAP).. Thirty-two rats were randomly divided into 4 groups: the Sham group, the Vehicle group, the HKC group and the RAP group. The early DN rat models were induced by unilateral nephrectomy combined with intraperitoneal injection of streptozotocin, and administered with HKC suspension or RAP suspension or vehicle after modeling for 4 weeks. Changes in the incipient renal lesions-related parameters in urine and blood were analyzed, respectively. Renal interstitial tissues were isolated for histomorphometry, immunohistochemistry and Western blotting at sacrifice.. For the early DN rat models, HKC at the suitable dose of 2 g/kg/day ameliorated the general condition and biochemical parameters partially including kidney weight (KW), urinary albumin (UAlb), serum creatinine (Scr) and serum albumin (Alb), attenuated renal tubular EMT significantly and inhibited the activation of NLRP3 inflammasome in the kidneys obviously, which was superior to RAP generally. In addition to these, HKC also suppressed TLR4/NF-κB signaling in the kidneys of the DN model rats accurately, which was different from RAP specifically.. The results of this study further indicated that HKC, different from RAP, can alleviate renal tubular EMT in the DN model rats, likely by inhibiting NLRP3 inflammasome activation and TLR4/NF-κB signaling in the kidneys. Our findings thus provide the more accurate information in vivo about a clinical value of HKC, a traditional anti-inflammatory phytomedicine, in the treatment of the early DN patients.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Diabetic Nephropathies; Disease Models, Animal; Drugs, Chinese Herbal; Epithelial-Mesenchymal Transition; Fibrosis; Inflammasomes; Kidney; Male; Nephrectomy; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Toll-Like Receptor 4

Podocyte injury, characterized by podocyte hypertrophy, apoptosis, and epithelial-mesenchymal transition (EMT), is the major causative factor of diabetic nephropathy (DN). Autophagy dysfunction is regarded as the major risk factor for podocyte injury including EMT and apoptosis. High mobility group box 1 (HMGB1) is involved in the progression of DN through the induction of autophagy. However, the underlying mechanism remains unknown.. Plasma HMGB1 concentrations were determined in DN patients using ELISA. Apoptosis of DN serum-treated podocytes was evaluated by flow cytometry. Podocyte autophagy flux was measured using immunofluorescence. Western blotting analysis was used to investigate HMGB1 expression, EMT, and autophagy-related signaling pathways.. Upregulation of HMGB1 was found in DN patients and DN serum-treated podocytes. Removal of HMGB1 inhibited DN serum-mediated podocyte apoptosis by inhibiting autophagy and activating AKT/mammalian target of rapamycin (mTOR) signaling. In addition, HMGB1 depletion repressed the progression of podocyte EMT by inhibiting transforming growth factor (TGF)-β/smad1 signaling in vitro and in vivo. The combination of HMGB1 short interference (si) RNA and the autophagy activator rapamycin protected against podocyte apoptosis and EMT progression by inhibiting the AKT/mTOR and TGF-β/smad signaling pathway, respectively.. Although HMGB1 siRNA and rapamycin treatment had opposite effects on autophagy and AKT/mTOR signaling, there was no contradiction about the role of HMGB1 siRNA and rapamycin on AKT/mTOR pathway because autophagy and AKT/mTOR signaling play dual roles in intracellular biological processes. Based on the findings of this study, we may assume that HMGB1-initiated autophagy is harmful, whereas rapamycin is beneficial to podocyte survival. Possibly combined treatment with HMGB1 siRNA and rapamycin improved podocyte damage and EMT by regulating autophagy homeostasis.. 摘要: 背景 以肥大、凋亡、上皮-间充质转化(EMT)为特征的足细胞损伤是糖尿病肾病(DN)的主要病因。自噬功能障碍被认为是包括EMT和凋亡在内的足细胞损伤的主要危险因素。HMGB1 (High mobility group box 1) 也通过自噬参与DN的发展进程，但具体机制尚未明确。 方法:采用ELISA法测定DN患者血浆HMGB1浓度。采用流式细胞术检测经DN患者血清处理后的足细胞的凋亡情况。用免疫荧光法测定足细胞自噬流。采用Western blotting分析HMGB1表达、EMT和自噬相关信号通路。 结果 在DN患者和经过DN患者血清处理的足细胞中均发现HMGB1表达量上升。通过抑制HMGB1可抑制自噬和激活AKT/mTOR信号通路，进而抑制DN患者血清介导的足细胞凋亡。此外，HMGB1的损耗可通过抑制体内、外的转化生长因子(TGF) -β/ smad1信号，抑制足细胞的EMT进程。HMGB1 siRNA和自噬激活剂雷帕霉素的结合可分别通过抑制 AKT/mTOR和TGF-β/smad信号通路而起到抑制足细胞凋亡和EMT进程的作用。 结论 虽然HMGB1 siRNA和雷帕霉素处理对自噬和AKT/mTOR信号通路有相反的作用，但因自噬和AKT/mTOR信号通路在细胞内的生物进程中起双重作用，所以二者对AKT/mTOR通路的作用并不矛盾。基于此项研究发现，我们可以推测HMGB1启动的自噬是有害的，而雷帕霉素对足细胞的存活有益。HMGB1 siRNA联合雷帕霉素可能通过调节自噬稳态而改善足细胞损伤和EMT。.

Topics: Animals; Apoptosis; Autophagy; Biomarkers; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Epithelial-Mesenchymal Transition; Female; Follow-Up Studies; HMGB1 Protein; Humans; Male; Mice, Inbred C57BL; Middle Aged; Podocytes; Prognosis; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Abnormal regulation of autophagy participates in the development of diabetic nephropathy. mTOR is the most common negative regulator of the autophagy signaling pathway. FBW7 constitutes the SCF (Skp1-Cullin1-F-box protein) recognition subunit of E3 ubiquitin ligase, and mTOR is a substrate of FBW7 that can be modified by ubiquitination and be degraded via proteasomes. In this study, we explored the relationship between FBW7 and autophagy and examined the effects of FBW7 on the occurrence of diabetic nephropathy in vitro.. We cultured mesangial cells induced by high glucose in vitro and used rapamycin as a specific mTOR inhibitor, performed FBW7 gene overexpression, and detected the expression of autophagy signal and inflammatory factors by WB, ELISA, RT-PCR, and immunofluorescence.. High glucose can downregulate the expression of FBW7 and activate mTOR signal, which leads to diminished autophagy in renal mesangial cells, as well as renal inflammatory cytokines and fibrotic factors. RAPA, as a specifically inhibitor of mTOR, can decrease inflammatory cytokines and fibrotic factors by inhibiting mTOR. Moreover, FBW7 gene overexpression can increase autophagy by inhibiting mTOR signal; at the same time, the inflammatory cytokines and fibrotic factors were decreased in mesangial cells.. FBW7 was decreased in renal mesangial cells induced by high glucose, and FBW7 gene overexpression can increase autophagy by inhibiting mTOR signaling and ameliorate inflammation and fibrosis.

Topics: Autophagy; Cell Line; Diabetic Nephropathies; F-Box-WD Repeat-Containing Protein 7; Gene Expression Regulation; Glucose; Humans; Mesangial Cells; Phosphorylation; Proteasome Endopeptidase Complex; Proteolysis; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Ubiquitination

An increased kidney oxygen consumption causing tissue hypoxia has been suggested to be a common pathway toward chronic kidney disease. The mammalian target of rapamycin (mTOR) regulates cell proliferation and mitochondrial function. mTOR inhibitors (e.g., rapamycin) are used clinically to prevent graft rejection. mTOR has been identified as a key player in diabetes, which has stimulated the use of mTOR inhibitors to counter diabetic nephropathy. However, the effect of mTOR inhibition on kidney oxygen consumption is unknown. Therefore, we investigated the effects of mTOR inhibition on in vivo kidney function, oxygen homeostasis, and glomerular permeability. Control and streptozotocin-induced diabetic rats were chronically treated with rapamycin, and the functional consequences were studied 14 days thereafter. In both groups, mTOR inhibition induced mitochondrial uncoupling, resulting in increased total kidney oxygen consumption and decreased intrarenal oxygen availability. Concomitantly, mTOR inhibition induced tubular injury, as estimated from urinary excretion of kidney injury molecule-1 (KIM-1) and reduced urinary protein excretion. The latter corresponded to reduced sieving coefficient for large molecules. In conclusion, mTOR inhibition induces mitochondrial dysfunction leading to decreased oxygen availability in normal and diabetic kidneys, which translates into increased KIM-1 in the urine. Reduced proteinuria after mTOR inhibition is an effect of reduced glomerular permeability for large molecules. Since hypoxia has been suggested as a common pathway in the development of chronic kidney disease, mTOR inhibition to patients with preexisting nephropathy should be used with caution, since it may accelerate the progression of the disease.

Topics: Animals; Capillary Permeability; Cell Adhesion Molecules; Cell Hypoxia; Diabetic Nephropathies; Disease Progression; Glomerular Filtration Rate; Kidney Glomerulus; Kidney Tubules; Male; Mitochondria; Oxidative Stress; Oxygen Consumption; Protein Kinase Inhibitors; Rats, Sprague-Dawley; Renal Insufficiency, Chronic; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Glomerular mesangial cell (MC) apoptosis is one of the important mechanisms of glomerulosclerosis, which induces an increased severity of albuminuria and promotes the development of diabetic nephropathy (DN). However, the mechanism by which high glucose (HG) induces MCs apoptosis is not fully understood. In the present study, we investigated the effects of mTOR signalling on apoptosis in cultured MCs exposed to HG and in type I diabetes, and tried to clarify the specific mechanisms underlying these effects. In vitro, exposure of MCs to HG stimulated ROS production, decreased the antioxidant enzyme superoxide dismutase (SOD) activity and glutathione (GSH) level, increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, upregulated P53 expression and Bax/Bcl-2 ratio and enhanced cleavage of caspase 3, resulting in an increase in programmed cell death. Pretreatment of the cells with rapamycin ameliorated oxidative stress, reduced the number of apoptotic cells induced by HG and caused the downstream effects of mTOR activation. In vivo, compared with control rats, diabetic rats had more apoptotic cells in glomeruli. Induction of diabetes increased the level of MDA and NADPH oxidase activity, decreased the SOD activity and GSH level, elevated the Bax/Bcl ratio and P53 expression and activated caspase 3. mTOR inhibitor rapamycin treatment prevented these changes further alleviated albuminuria and improved renal function. Taken together, our data suggest that mTOR plays a key role in mediating ROS-induced MC apoptosis in diabetic nephropathy, and these effects have been associated with the promotion of ROS production by upregulating the antioxidant enzyme and downregulating the NADPH oxidase activity.

Topics: Animals; Apoptosis; Cell Line; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Glucose; Kidney Cortex; Kidney Function Tests; Male; Mesangial Cells; Mitochondria; Models, Biological; NADPH Oxidase 4; Oxidative Stress; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Schwann cell apoptosis is one of the characteristics of diabetic peripheral neuropathy (DPN). The mammalian target of rapamycin (mTOR) is a multifunctional signaling pathway that regulates cell apoptosis in various types of tissues and cells. To investigate whether the mTOR pathway is involved in cell apoptosis in the Schwann cells of DPN, diabetic mice and rat Schwann cells (RSC96) were chosen to detect phospho-mTOR (Ser 2448), phospho-S6K1 (Thr 389), phospho-4EBP1 (Thr 37/46), Bcl-2, Bax and cleaved caspase-3 by diverse pathological and biological techniques. The results showed that phospho-mTOR (Ser 2448) was decreased in the sciatic nerves of diabetic mice, concomitant with decreased Bcl-2, increased Bax, cleaved caspase-3 and cell apoptosis. In addition, high glucose treatment for 72 h caused a 35.95% decrease in the phospho-mTOR (Ser 2448)/mTOR ratio, a 65.50% decrease in the phospho-S6K1 (Thr 389)/S6K1 ratio, a 3.67-fold increase in the Bax/Bcl-2 ratio and a 1.47-fold increase in the cleaved caspase-3/caspase-3 ratio. Furthermore, mTORC1 inhibition, rather than mTORC2 inhibition, resulted in mitochondrial controlled apoptosis in RSC96 cells by silencing RAPTOR or RICTOR. Again, suppression of the mTORC1 pathway by a chemical inhibitor led to mitochondrial controlled apoptosis in cultured RSC96 cells in vitro. By contrast, activation of the mTORC1 pathway with MHY1485 prevented decreased phospho-S6K1 (Thr 389) levels caused by high glucose and cell apoptosis. Additionally, constitutive activation of S6K1 avoided high glucose-induced cell apoptosis in RSC96 cells. In summary, our findings suggest that activating mTORC1/S6K1 signaling in Schwann cells may be a promising strategy for the prevention and treatment of DPN.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line; Diabetic Nephropathies; Glucose; Mechanistic Target of Rapamycin Complex 1; Mice; Mitochondria; Naphthyridines; Proto-Oncogene Proteins c-bcl-2; Rapamycin-Insensitive Companion of mTOR Protein; Rats; Regulatory-Associated Protein of mTOR; Ribosomal Protein S6 Kinases; Schwann Cells; Sciatic Nerve; Sirolimus; TOR Serine-Threonine Kinases

Previous studies have shown that podocyte autophagy is an important trigger for proteinuria and glomerulosclerosis. The mammalian rapamycin target protein (mTOR) occupies a pivotal position in the autophagy pathway. In this study, we planned to clarify the mechanism of mTOR regulation of podocyte autophagy and the effect of rapamycin (RAPA).. All rats were randomly divided into normal control group (n = 8), DN group (n = 8), and RAPA group (n = 8). Blood and urine samples were collected at the 4th, 8th, and 12th weeks of the experiment. The serum creatinine (Scr), urine volume levels, and the 24 h urine protein (UP) levels were examined. The nephrin, podocin, mTOR, ribosomal S6 kinase 1 (S6K1), and autophagy marker light chain 3 (LC3II) expression levels were evaluated by immunohistochemistry, quantitative PCR, and immunoblotting.. The urine volume, 24 h UP, and Scr of the DN and RAPA groups increased significantly compared with the NC group (p < .05). Nephrin and podocin expression was decreased in the kidney tissues of the DN and RAPA groups compared with the NC group (p < .05). The expression levels of mTOR and S6K1 increased and LC3II expression decreased in the renal tissues of the DN and RAPA groups compared with the NC group (p < .05). After RAPA treatment, all the above indexes were improved compared with the DN group (p < .05), but were significantly abnormal compared with the NC group (p < .05).. The proteinuria and kidney function had improved after RAPA treatment. These results confirmed that RAPA specifically binds to mTOR kinase, and inhibits mTOR activity, thereby regulating the pathological autophagic process.

Topics: Animals; Autophagy; Creatinine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; Streptozocin; TOR Serine-Threonine Kinases

Mammalian target for rapamycin complex 1 (mTORC1) is a common target for the action of immunosuppressant macrolide rapamycin and glucose-lowering metformin. Inhibition of mTORC1 can exert both beneficial and detrimental effects in different pathologies. Here, we investigated the differential effects of rapamycin (1.2 mg/kg per day delivered subcutaneously for 6 weeks) and metformin (300 mg/kg per day delivered orally for 11 weeks) treatments on male Zucker diabetic fatty (ZDF) rats that mimic the cardiorenal pathology of type 2 diabetic patients and progress to insulin insufficiency. Rapamycin and metformin improved proteinuria, and rapamycin also reduced urinary gamma glutamyl transferase (GGT) indicating improvement of tubular health. Metformin reduced food and water intake, and urinary sodium and potassium, whereas rapamycin increased urinary sodium. Metformin reduced plasma alkaline phosphatase, but induced transaminitis as evidenced by significant increases in plasma AST and ALT. Metformin also induced hyperinsulinemia, but did not suppress fasting plasma glucose after ZDF rats reached 17 weeks of age, and worsened lipid profile. Rapamycin also induced mild transaminitis. Additionally, both rapamycin and metformin increased plasma uric acid and creatinine, biomarkers for cardiovascular and renal disease. These observations define how rapamycin and metformin differentially modulate metabolic profiles that regulate cardiorenal pathology in conditions of severe type 2 diabetes.

Topics: Animals; Biomarkers; Cardiovascular Diseases; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Disease Models, Animal; Disease Progression; Hypoglycemic Agents; Insulin Resistance; Liver; Male; Mechanistic Target of Rapamycin Complex 1; Metformin; Multiprotein Complexes; Protein Kinase Inhibitors; Proteinuria; Rats, Zucker; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases

There is increasing evidence showing that innate immune responses and inflammatory processes play an important role in the development and progression of diabetic nephropathy (DN). The potential effect of innate immunity in the early stage of DN is still unclear. Toll-Like-Receptor 4 (TLR4) is vigorously involved in the progress of kidney diseases in a sterile environment. The activation of the interleukin 17 (IL-17) pathway produces inflammatory cytokines, appearing in various kidney diseases. Unfortunately the relationship between TLR4 and IL-17 has not been investigated in diabetic nephropathy to date. The aim of this study is to investigate whether mammalian target of rapamycin (mTOR) inhibition may be dependent on TLR4 signaling and the pro-inflammatory factor IL-17 to delay the progression of DN.. Streptozotocin (STZ)-induced diabetic rats were randomly assigned to 3 experimental groups: a diabetic nephropathy group (DN, n = 6); and a diabetic nephropathy treated with rapamycin group (Rapa, n = 6) and a control group (Control, n =6). Body weight, fasting blood sugar, and 24h urine albumin were assessed at week 2, week 4 and week 8. Renal tissues were harvested for H&E, PAS staining, as well as an immunohistochemistry assay for TLR4 and IL-17. TLR4 quantitative expression was measured by Western-Blot analysis and RT-PCR.. Our results demonstrated that the expression of both TLR4 and IL-17 were upregulated in early stage DN and reduced by rapamycin. TLR4 and IL-17 both increased and positively related to 24h urinary albumin and kidney/weight ratio. However, neither TLR4 nor IL-17 made a significant difference on fasting blood sugar.. Taken together, our results confirm and extend previous studies identifying the significance of the TLR4 and Th17 pathways in development of early stage DN. Furthermore, we suggest this overexpression of TLR4 might be involved in the immunopathogenesis of DN through activation of Th17 cells. Rapamycin may attenuate DN via reduction of the TLR4 signaling pathway and Th17 cells signaling. Although the underlying mechanisms need to be explored, the observed increase of TLR4 and IL-17 during the early stages of DN and their suppression with rapamycin treatment suggest the importance of TLR4 and IL-17 in DN pathophysiology.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Progression; Interleukin-17; Male; Rats; Rats, Sprague-Dawley; Sirolimus; Toll-Like Receptor 4

The mammalian target of rapamycin (mTOR) is a key regulator of mRNA translation and protein synthesis, and it is specifically inhibited by rapamycin. In chronic pain conditions, mTOR-mediated local protein synthesis is crucial for neuronal hyperexcitability and synaptic plasticity. The tetrodotoxin-resistant (TTX-R) sodium channel Nav1.8 plays a major role in action potential initiation and propagation and cellular excitability in DRG (dorsal root ganglion) neurons. In this study, we investigated if mTOR modulates the phosphorylation of Nav1.8 that is associated with neuronal hyperexcitability and behavioral hypersensitivity in STZ-induced diabetic rats. Painful diabetic neuropathy (PDN) was induced in Sprague-Dawley rats by intraperitoneal injection with streptozotocin (STZ) at 60mg/kg. After the onset of PDN, the rats received daily intrathecal administrations of rapamycin (1μg, 3μg, or 10μg/day) for 7 days; other diabetic rats received the same volumes of dimethyl sulfoxide (DMSO). Herein, we demonstrate a marked increase in protein expression of total mTOR and phospho-mTOR (p-mTOR) together with the up-regulation of phosphor-Nav1.8 (p-Nav1.8) prior to the mechanical withdrawal threshold reaching a significant reduction in dorsal root ganglions (DRGs). Furthermore, the intrathecal administration of rapamycin, inhibiting the activity of mTOR, suppressed the phosphorylation of DRG Nav1.8, reduced the TTX-R current density, heightened the voltage threshold for activation and lowered the voltage threshold for inactivation and relieved mechanical hypersensitivity in diabetic rats. An intrathecal injection (i.t.) of rapamycin inhibited the phosphorylation and enhanced the functional availability of DRG Nav1.8 attenuated STZ-induced hyperalgesia. These results suggest that rapamycin is a potential therapeutic intervention for clinical PDN.

Topics: Animals; Diabetic Nephropathies; Ganglia, Spinal; Hyperalgesia; Injections, Spinal; Male; NAV1.8 Voltage-Gated Sodium Channel; Neurons; Phosphorylation; Physical Stimulation; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Streptozocin; TOR Serine-Threonine Kinases; Touch

Mechanical stress which would cause deleterious adhesive effects on podocytes is considered a major contributor to the early progress of diabetic nephropathy (DN). Our previous study has shown that spironolactone could ameliorate podocytic adhesive capacity in diabetic rats. Autophagy has been reported to have a protective role against renal injury. The present study investigated the underlying mechanisms by which spironolactone reduced adhesive capacity damage in podocytes under mechanical stress, focusing on the involvement of autophagy. Human conditional immortalized podocytes exposed to mechanical stress were treated with spironolactone, LY294002 or rapamycin for 48 h. The accumulation of LC3 puncta was detected by immunofluorescence staining. Podocyte expression of mineralocorticoid receptor (MR), integrin β1, LC3, Atg5, p85-PI3K, p-Akt, p-mTOR were detected by Western blotting. Podocyte adhesion to collagen type IV was also performed with spectrophotometry. Immunofluorescence staining showed that the normal level of autophagy was reduced in podocytes under mechanical stress. Decreased integrin β1, LC3, Atg5 and abnormal activation of the PI3K/Akt/mTOR pathway were also detected in podocytes under mechanical stress. Spironolactone up-regulated integrin β1, LC3, Atg5 expression, down-regulated p85-PI3K, p-Akt, p-mTOR expression and reduced podocytic adhesive capacity damage. Our data demonstrated that spironolactone inhibited mechanical-stress-induced podocytic adhesive capacity damage through blocking PI3K/Akt/mTOR pathway and restoring autophagy activity.

Topics: Autophagy; Autophagy-Related Protein 5; Cell Adhesion; Cell Line; Chromones; Diabetic Nephropathies; Down-Regulation; Humans; Integrin beta1; Microtubule-Associated Proteins; Morpholines; Phosphatidylinositol 3-Kinases; Podocytes; Proto-Oncogene Proteins c-akt; Receptors, Mineralocorticoid; Signal Transduction; Sirolimus; Spironolactone; Stress, Mechanical; TOR Serine-Threonine Kinases; Up-Regulation

Hyperglycemia upregulates thioredoxin interacting protein (TXNIP) expression, which in turn induces ROS production, inflammatory and fibrotic responses in the diabetic kidney. Dysregulation of autophagy contributes to the development of diabetic nephropathy. However, the interaction of TXNIP with autophagy/mitophagy in diabetic nephropathy is unknown. In this study, streptozotocin-induced diabetic rats were given TXNIP DNAzyme or scrambled DNAzyme for 12 weeks respectively. Fibrotic markers, mitochondrial function and mitochondrial reactive oxygen species (mtROS) were assessed in kidneys. Tubular autophagy and mitophagy were determined in kidneys from both human and rats with diabetic nephropathy. TXNIP and autophagic signaling molecules were examined. TXNIP DNAzyme dramatically attenuated extracellular matrix deposition in the diabetic kidneys compared to the control DNAzyme. Accumulation of autophagosomes and reduced autophagic clearance were shown in tubular cells of human diabetic compared to non-diabetic kidneys, which was reversed by TXNIP DNAzyme. High glucose induced mitochondrial dysfunction and mtROS production, and inhibited mitophagy in proximal tubular cells, which was reversed by TXNIP siRNA. TXNIP inhibition suppressed diabetes-induced BNIP3 expression and activation of the mTOR signaling pathway. Collectively, hyperglycemia-induced TXNIP contributes to the dysregulation of tubular autophagy and mitophagy in diabetic nephropathy through activation of the mTOR signaling pathway.

Topics: Animals; Autophagy; Carrier Proteins; Cell Cycle Proteins; Cell Line; Collagen Type I; Diabetic Nephropathies; Female; Gene Silencing; Glucose; Kidney Tubules; Membrane Proteins; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Proteins; Mitophagy; Rats; Sequestosome-1 Protein; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Up-Regulation

Accumulation of extracellular matrix (ECM) components is an early sign of diabetic nephropathy. Also the glycosaminoglycan hyaluronan (HA) is elevated in the renal interstitium during experimental diabetes. The mammalian target of rapamycin (mTOR) pathway participates in the signaling of hyperglycemia-induced ECM accumulation in the kidney, but this has not yet been investigated for HA. We hypothesized that interstitial HA accumulation during diabetes may involve mTOR activation.. Diabetic rats (6 weeks post-streptozotocin (STZ)) were treated with rapamycin to inhibit mTOR or vehicle for 2 additional weeks. Kidney function (glomerular filtration rate, renal blood flow, urine output) and regional renal HA content were thereafter analyzed. The ability of the animals to respond to desmopressin was also tested.. Diabetic animals displayed hyperglycemia, proteinuria, hyperfiltration, renal hypertrophy, increased diuresis with reduced urine osmolality, and reduced weight gain. Cortical and outer medullary HA was elevated in diabetic rats. Urine hyaluronidase activity was almost doubled in diabetic rats compared with controls. The ability to respond to desmopressin was absent in diabetic rats. Renal blood flow and arterial blood pressure were unaffected by the diabetic state. In diabetic rats treated with rapamycin the proteinuria was reduced by 32%, while all other parameters were unaffected.. Regional renal accumulation of the ECM component HA is not sensitive to mTOR inhibition by rapamycin, while proteinuria is reduced in established STZ-induced diabetes. Whether the diabetes-induced renal accumulation of HA occurs through different pathways than other ECM components, or is irreversible after being established, remains to be shown.

Topics: Analysis of Variance; Animals; Creatinine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Glomerular Filtration Rate; Hyaluronic Acid; Male; Proteinuria; Random Allocation; Rats; Reference Values; Risk Factors; Sensitivity and Specificity; Sirolimus; Streptozocin; TOR Serine-Threonine Kinases; Urodynamics

Many synthesized drugs with clinical severe side effects have been used for diabetic nephropathy (DN) treatment. Therefore, it is urgent and necessary to identify natural and safe agents to remedy DN. Timosaponin B-II (TB-II), a major steroidal saponin constituent in Anemarrhena asphodeloides Bunge, exhibits various activities, including anti-inflammatory and hypoglycemic functions. However, the anti-DN effects and potential mechanism(s) of TB-II have not been previously reported.. To investigate the effect of TB-II on DN in alloxan-induced diabetic mice.. TB-II was isolated and purified from A. asphodeloides Bunge using macroporous adsorption resin and preparative high-performance liquid chromatography. The effect of TB-II on DN was evaluated in alloxan-induced diabetic mice using an assay kit and immunohistochemical determination in vivo. The expression of mammalian target of rapamycin (mTOR), thioredoxin-interacting protein (TXNIP), and nuclear transcription factor-κB (NF-κB) signaling pathways was also measured using Western blot analysis.. TB-II significantly decreased the blood glucose levels and ameliorated renal histopathological injury in alloxan-induced diabetic mice. In addition, TB-II remarkably decreased the levels of renal function biochemical factors, such as kidney index, blood urea nitrogen, serum creatinine, urinary uric acid, urine creatinine, and urine protein, and it reduced lipid metabolism levels of total cholesterol and triglycerides and the levels of inflammatory cytokines interleukin-6 and tumor necrosis factor-α in alloxan-induced mice. Furthermore, TB-II inhibited the expression of mTOR, TXNIP, and NF-κB.. The results revealed that TB-II plays an important role in DN via TXNIP, mTOR, and NF-κB signaling pathways. Overall, TB-II exhibited a prominently ameliorative effect on alloxan-induced DN.

Topics: Alloxan; Anemarrhena; Animals; Blood Glucose; Carrier Proteins; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Kidney; Male; Mice; Mice, Inbred ICR; NF-kappa B; Saponins; Signal Transduction; Sirolimus; Steroids; Thioredoxins

Diabetic nephropathy is associated with premature senescence. Our previous study showed that glomerular mesangial cells (GMCs) appeared to take on senescent phenotypes under high-glucose conditions in conjunction with the downregulation of connexin43 (Cx43). In this study, we investigated whether AMPK-mediated Cx43 expression and premature senescence in diabetic nephropathy are associated with mTOR activation. From in vivo and in vitro studies, we found decreased expression of Cx43 and p-AMPK but increased expression of p21 both in the glomeruli of diabetic nephropathy and in primary GMCs cultured in high glucose. Activating AMPK or inhibiting mTOR prevented the downregulation of Cx43 and reversed GMC senescence. Dominant-negative AMPK expression both reduced Cx43 expression and induced GMC senescence. Furthermore, AMPK regulated Cx43 expression and GMC senescence mainly through the inhibition of mTOR, although other pathways cannot be ruled out. This study demonstrated that AMPK signaling pathways play an important role in the regulation of the Cx43 expression that accompanies GMC senescence under high-glucose conditions.

Topics: AMP-Activated Protein Kinases; Cells, Cultured; Cellular Senescence; Connexin 43; Cyclin-Dependent Kinase Inhibitor p21; Diabetic Nephropathies; Down-Regulation; Glucose; Humans; Hypoglycemic Agents; Kidney Glomerulus; Mesangial Cells; Metformin; Signal Transduction; Sirolimus; Sweetening Agents; TOR Serine-Threonine Kinases

The aim was to explore the effects of rapamycin on autophagy and injury of podocytes in streptozocin (STZ)-induced type 1 diabetic mice, and its role in delaying progression of diabetic nephropathy. In this study, male Balb/c mice were divided into three groups: control (n = 12), STZ-induced diabetic (n = 12), and rapamycin-treated diabetic (DM + Rapa) (n = 12), which received intraperitoneal injection of rapamycin (2 mg/kg/48 h) after induction of DM. Levels of urinary albumin (UA), blood urea nitrogen, serum creatinine, and kidney weight/body weight were measured at week 12. Renal pathologic changes, number of podocytes autophagy, and organelles injury were investigated by PAS staining, transmission electron microscopy, and immunofluorescence staining, respectively. Western blot was performed to determine the expression of LC3 (a podocyte autophagy marker), phosphorylated mammalian target of rapamycin, p-p70S6K, bax, and caspase-3 protein. Podocytes count was evaluated by immunofluorescence staining and Wilms tumor 1 immunohistochemistry, and Western blot of nephrin and podocin. The results indicated that rapamycin could reduce the kidney weight/body weight and UA secretion. It could alleviate podocyte foot process fusion, glomerular basement membrane thickening, and matrix accumulation, and increase the number of autophagosomes, and LC3-expressing podocytes. Down-regulation of bax and caspase-3 protein, and up-regulation of nephrin and podocin protein were observed in the glomeruli of diabetic mice after administration of rapamycin. In conclusion, rapamycin can ameliorate renal injury in diabetic mice by increasing the autophagy activity and inhibition of apoptosis of podocytes.

Topics: Albuminuria; Animals; Autophagy; bcl-2-Associated X Protein; Biomarkers; Blood Urea Nitrogen; Caspase 3; Cytoprotection; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mice, Inbred BALB C; Microtubule-Associated Proteins; Podocytes; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; Time Factors

While rapamycin treatment has been reported to have a putatively negative effect on glucose homeostasis in mammals, it has not been tested in polygenic models of type 2 diabetes. One such mouse model, NONcNZO10/LtJ, was treated chronically with rapamycin (14 ppm encapsulated in diet) and monitored for the development of diabetes. As expected, rapamycin treatment accelerated the onset and severity of hyperglycemia. However, development of nephropathy was ameliorated, as both glomerulonephritis and IgG deposition in the subendothelial tuft were markedly reduced. Insulin production and secretion appeared to be inhibited, suppressing the developing hyperinsulinemia present in untreated controls. Rapamycin treatment also reduced body weight gain. Thus, rapamycin reduced some of the complications of diabetes despite elevating hyperglycemia. These results suggest that multiple factors must be evaluated when assessing the benefit vs. hazard of rapamycin treatment in patients that have overt, or are at risk for, type 2 diabetes. Testing of rapamycin in combination with insulin sensitizers is warranted, as such compounds may ameliorate the putative negative effects of rapamycin in the type 2 diabetes environment.

Topics: Animals; Blood Glucose; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Disease Models, Animal; Female; Humans; Hyperglycemia; Hyperinsulinism; Insulin; Insulin Secretion; Male; Mice; Sirolimus

Podocyte apoptosis is a critical mechanism for excessive loss of urinary albumin that eventuates in kidney fibrosis. Pharmacological doses of the mammalian target of rapamycin (mTOR) inhibitor rapamycin reduce albuminuria in diabetes. We explored the hypothesis that mTOR mediates podocyte injury in diabetes. High glucose (HG) induces apoptosis of podocytes, inhibits AMP-activated protein kinase (AMPK) activation, inactivates tuberin, and activates mTOR. HG also increases the levels of Nox4 and Nox1 and NADPH oxidase activity. Inhibition of mTOR by low-dose rapamycin decreases HG-induced Nox4 and Nox1, NADPH oxidase activity, and podocyte apoptosis. Inhibition of mTOR had no effect on AMPK or tuberin phosphorylation, indicating that mTOR is downstream of these signaling molecules. In isolated glomeruli of OVE26 mice, there is a similar decrease in the activation of AMPK and tuberin and activation of mTOR with increase in Nox4 and NADPH oxidase activity. Inhibition of mTOR by a small dose of rapamycin reduces podocyte apoptosis and attenuates glomerular injury and albuminuria. Our data provide evidence for a novel function of mTOR in Nox4-derived reactive oxygen species generation and podocyte apoptosis that contributes to urinary albumin excretion in type 1 diabetes. Thus, mTOR and/or NADPH oxidase inhibition may represent a therapeutic modality of diabetic kidney disease.

Topics: Adenylate Kinase; Aminoimidazole Carboxamide; Animals; Apoptosis; Cell Line; Diabetic Nephropathies; Glucose; Hypoglycemic Agents; Mice; NADPH Oxidase 4; NADPH Oxidases; Phosphorylation; Podocytes; Reactive Oxygen Species; Ribonucleotides; Sirolimus; TOR Serine-Threonine Kinases

Exposure of proximal tubular epithelial cells to high glucose contributes to the accumulation of tubulointerstitial and matrix proteins in diabetic nephropathy, but how this occurs is not well understood. We investigated the effect of the signaling molecule tuberin, which modulates the mammalian target of rapamycin pathway, on renal hypertrophy and fibronectin expression. We found that the kidney mass was significantly greater in partially tuberin-deficient (TSC2(+/-) ) diabetic rats than wild-type diabetic rats. Furthermore, TSC2(+/-) rats exhibited significant increases in the basal levels of phospho-tuberin and fibronectin expression in the kidney cortex. Increased levels of phosphorylated tuberin associated with an increase in fibronectin expression in both wild-type and TSC2(+/-) diabetic rats. Treatment with insulin abrogated the diabetes-induced increase in fibronectin expression. In vitro, high glucose enhanced fibronectin expression in TSC2(+/-) primary proximal tubular epithelial cells; both inhibition of Akt and inhibition of the mammalian target of rapamycin could prevent this effect of glucose. In addition, forced expression of tuberin in tuberin-null cells abolished the expression of fibronectin protein. Taken together, these data suggest that tuberin plays a central role in the development of renal hypertrophy and in modulating the production of the matrix protein fibronectin in diabetes.

Topics: Animals; Base Sequence; Cells, Cultured; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Fibronectins; Gene Expression; Gene Targeting; Glucose; Hypertrophy; Kidney; Male; Models, Biological; Phosphorylation; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Rats; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Messenger; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Abnormal lipid metabolism contributes to the pathogenesis of diabetes, but it is uncertain whether it plays a role in the development of diabetic nephropathy (DN). While rapamycin was shown to prevent DN development in streptozotocin (STZ)-induced diabetic rats in our previous studies, it is unknown if it intervenes with lipid metabolism.. We divided the rats into four groups: normal control rats, rapamycin-treated normal rats, diabetic rats and rapamycin-treated DN rats. The apoptosis was evaluated by immunohistochemistry. The crude lipid and sphingolipid were extracted from rat renal cortex and analysed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The expression of the three key enzymes in sphingolipid metabolism including serine palmitoyltransferase, acid sphingomyelinase and sphingomyelin synthase was measured by western blot and immunohistochemistry in rat renal cortex.. The level of apoptosis was increased in diabetic rats, and rapamycin treatment reduced apoptosis. STZ treatment significantly increased formation of many sphingolipids species through elevated de novo synthesis. These changes were inhibited by treatment with rapamycin.. Accumulation of sphingolipids contributes to STZ-induced diabetes, and the therapeutic effect of rapamycin on diabetic nephropathy is partly through suppression of sphingolipid abnormality.

Topics: Animals; Apoptosis; Blotting, Western; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Immunosuppressive Agents; Kidney Cortex; Male; Rats; Rats, Sprague-Dawley; Sirolimus; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sphingolipids; Streptozocin

Chronic glomerular diseases, associated with renal failure and cardiovascular morbidity, represent a major health issue. However, they remain poorly understood. Here we have reported that tightly controlled mTOR activity was crucial to maintaining glomerular podocyte function, while dysregulation of mTOR facilitated glomerular diseases. Genetic deletion of mTOR complex 1 (mTORC1) in mouse podocytes induced proteinuria and progressive glomerulosclerosis. Furthermore, simultaneous deletion of both mTORC1 and mTORC2 from mouse podocytes aggravated the glomerular lesions, revealing the importance of both mTOR complexes for podocyte homeostasis. In contrast, increased mTOR activity accompanied human diabetic nephropathy, characterized by early glomerular hypertrophy and hyperfiltration. Curtailing mTORC1 signaling in mice by genetically reducing mTORC1 copy number in podocytes prevented glomerulosclerosis and significantly ameliorated the progression of glomerular disease in diabetic nephropathy. These results demonstrate the requirement for tightly balanced mTOR activity in podocyte homeostasis and suggest that mTOR inhibition can protect podocytes and prevent progressive diabetic nephropathy.

Topics: Adaptor Proteins, Signal Transducing; Adult; Animals; Carrier Proteins; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Progression; Gene Dosage; Genetic Predisposition to Disease; Humans; Kidney Glomerulus; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Knockout; Mice, Transgenic; Multiprotein Complexes; Nephrosis, Lipoid; Podocytes; Proteins; Proteinuria; Rapamycin-Insensitive Companion of mTOR Protein; Regulatory-Associated Protein of mTOR; Sirolimus; TOR Serine-Threonine Kinases; Trans-Activators; Transcription Factors

Diabetic nephropathy (DN) is among the most lethal complications that occur in type 1 and type 2 diabetics. Podocyte dysfunction is postulated to be a critical event associated with proteinuria and glomerulosclerosis in glomerular diseases including DN. However, molecular mechanisms of podocyte dysfunction in the development of DN are not well understood. Here we have shown that activity of mTOR complex 1 (mTORC1), a kinase that senses nutrient availability, was enhanced in the podocytes of diabetic animals. Further, podocyte-specific mTORC1 activation induced by ablation of an upstream negative regulator (PcKOTsc1) recapitulated many DN features, including podocyte loss, glomerular basement membrane thickening, mesangial expansion, and proteinuria in nondiabetic young and adult mice. Abnormal mTORC1 activation caused mislocalization of slit diaphragm proteins and induced an epithelial-mesenchymal transition-like phenotypic switch with enhanced ER stress in podocytes. Conversely, reduction of ER stress with a chemical chaperone significantly protected against both the podocyte phenotypic switch and podocyte loss in PcKOTsc1 mice. Finally, genetic reduction of podocyte-specific mTORC1 in diabetic animals suppressed the development of DN. These results indicate that mTORC1 activation in podocytes is a critical event in inducing DN and suggest that reduction of podocyte mTORC1 activity is a potential therapeutic strategy to prevent DN.

Topics: Adaptor Proteins, Signal Transducing; Animals; Carrier Proteins; Cell Differentiation; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Disease Models, Animal; Endoplasmic Reticulum; Enzyme Activation; Glomerular Basement Membrane; Glomerular Mesangium; Male; Mechanistic Target of Rapamycin Complex 1; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Mutant Strains; Multiprotein Complexes; Phosphorylation; Podocytes; Protein Processing, Post-Translational; Proteins; Regulatory-Associated Protein of mTOR; Ribosomal Protein S6 Kinases; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

The podocyte plays a key role both in maintenance of the glomerular filtration barrier and in glomerular structural integrity. Podocyte injury and loss contribute to proteinuria and progressive sclerosis. Inhibitors of mammalian target of rapamycin (mTOR) have variably decreased or caused proteinuria and sclerosis in human disease and experimental settings. In this issue of the JCI, two interesting studies of podocyte-specific manipulation of the mTOR system shed light on the complexity of this pathway in the podocyte.

Topics: Adaptor Proteins, Signal Transducing; Adult; Animals; Carrier Proteins; Child; Diabetic Nephropathies; Disease Models, Animal; Genetic Predisposition to Disease; Humans; Kidney Glomerulus; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Multiprotein Complexes; Podocytes; Proteins; Rapamycin-Insensitive Companion of mTOR Protein; Regulatory-Associated Protein of mTOR; Sirolimus; TOR Serine-Threonine Kinases; Trans-Activators; Transcription Factors

In order to evaluate the effectiveness of everolimus vs. rapamycin in the treatment of diabetic nephropathy, 8-week old diabetic (db/db) mice received everolimus (2 mg/kg every day) or rapamycin (2 mg/kg every day) for 4 weeks or 12 weeks respectively. Blood and 24-h urine samples were collected for biochemical tests. One kidney from each mouse was homogenized for protein analysis and the other was removed for histological analysis. The expression levels of transforming growth factor-β1 (TGF-β1)and phospho-p70s6k were detected by using ELISA and Western blot, respectively in the renal tissue as well as in mesengial cell culture samples. Everolimus was significantly more effective than rapamycin in improving indexes of renal function and glomerular hypertrophy, and in decreasing accumulation and expansion of the extracellular matrix. However, everolimus inhibited TGF-β1 secretion and p70s6k phosphorylation induced by high glucose in vitro less efficiently than rapamycin at the same dose. Everolimus was more effective than rapamycin in preventing diabetic nephropathy in vivo, which may be contributed to the fact that everolimus has better bioavailability and a higher oral absorption rate.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Models, Animal; Everolimus; Male; Mice; Sirolimus

Topics: Animals; Diabetic Nephropathies; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Knockout; Monomeric GTP-Binding Proteins; Multiprotein Complexes; Neuropeptides; Podocytes; Proteins; Ras Homolog Enriched in Brain Protein; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists and mammalian target of rapamycin (mTOR) inhibitors share mechanisms concerning cell growth and reduction of extracellular matrix accumulation. The purpose of this study was to evaluate the potential synergistic effect of this combination on diabetic kidney disease in rats.. Diabetes was induced by streptozotocin in 42 male Sprague-Dawley rats. Sixteen weeks after diabetes induction, animals were divided into four groups: diabetic animals without intervention (D), diabetic animals with administration of sirolimus (D+SRL), diabetic animals with administration of rosiglitazone (D+RGT), and diabetic animals with administration of sirolimus and rosiglitazone (D+SRL+RGT).. At a 30-day follow up, diabetic rats showed higher kidney weight, mean glomerular volume, mesangial expansion and albuminuria compared with non-diabetic rats. mTOR downstream proteins, p-T389-S6K and p-T37/46-4EBP1, were higher in diabetic than non-diabetic kidneys, whereas p-S473-AKT was not, suggesting that hyperglycemia mainly activated the mTORC1 pathway in vivo. Moreover, the catalytic subunit of protein phosphatase 2A (PP2Ac) was down-regulated in the diabetic kidney. Sirolimus inhibited the mTORC1 pathway, while the PPAR-gamma agonist rosiglitazone enhanced PP2Ac and reduced p70S6K. Both drugs were associated with a reduction in albuminuria, renal enlargement and mesangial expansion, but without any improvement in glycemic control. Sirolimus and rosiglitazone in combination down-regulated the mTORC1 pathway and over-activated PP2Ac in diabetic kidney. This effect may account for the synergistic reduction of renal hypertrophy, albuminuria and renal TGF-beta1 observed in diabetic rats treated with SRL+RGT.. The combination of sirolimus and rosiglitazone is renoprotective with respect to diabetic nephropathy.

Topics: Albuminuria; Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Down-Regulation; Drug Synergism; Drug Therapy, Combination; Hypoglycemic Agents; Immunosuppressive Agents; Male; Protein Phosphatase 2; Rats; Rats, Sprague-Dawley; Rosiglitazone; Sirolimus; Thiazolidinediones; Transcription Factors; Transforming Growth Factor beta1

Diabetic nephropathy (DN) associated with type 2 diabetes is the most common cause of end-stage renal disease (ESRD) and a serious health issue in the world. Currently, molecular basis for DN has not been established and only limited clinical treatments are effective in abating the progression to ESRD associated with DN. Here we found that diabetic db/db mice which lack the leptin receptor signaling can be used as a model of ESRD associated with DN. We demonstrated that p70S6-kinase was highly activated in mesangial cells in diabetic obese db/db mice. Furthermore, systemic administration of rapamycin, a specific and potent inhibitor of mTOR, markedly ameliorated pathological changes and renal dysfunctions. Moreover, rapamycin treatment shows a significant reduction in fat deposits and attenuates hyperinsulinemia with few side effects. These results indicate that mTOR activation plays a pivotal role in the development of ESRD and that rapamycin could be an effective therapeutic agent for DN.

Topics: Animals; Carrier Proteins; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Kidney; Kidney Failure, Chronic; Mice; Mice, Inbred Strains; Phosphotransferases (Alcohol Group Acceptor); Restriction Mapping; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; TOR Serine-Threonine Kinases

Not just de novo induction of diabetes mellitus, but also the progression of diabetic nephropathy may be enhanced under immunosuppressive therapy after organ transplantation. We evaluated whether sirolimus (SRL) or cyclosporine A (CsA) therapy would be a superior immunosuppressant in streptozotocin-induced diabetic nephropathy.. Diabetes was induced by intravenous injection of streptozotozin (60 mg/kg body weight) in 26 male Sprague-Dawley rats. Eight days after diabetes induction, animals were divided into three groups, which were treated with placebo (n=8), SRL (n=9), or CsA (n=9). Six nondiabetic placebo-treated rats were included as controls.. After 19 weeks of diabetes, SRL significantly decreased fibrosis as assessed by periodic acid Schiff staining and by specific extracellular matrix proteins such as fibronectin and laminin at messenger RNA and protein level compared with the diabetic placebo group. SRL ameliorated renal inflammation, glomerular hypertrophy, and podocyte loss as indicated by morphometric and immunohistological analysis. SRL lowered expression and activity of glomerular transforming growth factor-beta1/2 and vascular endothelial growth factor, all of which are considered central cytokines in the pathogenesis of diabetic nephropathy. In contrast, calcineurin phosphatase inhibition through CsA did not ameliorate any of the features of diabetic nephropathy compared with placebo treatment but slightly aggravated glomerular fibrosis without affecting transforming growth factor-beta1/2 or vascular endothelial growth factor.. Compared with CsA, SRL by anti-inflammatory, antifibrotic, and podocyte-protective effects clearly seems to be the superior treatment of prevention or amelioration of diabetic nephropathy in the rat.

Topics: Animals; Cyclosporine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; DNA Primers; Fibronectins; Immunosuppressive Agents; Laminin; Male; Nephrogenic Fibrosing Dermopathy; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sirolimus; Vascular Endothelial Growth Factor A

Removal of one kidney stimulates synthesis of RNA and protein, with minimal DNA replication, in all nephron segments of the remaining kidney, resulting in cell growth (increase in cell size) with minimal cell proliferation (increase in cell number). In addition to the compensatory renal hypertrophy caused by nephron loss, pathophysiological renal hypertrophy can occur as a consequence of early uncontrolled diabetes. However, the molecular mechanism underlying renal hypertrophy in these conditions remains unclear. In the present study, we report that deletion of S6 kinase 1 (S6K1) inhibited renal hypertrophy seen following either contralateral nephrectomy or induction of diabetes. In wild-type mice, hypertrophic stimuli increased phosphorylation of 40S ribosomal protein S6 (rpS6), a known target of S6K1. Immunoblotting analysis revealed that S6K1(-/-) mice exhibited moderately elevated basal levels of rpS6, which did not increase further in response to the hypertrophic stimuli. Northern blotting indicated a moderate upregulation of S6K2 expression in the kidneys of S6K1(-/-) mice. Phosphorylation of the eukaryotic translation initiation factor 4E-binding protein 1, another downstream target of the mammalian target of rapamycin (mTOR), was stimulated to equivalent levels in S6K1(-/-) and S6K1(+/+) littermates during renal hypertrophy, indicating that mTOR was still activated in the S6K1(-/-) mice. The highly selective mTOR inhibitor, rapamycin, inhibited increased phosphorylation of rpS6 and blocked 60-70% of the hypertrophy seen in wild-type mice but failed to prevent the approximately 10% hypertrophy seen in S6K1(-/-) mice in response to uninephrectomy (UNX) although it did inhibit the basal rpS6 phosphorylation. Thus the present study provides the first genetic evidence that S6K1 plays a major role in the development of compensatory renal hypertrophy as well as diabetic renal hypertrophy and indicates that UNX- and diabetes-mediated mTOR activation can selectively activate S6K1 without activating S6K2.

Topics: Adaptor Proteins, Signal Transducing; Animals; Blood Glucose; Carrier Proteins; Cell Cycle Proteins; Cell Proliferation; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Models, Animal; Enzyme Inhibitors; Eukaryotic Initiation Factors; Gene Expression Regulation, Enzymologic; Hypertrophy; Kidney; Male; Mice; Mice, Knockout; Nephrectomy; Phosphoproteins; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Ribosomal Protein S6; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases

Topics: Animals; Cell Proliferation; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Enzyme Inhibitors; Eukaryotic Initiation Factors; Gene Expression Regulation, Enzymologic; Humans; Hypertrophy; Kidney; Mice; Mice, Knockout; Phosphorylation; Protein Kinases; Ribosomal Protein S6; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Calcineurin inhibitors (CNI) have brought dramatic improvements in early renal allograft survival. However, CNI are associated with posttransplant hypertension (PTHTN), a risk factor for mortality from cardiovascular disease and graft failure. Sirolimus (SRL) is emerging as an alternative to CNI. SRL effects on blood pressure (BP) in humans are unclear. We compared the prevalence of PTHTN among patients receiving SRL as maintenance immunosuppression with a group receiving CNI by using 24-hour ambulatory BP (AMBP). AMBP has been shown to predict cardiovascular events and progression of kidney disease better than casual office BP measurements in chronic kidney disease (CKD) patients.. Renal transplant recipients with office hypertension (defined as BP > 130/80 or on antihypertensive medications), receiving stable immunosuppression and displaying consistent serum creatinine values for > or =6 months were eligible. We enrolled the first 40 patients to consent. Office BP was measured twice using a BP-Tru machine. AMBP was then analyzed for systolic BP (SBP), diastolic BP (DBP), and nocturnal blood pressure fall (NF; "dipping"). Patients were placed in the SRL group (n = 18) and the CNI group (n = 20) based on their maintenance immunosuppressive protocol. Two patients were excluded because of incomplete data. All patients received mycophenolate mofetil, and 14/38, maintenance steroids. We collected, demographics as well as type and date of renal allograft, medications, comorbidities, CKD stage, proteinuria, and plasma creatinine at the time of study enrollment.. Patients in the SRL group displayed lower 24-hour SBP than the CNI group (128.0 +/- 10.8 vs 137.7 +/- 14; P = .029). Nightime MAP, nightime SBP, and nighttime DBP were all lower in the SRL group. NF did not reach significance between the SRL and CNI groups (44% vs 15%; P = .074). Patient demographics and number of antihypertensive medications did not differ.. The lower 24-hour SBP seen in the SRL group by AMBP may lead to improved cardiovascular and renal outcomes over time. Long-term patient follow-up will be needed to clarify the effect of the lower 24-hour SBP.

Topics: Adult; Aged; Antihypertensive Agents; Blood Pressure; Body Mass Index; Cadaver; Calcineurin; Cardiovascular Diseases; Diabetic Nephropathies; Female; Humans; Hypertension; Immunosuppression Therapy; Kidney Failure, Chronic; Kidney Transplantation; Living Donors; Male; Middle Aged; Monitoring, Ambulatory; Postoperative Complications; Postoperative Period; Sirolimus; Tissue Donors

Reports on the use of sirolimus (SRL) in pancreas transplantation are still limited. The aim of this study was to evaluate the outcome of SRL conversion in pancreas transplant patients. Among 247 patients undergoing simultaneous kidney-pancreas or solitary pancreas transplantation, 33 (13%) were converted to SRL. The reasons for conversion were calcineurin inhibitors (CNI) nephrotoxicity (n = 24; 73%), severe neurotoxicity owing to CNI (n = 1; 3%), severe and/or recurrent acute rejection episodes (n = 7; 21%), gastrointestinal (GI) side effects of mycophenolate mofetil (MMF; n = 5; 15%), and hyperglycemia (n = 4; 12%). Before conversion, all patients were maintained on a CNI, MMF, and low-dose steroids. They were gradually converted to SRL associated with either CNI or MMF withdrawal. Sixty-three percent (n = 15) of patients who were converted owing to CNI nephrotoxicity, showed stable or improved renal function. At 12 months after conversion, serum creatinine levels were significantly decreased in this group (2.2 +/- 0.5 vs 1.6 +/- 0.3 mg/dL; P = .001) and C-peptide values increased (2.9 +/- 1.1.1 vs 3.1 +/- 1.3 nmol/L; P = .018). The only patient with leucoencephalopathy showed improved neurologic status after SRL conversion. All patients converted to SRL because of GI side effects of MMF showed improvements, and none of those converted because of hyperglycemia experienced improvement. There were no episodes of acute rejection after conversion. We concluded that conversion to SRL in pancreas transplantation should be considered an important alternative strategy, particularly for CNI nephrotoxicity and neurotoxicity, and in cases of severe diarrhea due to MMF.

Topics: Adult; Calcineurin Inhibitors; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Female; Humans; Hyperglycemia; Immunosuppressive Agents; Kidney Transplantation; Male; Middle Aged; Mycophenolic Acid; Pancreas Transplantation; Retrospective Studies; Sirolimus; Young Adult

Incisional surgical site infections are common bacterial infections in kidney transplantation. The purpose of this study was to determine the incidence, timing, etiology, and risk factors for incisional surgical site infections.. We performed a prospective study that included a population of 1400 consecutive patients (58.4% males) who underwent kidney transplantation in Spanish hospitals pertaining to the RESITRA research network.. A total of 55 patients developed 63 episodes of incisional surgical site infections. Median time from transplant to incisional surgical site infections was 20 days (range, 2 to 76 days). All infected patients recovered from incisional surgical site infections. The most frequently isolated pathogens were Escherichia coli (31.7%), Pseudomonas aeruginosa (13.3%), Enterococcus faecalis (11.6%), Enterobacter spp. (10%), and coagulase-negative staphylococci (8.3%). Diabetic patients had an increased risk of incisional surgical site infections (7.5%, P = 0.013). We used several different regimens of antimicrobial prophylaxis. None were found to be associated with an increased risk of incisional surgical site infections. The use of sirolimus was associated with an increased risk of incisional surgical site infections (7.4%, P = 0.018).. Diabetic patients, and those who received sirolimus-based immunosuppressive regimens, showed an increased risk of developing incisional surgical site infections after kidney transplantation.

Topics: Adult; Aged; Diabetic Nephropathies; Female; Humans; Immunocompromised Host; Immunosuppressive Agents; Kidney Transplantation; Male; Middle Aged; Risk Factors; Sirolimus; Surgical Wound Infection

Islet transplantation alone is an alternative for the replacement of pancreatic endocrine function in patients with type 1 diabetes. The aim of our study was to assess the impact of the Edmonton immunosuppressive protocol (tacrolimus-sirolimus association) on kidney function.. Nineteen patients with type 1 diabetes and metabolic instability received islet transplantation alone and immunosuppressive therapy according to the Edmonton protocol. Serum creatinine (sCr), creatinine clearance (CrCl), and 24-h urinary protein excretion (UPE) were assessed at baseline and during a follow-up of 339 patient-months.. After islet transplantation we observed 1) sCr within the normal range in all but two patients in whom sCr increased immediately after islet transplantation, and despite withdrawal of immunosuppression, patients progressed to end-stage renal disease (ESRD); 2) CrCl remained within the normal range for those patients who had normal baseline values and decreased, progressing to ESRD in two patients with a decreased baseline CrCl; and 3) 24-h UPE worsened (>300 mg/24 h) in four patients. In the two patients who progressed to ESRD, the worsening of 24-h UPE occurred immediately after islet transplantation. In one patient 24-h UPE worsening occurred at 18 months, and, after withdrawal of immunosuppression, it returned to the normal range. In another patient 24-h UPE increased at 24 months and remained stable while immunosuppression was continued.. In type 1 diabetic patients receiving islet transplantation alone, the association of tacrolimus and sirolimus should be used only in patients with normal kidney function. Alternative options for immunosuppressive treatment should be considered for patients with even a mild decrease of kidney function.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Creatinine; Daclizumab; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Disease Progression; Follow-Up Studies; Glycated Hemoglobin; Humans; Hypoglycemic Agents; Immunoglobulin G; Immunosuppressive Agents; Insulin; Islets of Langerhans Transplantation; Kidney Function Tests; Retrospective Studies; Sirolimus; Tacrolimus

Recent studies suggested the involvement of the Akt/mammalian target of rapamycin (mTOR) pathway in the pathogenesis of diabetic nephropathy. The effect of mTOR blockade by rapamycin in diabetic nephropathy was investigated, but in vivo study of rapamycin treatment in the course of early diabetes is still insufficient. This study was designed to determine the therapeutic effects of rapamycin on diabetic nephropathy at an early stage.. Diabetes was induced in Sprague-Dawley rats with streptozotocin, and rapamycin (1 mg/kg) was administered by daily gavage for 4 weeks. Renal structural changes and some factors involved in the early pathogenesis of diabetic nephropathy were tested. The activation level of the Akt/mTOR pathway was also determined.. Rapamycin treatment reduced albuminuria, glomerular enlargement, glomerular basement membrane thickening, renal macrophage recruitment, and levels of renal mRNA expression of proliferating cell nuclear antigen, transforming growth factor-beta1, vascular endothelial growth factor, and monocyte chemoattractant protein-1 without change in blood glucose level and blood pressure in experimental diabetic rats. In addition, treatment with rapamycin also down-regulated the enhanced levels of renal p-Akt, phospho-p70S6 kinase, and phospho-ribosomal S6 protein in diabetic rats.. Rapamycin treatment can prevent the early renal structural changes of diabetes in experimental rats, and thus halt the early steps of the development of diabetic nephropathy. mTOR blockade might be beneficial for the treatment of diabetic nephropathy.

Topics: Albuminuria; Animals; Anti-Inflammatory Agents; Cell Proliferation; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Glomerular Basement Membrane; Hypertrophy; Kidney; Kidney Glomerulus; Male; Protein Kinases; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Chronic allograft nephropathy, cardiovascular mortality, and posttransplant malignancy are complications of conventional immunosuppression after kidney transplantation. We reported the feasibility of maintenance monotherapy with sirolimus (SRL) in a pilot experience. The aim was to study safety and feasibility of SRL maintenance monotherapy in 50 kidney transplant patients.. All patients from our center with at least 6 months follow-up on SRL monotherapy were included. During the first month after start of SRL monotherapy, follow-up visits were performed weekly, then each month for the following 2 months. Each follow-up visit included a physical exam and laboratory screening.. Mean follow-up on SRL monotherapy was 34.7 +/- 14.9 months. The time between transplantation until start of monotherapy was 7.7 +/- 3.3 years. No rejections occurred. During follow-up, two patients died of cardiovascular disease (already diagnosed before monotherapy); one, of previously diagnosed posttransplant malignancy and one, of hepatitis C-related liver failure. Glomerular filtration rate (GFR) was 53 mL/min x 1.73 m2 at start of monotherapy and 50 mL/min x 1.73 m2 after 4 years. Proteinuria was 632 +/- 562 mg/24 hours at 4 years. During the follow-up, no significant changes in the lipid profile, glycemia, or hemoglobin occurred.. Sirolimus monotherapy is safe in a selected group of immunological low-risk patients without increasing the risk of rejection.

Topics: Diabetes Mellitus, Type 2; Diabetic Nephropathies; Female; Follow-Up Studies; Humans; Immunosuppression Therapy; Immunosuppressive Agents; Kidney Failure, Chronic; Kidney Transplantation; Male; Reoperation; Sirolimus; Time Factors

High glucose and high insulin, pathogenic factors in type 2 diabetes, induce rapid synthesis of the matrix protein laminin-beta1 in renal proximal tubular epithelial cells by stimulation of initiation phase of mRNA translation. We investigated if elongation phase of translation also contributes to high glucose and high insulin induction of laminin-beta1 synthesis in proximal tubular epithelial cells. High glucose or high insulin rapidly increased activating Thr56 dephosphorylation of eEF2 and inactivating Ser366 phosphorylation of eEF2 kinase, events that facilitate elongation. Studies with inhibitors showed that PI3 kinase-Akt-mTOR-p70S6 kinase pathway controlled changes in phosphorylation of eEF2 and eEF2 kinase induced by high glucose or high insulin. Renal cortical homogenates from db/db mice in early stage of type 2 diabetes showed decrease in eEF2 phosphorylation and increment in eEF2 kinase phosphorylation in association with renal hypertrophy and glomerular and tubular increase in laminin-beta1 content. Rapamycin, an inhibitor of mTOR, abolished diabetes-induced changes in phosphorylation of eEF2, eEF2 kinase, and p70S6 kinase and ameliorated renal hypertrophy and laminin-beta1 protein content, without affecting hyperglycemia. These data show that mTOR is an attractive target for amelioration of diabetes-induced renal injury.

Topics: Animals; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Elongation Factor 2 Kinase; Glucose; Insulin; Kidney Cortex; Laminin; Mice; Mice, Mutant Strains; Phosphorylation; Protein Biosynthesis; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases

Early diabetic nephropathy is characterized by renal hypertrophy that is mainly due to proximal tubular hypertrophy. Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase, and its signaling has been reported to regulate protein synthesis and cellular growth, specifically, hypertrophy. Therefore, we examined the effect of mTOR signaling on diabetic renal hypertrophy by using the specific inhibitor for mTOR, rapamycin. Ten days after streptozotocin-induced diabetes, mice showed kidney hypertrophy with increases in the phosphorylation of p70S6kinase and the expression of cyclin kinase inhibitors, p21(Cip1) and p27(Kip1), in the kidneys. The intraperitoneal injection of rapamycin (2 mg/kg/day) markedly attenuated the enhanced phosphorylation of p70S6kinase, the increment of cyclin-dependent kinase inhibitors, and renal enlargement without any changes of clinical parameters, including blood glucose, blood pressure, and food intake. Overexpression of a constitutive active form of p70S6kinase resulted in increased cell size of cultured mouse proximal tubule cells; thus, activation of p70S6kinase causes hypertrophy of proximal tubular cells. Our findings suggest that activation of mTOR signaling causes renal hypertrophy at the early stage of diabetes.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Progression; Hypertrophy; Immunosuppressive Agents; Injections, Intraperitoneal; Kidney; Male; Mice; Mice, Inbred C57BL; Organ Size; Protein Kinases; Severity of Illness Index; Signal Transduction; Sirolimus; Streptozocin; TOR Serine-Threonine Kinases; Treatment Outcome

Recent data suggest that the phosphatidylinositol 3-kinase (PI3-K)/Akt/mammalian target of rapamycin (mTOR) pathway is important in diabetic nephropathy. The effect of mTOR blockade by sirolimus (SRL) in diabetic kidney disease in rats was investigated. Diabetes was induced by streptozotocin in male Sprague-Dawley rats. Sixteen weeks later, diabetic animals were divided into the following groups: diabetes (D; n = 8), diabetes + SRL at 1 mg/kg per d, SRL trough level 2.3 +/- 0.25 ng/ml (D+SRL; n = 7); and diabetes + normoglycemia maintained by insulin implants (D+NG; n = 5). There was an age-matched nondiabetic group (ND; n = 6). All animals were followed for 4 wk. The D group showed glomerular hypertrophy (mean glomerular volume 5.0 +/- 0.4 in D versus 3.3 +/- 0.2 10(6) mu(3) in ND; P < 0.05) without renal hyperplasia (calculated by reverse transcription-PCR of proliferative cell nuclear antigen) and albuminuria (29 +/- 4 in D versus 1.4 +/- 1.5 mg/24 h in ND; P < 0.05). Both D+NG and D+SRL groups had a significant reduction of albuminuria, although glomerular hypertrophy was still present. SRL treatment did not modify the number of infiltrating renal ED1(+) cells. Diabetic animals had greater expression of p-Akt and mTOR, unlike ND rats. NG and SRL treatment reduced p-Akt and normalized mTOR. It is interesting that D+SRL was associated with a significant reduction of renal TGF-beta1 and glomerular connective tissue growth factor. SRL treatment reduced glomerular alpha-smooth muscle actin overexpression and reduced significantly the mesangial matrix accumulation that is characteristic of diabetic nephropathy. In conclusion, mTOR blockade by low-dose SRL has a beneficial effect in diabetic kidney disease, suggesting that the mTOR pathway has an important pathogenic role in diabetic nephropathy.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Progression; Immunosuppressive Agents; Kidney; Male; Protein Kinases; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Streptozocin; TOR Serine-Threonine Kinases

We have already reported Gas6 is involved in glomerular hypertrophy observed in diabetic nephropathy. However, the molecular mechanisms involved in glomerular hypertrophy are still unknown, especially in vivo.. In vivo, diabetes was induced in rats and mice by streptozotocin (STZ) and the activation of the Akt/mTOR pathway in glomeruli was examined. In vitro, mesangial hypertrophy was assessed by [(3)H]leucine incorporation and measuring cell areas.. Akt, p70 S6 kinase, and 4E-BP-1 were induced and phosphorylated in rat glomerular lysates after 12 weeks of STZ injection when mesangial and glomerular hypertrophy was observed. We then examined the role of Gas6 by treating STZ-rats with warfarin, and found that warfarin treatment inhibited the phosphorylation of these molecules as well as the hypertrophy. We next examined whether high glucose stimulation can induce the expression of Gas6/Axl in mesangial cells. Stimulation of the cells with 25 mmol/L of glucose increased the expression of Gas6/Axl and mesangial cell size compared with that with 5.6 mmol/L of glucose. This hypertrophic effect was abolished in mesangial cells derived from Gas6 knockout mice. We also found that LY294002 and rapamycin blocked Gas6-induced activation of the Akt/mTOR pathway and mesangial hypertrophy. Furthermore, less phosphorylated Akt-positive or 4E-BP-1-positive areas were found in STZ-treated Gas6 knockout mice than in STZ-treated wild-type mice.. Our study indicates that the Akt/mTOR pathway is a key signaling cascade in Gas6-mediated mesangial and glomerular hypertrophy and revealed a crucial role of Gas6/Axl and the Akt/mTOR pathway in the development of diabetic nephropathy.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antibiotics, Antineoplastic; Butadienes; Carrier Proteins; Cell Cycle Proteins; Cells, Cultured; Chromones; Cyclin-Dependent Kinase Inhibitor p27; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Enzyme Inhibitors; Eukaryotic Initiation Factors; Female; Glomerular Mesangium; Glucose; Hypertrophy; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Morpholines; Nitriles; Phosphoproteins; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; TOR Serine-Threonine Kinases; Tumor Suppressor Proteins

Conversion from cyclosporine (CsA) to sirolimus (SRL) has mainly been done in clinical conditions warranting calcineurin inhibitor discontinuation. Little is known about the clinical outcome of conversion in renal transplant recipients without transplant dysfunction.. This prospective, open-label, multicentric pilot study evaluates the safety and efficacy of converting patients with stable renal function from CsA to SRL.. Forty stable patients on CsA, mycophenolate mofetil (MMF) (1.5 g/day), and steroids (ST) were converted at 7.6+/-1.4 months after renal transplantation. At 1 year, graft and patient survival was 100% and the incidence of acute rejection 5%. Calculated glomerular filtration rate (GFR) increased from 54+/-18 to 66+/-16 ml/min (P<0.0001). Blood pressure remained unchanged. A gradual increase in the incidence and severity of proteinuria was observed from month 6 onwards with de novo proteinuria in 30% of the patients at 1 year. Protein excretion was below 1 g/day in 12.5%, between 1 and 3 g/day in 17.5% and above 3 g/day in 7.5% of the proteinuric cohort (P=0.0043, compared to baseline). No predictors could be identified for the development of proteinuria. All patients had a reduction in protein excretion following renin-angiotensin blockade and were continued on SRL.. Conversion of stable renal transplant recipients from a CsA-MMF-ST to a SRL-MMF-ST regimen is safe and results in improved renal function but is associated with the development of proteinuria in 30% of the patients requiring renin-angiotensin blockade.

Topics: Adult; Aged; Creatinine; Cyclosporine; Diabetic Nephropathies; Drug Therapy, Combination; Female; Glomerular Filtration Rate; Graft Rejection; Humans; Immunosuppressive Agents; Kidney Failure, Chronic; Kidney Function Tests; Kidney Transplantation; Male; Middle Aged; Mycophenolic Acid; Sirolimus

Topics: Anti-Inflammatory Agents; Body Temperature; Bronchoalveolar Lavage Fluid; Bronchoscopy; Coronary Artery Bypass; Cough; Cyclosporine; Diabetic Nephropathies; Diagnosis, Differential; Female; Humans; Immunosuppression Therapy; Immunosuppressive Agents; Kidney Transplantation; Lung Diseases, Interstitial; Middle Aged; Prednisone; Radiography, Thoracic; Risk Factors; Sirolimus; Tomography, X-Ray Computed; Xenobiotics

The increased incidence of Kaposi's sarcoma (KS) in organ transplantation has been related to the KS herpesvirus and the permissive effect of immunosuppressive therapy. Calcineurin inhibitors are the cornerstone of immunosuppression in organ transplantation, although they could promote tumor progression. In contrast, sirolimus, a new immunosuppressive agent, exhibits potent antitumor activity. We postulated that conversion from cyclosporine to sirolimus in patients with KS could favor regression of KS lesions without increasing the risk of graft rejection. Two renal transplant recipients with KS underwent conversion from cyclosporine to sirolimus. Both patients showed complete regression of KS lesions and excellent clinical and functional results. Sirolimus offers a new and promising approach to the management of posttransplantation KS and probably to other types of malignancies in organ transplant recipients.

Topics: Aged; Antibiotics, Antineoplastic; Cyclosporine; Diabetic Nephropathies; Glomerulonephritis; Humans; Immunosuppressive Agents; Kidney Transplantation; Male; Postoperative Complications; Sarcoma, Kaposi; Sirolimus

Among the numeruos adverse side effects of tacrolimus (TAC), de novo thrombotic microangiopathy stands out as an infrecuente but severe complication. Renal dysfunction is the only alteration that should lead to suspicion of thrombotic microangiopathy, because the clinical features of intravascular hemolysis are not always found. The definitive diagnosis can usually be made with kidney biopsy. Patientes with TAC induced thrombotic microangiopathy usually promptly recover after treatment withdrawal or reduction in the dose of TAC and a short course of plasma therapy, but the risk of rejection increases. Switching from TAC to cyclosporine has also been tried with resolution of the hemolysis but thrombotic microangiopathy has been noted with both and this condition may later recur. We present a 29-year-old man who received a kidney-pancreas transplant for end-stage diabetic nephropathy. After initial induction with basiliximab, the immunosuppression consisted of prednisone, tacrolimus and mycophenolate mofetil. Twenty four days posttransplantation his renal function declined with a peak creatine level of 2.35 mg/dl. Laboratory studies showed thrombocytopenia and features of intravascular hemolysis. TAC associated hemolytic uremic syndrome was suspected and drug was immediately stopped and converted to sirolimus. Also he was treated with plasma infusion. The allograft biopsy showed focal glomerular and arteriolar acute thrombosis without evidence of rejection. Our experience demostrate that switching from tacrolimus to sirolimus could be an adecuate strategy for patients who develop FK506-associated de novo thrombotic microangiopathy without increase risk of acute rejection.

Topics: Adult; Antibodies, Monoclonal; Basiliximab; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Drug Therapy, Combination; Duodenostomy; Hemolytic-Uremic Syndrome; Humans; Immunosuppressive Agents; Jejunostomy; Kidney Failure, Chronic; Kidney Transplantation; Kidney Tubular Necrosis, Acute; Male; Mycophenolic Acid; Pancreas Transplantation; Pancreatic Fistula; Plasma; Postoperative Complications; Prednisone; Recombinant Fusion Proteins; Sirolimus; Tacrolimus

Topics: Adult; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Drug Therapy, Combination; Female; Humans; Immunosuppressive Agents; Kidney Failure, Chronic; Kidney Transplantation; Mycophenolic Acid; Necrosis; Sirolimus; Skin; Treatment Outcome; Vasculitis, Leukocytoclastic, Cutaneous

The emerging anti-cancer approach is based on combining a 'traditional' cytotoxic drug with a 'signaling' blocking agent. Such combination, if designed and applied properly, may increase selectivity towards tumor cells. The use of such combinations requires smart planning and choice of the drugs to be combined, their proper dosing as well as correct sequence and schedule of application. The combination of the anti-metabolite gemcitabine and the mTOR blocker, rapamycin, has achieved an impressive response in a patient with metastatic leiomyosarcoma.

Topics: Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Deoxycytidine; Diabetic Nephropathies; Gemcitabine; Humans; Kidney Failure, Chronic; Kidney Neoplasms; Kidney Transplantation; Leiomyosarcoma; Liver Neoplasms; Male; Middle Aged; Sirolimus; Tomography, X-Ray Computed; Treatment Outcome

We report a diabetic renal transplant recipient who experienced an episode of acute allograft rejection in the 6th month posttransplant when there was an attempt at steroid withdrawal. The acute rejection was steroid resistant. Furthermore calcineurin inhibitor nephrotoxicity was exacerbated by rescue therapy with tacrolimus conversion. The allograft dysfunction ultimately stabilized upon institution of sirolimus and minimization of tacrolimus.

Topics: Adult; Calcineurin Inhibitors; Cyclosporine; Diabetic Nephropathies; Humans; Immunosuppressive Agents; Kidney Failure, Chronic; Kidney Transplantation; Male; Reoperation; Sirolimus; Tacrolimus; Treatment Outcome

Withdrawal of corticosteroids from the immunosuppressive regimens of kidney transplant recipients has been associated with an increased risk of acute and chronic allograft rejection. Previous studies indicate that the risk of rejection is particularly high in African Americans.. We prospectively enrolled 44 African American kidney transplant recipients to participate in an uncontrolled trial in which they were initially treated with sirolimus, tacrolimus, and corticosteroids. No patient received antibody induction therapy. Prednisone was withdrawn from eligible patients free of acute rejection beginning as early as 3 months posttransplant, and followed for a minimum of 9 months posttransplant. Patients were followed for acute rejection and for changes in blood pressure, body weight, and serum creatinine concentrations before and after withdrawal of steroids.. Thirty of 44 patients (68%) were weaned off of prednisone. Follow-up after withdrawal of prednisone ranged from 3 to 26 months (mean, 14.3+/-7.7 months). Two of 30 patients (6.7%) developed acute rejection. At last follow-up, 27 of 30 patients (90%) remain steroid-free. Steroid withdrawal was associated with significant reductions in blood pressure.. Use of sirolimus and tacrolimus, without the use of induction antibody therapy, allows withdrawal of prednisone as early as 3 months posttransplant with low rates of subsequent acute rejection in African American kidney transplant recipients. Withdrawal of prednisone was associated with lower blood pressures and the need for fewer antihypertensive medications.

Topics: Adrenal Cortex Hormones; Adult; Aged; Black or African American; Diabetic Nephropathies; Drug Administration Schedule; Female; Humans; Hypertension, Renal; Immunosuppressive Agents; Kidney Failure, Chronic; Kidney Transplantation; Male; Middle Aged; Ohio; Retrospective Studies; Sirolimus; Tacrolimus

Topics: Adult; Age of Onset; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Drug Resistance; Female; Follow-Up Studies; Graft Rejection; Humans; Immunosuppressive Agents; Kidney Failure, Chronic; Kidney Transplantation; Male; Middle Aged; Muromonab-CD3; Pancreas Transplantation; Renal Dialysis; Retrospective Studies; Sirolimus; Steroids; Time Factors; Tissue Donors