Cyclosporine Nephrotoxicity Research Articles

Interaction of endothelin-1 and nitric oxide pathways in human tubular epithelial cells under the influence of cyclosporine-A

Background: The exact mechanism of cyclosporine (CsA) nephrotoxicity has not been clarified. In this study, we investigated the effect of pharmacological doses of CsA on the production of nitric oxide synthases (NOSs) and endothelin (ET) receptors (ETR-A, ETR-B), in human tubular cells [human kidney (HK)-2], to identify any implication of these pathways in CsA nephrotoxicity. Methods: Human tubular epithelial cells (HK-2) were cultured in the presence of CsA at various concentrations (0–1000 ng/mL). Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine mRNA synthesis of NOSs (eNOS, iNOS) and ET receptors (ETR-A, ETR-B) and western blot analysis for the subsequent proteins. Results: A dose-dependent induction of synthesis of NO synthases eNOS and iNOS and ET receptors ETR-A and ETR-B was observed, even at therapeutic doses of CsA. An interaction between NO and ET-1 systems under the influence of CsA was also observed. Blockage of NO production was followed by down-regulation of ETR-B whereas blockade of ET pathway with ET receptor antagonists was followed by down-regulation of eNOS expression. Conclusion: CsA induces NOSs as well as ET receptor mRNA and protein synthesis in tubular epithelial cells. The up-regulation of NO and ET-1 pathways is probably implicated in the nephrotoxic action of CsA, whereas an interplay between ETR-B and eNOS seems to be involved.

Treatment with microemulsified cyclosporine in children with frequently relapsing nephrotic syndrome

We previously established a treatment protocol for conventional cyclosporine (Sandimmune, Novartis, Basel, Switzerland) in children with frequently relapsing nephrotic syndrome; ∼50% of patients remained relapse free for 2 years, without serious adverse events. Recently, microemulsified cyclosporine (Neoral, Novartis), which has a more stable absorption profile than conventional cyclosporine, has been developed. We tested the hypothesis that microemulsified cyclosporine is at least as effective as conventional cyclosporine. To evaluate the safety and efficacy of microemulsified cyclosporine, a prospective, multicentre trial was conducted according to the previously established protocol, using microemulsified cyclosporine instead of conventional cyclosporine. The duration of treatment was 24 months. During the first 6 months, patients received microemulsified cyclosporine in a dose that maintained the trough level between 80 and 100 ng/mL of cyclosporine. For the next 18 months, the dose was adjusted to maintain a level between 60 and 80 ng/mL. A total of 62 patients (median age, 5.4 years; 48 males, 14 females) were studied. The frequency of relapse decreased from 4.6 ± 1.4 to 0.7 ± 1.5 times per year (P < 0.0001). The probability of relapse-free survival at Month 24 was 58.1% (95% confidence interval, 45.8-70.3%). The probability of progression (to frequently relapsing nephrotic syndrome)-free survival at Month 24 was 88.5% (95% confidence interval, 80.4-96.5%). Cyclosporine nephrotoxicity was detected in only 8.6% of patients who underwent renal biopsy after 2 years of treatment. Antihypertensive agents were administered to 12.9% of the patients to control hypertension without severe sequelae. Microemulsified cyclosporine administered according to our treatment protocol is safe and effective in children with frequently relapsing nephrotic syndrome.

Paricalcitol attenuates cyclosporine-induced kidney injury in rats

Despite its benefits, the clinical use of cyclosporine A (CsA) is limited by its nephrotoxic properties. Because paricalcitol (19-nor-1,25-hydroxyvitamin D(2)) has renoprotective effects, we tested whether it can blunt renal dysfunction and fibrosis in a rat model of CsA-induced nephropathy. Treatment with CsA decreased creatinine clearance, increased monocyte/macrophage infiltration, and increased the expression of inflammatory cytokines within the kidney. Paricalcitol reduced the decline in kidney function and pro-fibrotic changes and also blunted the increased transforming growth factor (TGF)-beta1 expression and Smad signaling. Using an in vitro model, we treated HK-2 cells with CsA and found that paricalcitol attenuated the CsA-induced increases in phosphorylated extracellular signal-regulated and c-Jun N-terminal kinases, and also prevented the activation of nuclear factor-kappaB. Paricalcitol effectively prevented TGF-beta1-induced epithelial-to-mesenchymal transitions and extracellular matrix accumulation as evidenced by attenuated collagen deposition and fibrosis in CsA-treated rats. In addition, paricalcitol decreased the number of TUNEL-positive nuclei and reduced the expression of pro-apoptotic markers in CsA-treated HK-2 cells. Thus, paricalcitol appears to attenuate CsA-induced nephropathy by suppression of inflammatory, pro-fibrotic, and apoptotic factors through inhibition of the nuclear factor-kappaB, Smad, and mitogen-activated protein kinase signaling pathways.

Combined Heart and Kidney Transplantation: Long-Term Analysis of Renal Function and Major Adverse Events at 20 Years

Background Combined heart-kidney transplantation (HKTx) is an accepted therapeutic option for patients with end-stage heart disease associated with severely impaired renal function. We report our long-term follow-up with this combined procedure. Patients and methods Between April 1989 to November 2009, nine patients underwent combined simultaneous (HKTx) at our center. Seven patients were males (mean age 45.2 ± 10.12 years); seven patients were on dialysis at the time of transplantation. Results Surgical procedures were uneventful in all patients. One patient died in the intensive care unit 41 days after transplantation. During long-term follow-up, three patients died: one due to infection and multiorgan failure 148 months after HKTx, one due to a lung neoplasm after 6 years, and one, a cerebral stroke at 34 months after transplantation. Only one patient experience renal allograft failure secondary to hypertension and cyclosporine nephrotoxicity at 10 years after HKTx with the need for renal replacement therapy. Last estimated glomerular filtration rates of all other patients was 61.3 ± 17.4 mL/min. Conclusions In selected patients, with coexisting end-stage cardiac and renal failure, combined HKTx with an allograft from the same donor proved to give satisfactory short- and long-term results, with a low incidence of both cardiac and renal allograft complications.

Serum indoxyl sulfate as an early marker for detecting chronic cyclosporine nephrotoxicity

Cyclosporine A (CsA) is an effective agent for frequently relapsing steroid-dependent nephrotic syndrome (FR-SDNS), but its use can also be complicated by renal toxicity. Because no biochemical markers from urine or blood samples have yet been established for detecting CsA-induced renal injury to date, repeated renal biopsies are therefore required for all patients with long-term CsA treatment. The purpose of the present study was therefore to detect early change of CsA nephropathy (CsAN) using blood samples. Several biochemical markers were analyzed in an attempt to examine the renal function in 24 patients with FR-SDNS who had been treated with CsA. Those included serum cystatin C and indoxyl sulfate, as well as creatinine and beta2-microglobulin. Renal biopsy findings indicated chronic CsAN in 13 of the 24 patients. Among those markers, only serum indoxyl sulfate was significantly elevated in patients with CsAN. It may be possible for measurement of serum indoxyl sulfate level to replace repeated renal biopsies in evaluation of chronic CsAN in pediatric patients with FR-SDNS.

Polymerized type I collagen reduces chronic cyclosporine nephrotoxicity

Polymerized type I collagen (P-collagen) has been successfully used to reduce human hypertrophic scars due to its anti-fibrotic and anti-inflammatory properties. We therefore carried out a study to determine if P-collagen reduces functional and structural injury in chronic cyclosporine [cyclosporine A (CsA)] nephropathy. Four groups of six male Wistar rats fed with a low sodium diet were treated with vehicle, P-collagen (0.8 mg/day, i.p.), CsA (15 mg/kg) or CsA + P-collagen for 15 days. Mean arterial pressure, renal blood flow and glomerular filtration rate were measured in all groups. Structural injury such as arteriolopathy, tubulo-interstitial fibrosis (TI-fibrosis) and positive apoptotic cells were quantified. The mRNA expression levels of transforming growth factor-beta (TGF-beta), kidney injury molecule (Kim-1), alpha-smooth muscle actin (alpha-SMA), glutathione peroxidase, catalase and Cu/Zn superoxide dismutase (SOD) as well as MnSOD were assessed. Antioxidant enzyme activity, renal lipoperoxidation and urinary excretion of oxygen peroxide (UH(2)O(2)V) were determined. Cyclosporine produced renal dysfunction and induced the development of arteriolopathy, TI-fibrosis and tubular apoptosis. These alterations were associated with increases in TGF-beta, Kim-1 and alpha-SMA mRNA levels as well as with a significant increase of oxidative stress and a reduction of SOD activity. P-Collagen partially ameliorated CsA-induced renal dysfunction and structural injury and prevented both tubular apoptosis and increased oxidative stress. This renoprotective effect was found to be associated with a reduction of TGF-beta, Kim-1 and alpha-SMA mRNA levels. This study has therefore demonstrated that P-collagen appears to have anti-fibrotic and anti-apoptotic properties and highlights the possibility that the compound might be useful in a strategy to reduce chronic CsA nephrotoxicity.

Neutrophil gelatinase-associated lipocalin (NGAL): a new marker of cyclosporine nephrotoxicity?

The aim of work was to investigate whether serum and urinary neutrophil gelatinase-associated lipocalin(sNGAL and uNGAL, respectively) are potential biomarkers of early cyclosporine A (CsA) nephrotoxicity in steroid-dependent nephrotic children (SDNS). The study group (I) consisted of 19 children with SDNS aged 9.46+/-5.52 years treated with CsA. The children were examined four times: at proteinuria relapse, prior to CsA treatment,then after 3, 6, and 12 months of CsA treatment. The control group (II) consisted of 18 healthy children aged 3-15 years. A commercial enzyme-linked immunosorbent assay method was used to measure NGAL concentration.The sNGAL level in SDNS children prior to the administration of CsA was similar to that in the healthy controls (p>0.05), but it increased significantly during the course of treatment (p<0.01). The uNGAL/creatinine (cr) ratio in SDNS patients was higher before the withdrawal of CsA therapy (p<0.05), and was also increased at the consecutive examinations (p<0.01). There was a positive correlation between both sNGAL and uNGAL levels and CsA serum level. However, based on the serum and urinary NGAL/cr receiver operating characteristic curve and area under the curve (AUC) analysis, it remains uncertain whether uNGAL is a good predictor of cyclosporine nephropathy. Both sNGAL and uNGAL concentrations increased during the course of CsA treatment. Further studies in larger groups of patients are therefore necessary to confirm our experimental data that increased NGAL levels may be a non-invasive marker for the early detection of tubulointerstitial damage in CsA nephrotoxicity.

Tranilast Attenuates Chronic Cyclosporine Nephrotoxicity in Rats

Our aim was to explore the effects of the anti-allergic and antifibrotic agent tranilast on chronic cyclosporine (CsA) nephrotoxicity in rats. Methods Eighteen Sprague-Dawley rats were randomized to be given the following daily treatments by gavage for 4 weeks: (1) controls, olive oil; (2) CsA group, CsA 25 mg/kg; (3) CsA plus tranilast group, CsA 25 mg/kg and tranilast 400 mg/kg. We examined the body weights and the effects of tranilast on histopathology, macrophage (Mϕ) infiltration, and expression of osteopontin (OPN). Results The administration of tranilast improved body weight gain by CsA-treated rats (232 ± 24 vs 203 ± 6 g; P < .05). This treatment reduced the expression of OPN protein and infiltration of macrophages (9.14 ± 2.7 vs 22.44 ± 5.68 ED-1 positive cells per high power field, P < .05). Furthermore compared with the CsA alone group, it ameliorated tubulointerstitial fibrosis scores (1.18 ± 0.08 vs 2.57 ± 0.21, P < .05). Conclusion Tranilast attenuated tubulointerstitial fibrosis through decreased expression of OPN protein and macrophage infiltration, showing renal protective effects in rats with chronic CsA nephrotoxicity.

Antiapoptotic properties of recombinant human erythropoietin protects against tubular cyclosporine toxicity

During the early post transplant period, the tubular epithelium is the main target of injuries including ischemia reperfusion and toxicity effects from calcineurin inhibitors. Taking into account the tissue protective effects of erythropoietin mediated through its antiapoptotic properties, we tested whether administration of recombinant human erythropoietin protects against acute cyclosporine nephrotoxicity. Four groups of five rats were intraperitoneally treated over 28 days with 100 UI/Kg/48 h Epoetin β (15 mg/kg/day CsA diluted in olive oil, 100 UI/Kg/48 h Epoetin β + 15 mg/kg/day CsA, or olive oil. Histological changes due to tubular necrosis were evaluated with Masson’Trichrome staining. Apoptotic nuclei in kidneys were detected using the Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) method. Phospho-Akt, Akt, cleaved caspase 3 and non cleaved caspase 3 expression were evaluated using immunblotting. We demonstrate that recombinant human erythropoietin (epoetin β) improves renal function and protects against acute tubular injury. Our data suggest that this nephroprotective effect is mediated by Akt activation and inhibition of tubular apoptosis. Indeed, western blotting analysis of caspase 3 cleavage and Akt phosphorylation demonstrates that rhEPO activate Akt signaling and inhibits caspase 3 cleavage induced by CsA. TUNEL staining confirms that rhEPO inhibits CsA-induced tubular apoptosis. In conclusion, we describe here a new potential target of recombinant human erythropoietin and our results provide an interesting framework for further nephroprotective therapies based on recombinant human erythropoietin.

Safety of Low-Dose Cyclosporine Therapy Before Transplantation in Kidney Allograft Recipients

Graft dysfunction immediately posttransplantation can vary from subtle slowing of the expected decrease in creatinine concentration to frank oliguria requiring dialysis therapy for days to weeks. Risk factors for slow and delayed graft function include prolonged preservation, older donor age, and high plasma renin activity in the recipient. Cyclosporine (CsA) nephrotoxicity is another cause of early kidney allograft dysfunction. To evaluate early kidney allograft function in patients who received low-dose CsA therapy for 48 hours before transplant surgery for comparison with that in recipients who received CsA therapy after improvement in allograft function. In a case-control comparative study, 66 kidney recipients were divided into 2 groups on the basis of time of initiation of CsA therapy. In group 1, patients received CsA, 100 mg twice a day, for 48 hours before surgery, and in group 2, patients received CsA therapy after surgery when allograft function had improved (serum creatinine concentration <or=3 mg/dL). Other immunosuppression medications were the same in both groups. Statistical analysis was performed to compare kidney allograft function in the first month posttransplantation. In group 1 vs group 2, at day 1 posttransplantation, mean (SD) blood urea concentration was 73.72 (31.00) mg/dL vs 87.52 (29.82) mg/dL, serum creatinine concentration was 5.11 (1.83) mg/dL vs 6.42 (3.64) mg/dL, and urine volume in 24 hours was 11,052 (4290) mL vs 9629 (45.30) mL. At the end of the study, blood urea concentration was 49.61 (12.18) mg/dL vs 69.11 (33.76) mg/dL, serum creatinine concentration was 1.22 (0.28) mg/dL vs 1.47 (0.79) mg/dL, and urine volume in 24 hours was 3202 (986) mL vs 3095 (726) mL. No significant difference was noted between the 2 groups for age, sex, and immunosuppression medications. Low-dose CsA therapy before transplant surgery preserves early allograft function without deleterious effects.

Renal tubular expression of Toll‐like receptor 4 in cyclosporine nephrotoxicity

Exploring the expression of Toll-like receptor (TLR) in cyclosporine (CsA)-induced renal injury in humans, we evaluated the expression of TLR4 in both biopsied renal tissue and cultured tubular cells. Immunohistochemical stains for TLR4, heat shock protein (HSP) 47, and HSP70 were performed in both pre- and post-treatment biopsies obtained from 18 patients of minimal change nephrotic syndrome or IgA nephropathy treated with CsA, and the percentage of positive tubules was compared in each case. For in vitro experiments, HK-2 cells were treated with CsA (2, 5, and 10 microg/ml) for 24, 48, and 72 h. TLR4 mRNA and protein were measured using real-time RT-PCR and Western blot. In addition, hypoxic effect was added by GasPak System. The tubular expressions of TLR4 (2.2 +/- 1.2% vs 4.4 +/- 2.0%, p < 0.001) and HSP70 (2.6 +/- 2.8% vs 6.1 +/- 4.2%, p = 0.002) were increased after CsA treatment. TLR4 mRNA and protein expression were also increased in a dose-dependent manner. Hypoxia enormously increased TLR4 expression. In summary, CsA increased tubular expression of TLR4 and its ligand HSP70. As hypoxia was shown to be a strong stimulus for TLR4 expression, it can be said that TLR4 is influenced by both direct toxicity and impediment of renal microcirculation in human CsA nephrotoxicity.

Metabolic Profiles in Urine Reflect Nephrotoxicity of Sirolimus and Cyclosporine following Rat Kidney Transplantation

Background: Cyclosporine and/or sirolimus impair recovery of renal transplants. This study examines the changes in urine metabolite profiles as surrogate markers of renal cell metabolism and function after cyclosporine and/or sirolimus treatment employing a rat kidney transplantation model. Methods: Using inbred Lewis rats, kidneys were transplanted into bilaterally nephrectomized recipients followed by treatment with either CsA (cyclosporine) 10, Rapa (sirolimus) 1, CsA10/Rapa1 or CsA25/Rapa1 mg/kg/day for 7 days. On day 7, urine was analyzed by ¹H-NMR spectroscopy. Blood and kidney tissue drug concentrations, tissue high-energy compounds (including ATP, ADP) and oxidative stress markers (15-F_2t-isoprostanes) in urine were measured by HPLC mass spectrometry. Results: Changes in urine metabolites followed the order Rapa1 < CsA10 < CsA10/Rapa1 < CsA25/Rapa1. Compared with controls, CsA25/Rapa1 showed the greatest changes (creatinine –36%, succinate –57%, citrate –89%, α-ketoglutarate –75%, creatine +498%, trimethylamine +210% and taurine +370%). 15-F_2t-isoprostane concentrations in urine increased in the combined immunosuppressant-treated animals ([CsA25/Rapa1]: 795 ± 222, [CsA10/Rapa1]: 475 ± 233 pg/mg/creatinine) as compared with controls (165 ± 78 pg/mg creatinine). Rapa concentration in blood and tissues increased in the combined treatment (blood: 31 ± 8 ng/ml, tissue: 1.3 ± 0.4 ng/mg) as compared with monotherapy (blood: 14 ± 8 ng/ml, tissue: 0.35 ± 0.15 ng/mg). Drug blood concentrations correlated with isoprostane urine concentrations, which correlated negatively with citrate, α-ketoglutarate and creatinine concentrations in urine. Only CsA25/Rapa1 significantly reduced high-energy metabolite concentrations in transplant kidney tissue (ATP –55%, ADP –24%). Conclusion: Immunosuppressant drugs induce changes in urine metabolite patterns, suggesting that immunosuppressant-induced oxidative stress is an early event in the development of nephrotoxicity. Urine 15-F_2t-isoprostane concentrations and metabolite profiles may be sensitive markers of immunosuppressant-induced nephrotoxicity.

TGF-β upregulation drives tertiary lymphoid organ formation and kidney dysfunction in calcineurin A-α heterozygous mice

Calcineurin is an important intracellular signaling molecule which can be inhibited by cyclosporin resulting in immune suppression and nephrotoxicity. Previously, we reported that homozygous loss of the alpha isoform of calcineurin impairs kidney development and function and mimics many features of cyclosporin nephrotoxicity. However, early lethality of null mice prevented further study of renal changes. Alternatively, we examined aged heterozygous (CnAalpha(+/-)) mice. In addition to renal dysfunction and inflammation, we find that CnAalpha(+/-) mice spontaneously develop tertiary lymphoid aggregates in the kidney, small intestine, liver, and lung. Lymphoid aggregates contain both T cells and B cells and exhibited organization suggestive of tertiary lymphoid organs (TLOs). Kidney function and TLO formation were highly correlated suggesting that this process may contribute to nephrotoxicity. Consistent with previous findings, transforming growth factor (TGF)-beta is significantly increased in CnAalpha(+/-) mice. Neutralization of TGF-beta attenuated TLO formation and improved kidney function. In conclusion, we report that haploinsufficiency of CnAalpha causes uregulation of TGF-beta which contributes to chronic inflammation and formation of TLOs. While the process that leads to TLOs formation in transplant allografts is unknown, TLOs are associated with poor clinical prognosis. This study suggests that calcineurin inhibition itself may lead to TLO formation and that TGF-beta may be a novel therapeutic target.

Late Conversion to Everolimus Complicated With Necrotizing Glomerulonephritis in a Renal Allograft Recipient: Case Report

Conversion from calcineurin inhibitors (CNI) to proliferation signal inhibitors (PSI), such as sirolimus or everolimus (EV), may improve the course of chronic allograft nephropathy. Herein we have presented a case of a kidney recipient with chronic cyclosporine (CsA) nephrotoxicity who was converted from CsA to EV at 5.5 years posttransplantation. There were no significant changes in immunofluorescence (IFL) or in electron microscopy (EM) in the preconversion biopsy. Two months after conversion, proteinuria and creatinine increased. The biopsy showed focal, segmental necrosis of the glomerular tuft with the formation of segmental cellular crescents and increased endocapillary cellularity. IFL showed granular deposits of IgG, IgM, and C3 mostly along the capillary walls; it was negative for C4d. EM revealed electron-dense deposits within the glomerular basement membrane (GBM) and in the subendothelial region with significant reduction in the capillary lumina due to GBM reduplication and widening of lamina rara interna with the formation of fibrillary structures therein: focal, segmental glomerulosclerosis. EV was withdrawn and we administered tacrolimus and steroid pulses with improvement. Five months after the withdrawal of EV, a third graft biopsy showed remission of the necrotizing glomerulonephritis. However, the patient demanded dialysis at 17 months after conversion to EV. We concluded that necrotizing glomerulonephritis with immune complex deposition in a renal allograft was possibly induced by late conversion from CNI to EV. Reconversion to CNI may be recommended in cases of PSI-associated posttransplantation glomerulonephritis but the long-term prognosis is uncertain.

Toll-like Receptor Expression in Patients With Renal Allograft Dysfunction

Toll-like receptor (TLR) is known to be a mediator of innate immunity, but recent reports have shown that TLR provides a link to adaptive immunity involved in allograft rejection. To explore the expression patterns in various conditions of renal transplantation, we examined TLR subunit mRNA expressions in renal allograft biopsies of acute rejection (AR; n = 11), chronic rejection (CR; n = 15), chronic cyclosporine nephrotoxicity (CsAN; n = 22), and immunoglobulin A nephropathy (IgAN; n = 9) patients. Control tissues (n = 7) were obtained from normal renal cortical tissue of renal cell carcinoma patients. The diagnosis was made according to the Banff 97 classification. The expressions of TLR 2, 3, 4, and 9 mRNA were analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) using SYBR green. Statistical analysis was performed using analysis of variance (ANOVA) and the Student t test. TLR 2 and 3 mRNA expressions were not significantly different in any group ( P > .05). In contrast, TLR 4 mRNA expression was significantly increased in all allograft groups compared with that of controls, and significantly higher in the CsAN than other transplant groups ( P < .05). TLR 9 mRNA expression was up-regulated in CsAN and IgAN compared with AR and CR ( P < .05). These results suggested that TLR4 mRNA expression was increased in renal allograft patients with chronic allograft dysfunction. Further studies are needed to correlate TLR subtypes with various causes of graft dysfunction among renal allograft patients.

Protective effect of Epo on oxidative renal injury in rats with cyclosporine nephrotoxicity

The aim of our study was to determine the effect of recombinant human erythropoietin (rhEPO) on cyclosporine (CsA) nephrotoxicity. Twenty-six female Wistar rats were injected with 15 mg/kg subcutaneous CsA and intraperitoneal saline/rhEPO for 28 days. Four groups were formed: Group 1 (n = 5), a control group; Group 2 (n = 7), CsA + saline; Group 3 (n = 7), CsA + low dose (20 U/kg per day) rhEPO; Group 4 (n = 7), CsA + high dose (100 U/kg per day) rhEPO. Body weights, creatinine clearance, urinary protein/creatinine, hematocrit, serum creatinine levels, histopathological parameters, apoptosis and lipid peroxidation tests were compared between the three groups. Body weights and renal functions were similar in Groups 2, 3 and 4 rats but significantly lower than the values found for the control group at the end of the study. The hematocrit was significantly different between the four groups, showing a positive association with the strength of the injected rhEPO doses. Tubular and arteriolar damage was significantly lower in Groups 3 and 4 rats than in Group 2 rats, while chronic changes were similar between the three groups. TUNEL-positive cells and thiobabarbituric acid reacting substance (TBARS) levels were significantly higher in Group 2 rats, whereas superoxide dismutase levels were significantly lower in Group 2 rats than in those of the other three groups. Low or high dose rhEPO had no significant protective effects on body weight, renal functions, chronic fibrotic changes, but both doses reduced tubular and arteriolar changes, apoptotis and oxidative stress.

Cyclosporine-Induced Endoplasmic Reticulum Stress Triggers Tubular Phenotypic Changes and Death

The molecular mechanisms by which cyclosporine induces chronic nephrotoxicity remain poorly understood. A previous transcriptomic study suggested that cyclosporine might induce endoplasmic reticulum (ER) stress in human tubular cells. The aim of the present study was to characterize the features of tubular ER stress induced by cyclosporine and to investigate its effects on cell differentiation and viability. Using primary cultures of human tubular cells, we confirmed that cyclosporine is responsible for ER stress in vitro. This was also confirmed in vivo in the rat. In vitro, cyclosporine and other ER stress inducers were responsible for epithelial phenotypic changes leading to the generation of protomyofibroblasts, independent of transforming growth factor-beta signaling. RNA interference directed against cyclophilin A supported the role of its inhibition in triggering ER stress as well as epithelial phenotypic changes induced by cyclosporine. Salubrinal, which is known to protect cells from ER stress, significantly reduced epithelial phenotypic changes and cytotoxicity induced by cyclosporine in vitro. Salubrinal also reduced cyclosporine nephrotoxicity in rat kidneys. Thus, we describe a novel mechanism that initiates dedifferentiation and tubular cell death upon cyclosporine treatment. These results provide an interesting framework for further nephroprotective therapies by targeting ER stress.

Possible Mechanism by Which Rapamycin Increases Cyclosporine Nephrotoxicity

It is well known that the combination of cyclosporine A (CsA) with rapamycin produces serious nephrotoxicity. Herein we suggest a mechanism by which rapamycin increases CsA nephrotoxicity. Previously, we demonstrated that activation of Akt/protein kinase B protects against cyclosporine nephrotoxicity and prevents apoptosis. Recently, it has been shown that Akt phosphorylation activates mammalian target of rapamycin (m-TOR) and inhibits programmed cell death including apoptosis and autophagy. Akt is believed to be an importance factor for cell survival. In theory, blockade of the Akt pathway through inhibition of m-TOR may increase cyclosporine-induced apoptosis. We added cyclosporine and rapamycin to cultures of ER52K proximal renal tubule cells, leading to a significantly decreased survival rate. The nephrotoxicity was associated with increased apoptosis by cleavage of caspase-3 and decreased phosphorylation of m-TOR and AktSer473, findings that support this hypothesis. This nephrotoxic effect may explain the clinical finding that patients treated with rapamycin alone exhibited better renal function than those treated with concomitant cyclosporine therapy.

Six-Month Clinical Outcome of Cyclosporine Microemulsion Formulation (Sigmasporin Microral) in Stable Renal Transplant Patients Previously Maintained on Sandimmun Neoral

The trial objective was to investigate the feasibility and safety of conversion to a generic microemulsion cyclosporine in stable renal transplant patients premaintained on Neoral. We enrolled 75 patients from seven centers in five Middle Eastern countries monitored them for 6 months after conversion to Sigmasporin Microral. Readings at 0, 0.5, 1, 2, 3, 4.5, and 6 months included cyclosporine blood level, serum creatinine, liver enzymes, lipid profile, blood sugar, blood pressure and adverse events. Patients included 54 men and 21 women of mean age 38.9 ± 10.7 years at 30.3 ± 29.3 months post-transplantation maintained on Sigmasporin Microral dose of 2.8 ± 1.0 mg/kg per day; they were observed to be stable throughout the study period as reflected by the therapeutic blood C 0 level of 181.6 ± 102.1 and C 2 of 759.2 ± 384.4. Their absorption profile as represented by C 2/C 0 was 4.9 ± 2.8, and C 2/cyclosporine dose of 282.3 ± 128.8. An average serum creatinine level of 116.1 ± 29.5 μmol/L denoted stable graft function and their liver enzymes did not change during the study. No new-onset cases of hypertension, diabetes mellitus, or hyperlipidemia were reported among the patients. Graft function was stable for all patients, except for two incidences of mild acute rejection and two of mild cyclosporine nephrotoxicity; graft and patient survival rates were both 100%. Results of this 6-month study showed that Sigmasporin Microral was effective to maintain stable renal function in kidney transplant patients converted from Neoral with similar safety and tolerability profiles as those reported in the literature.

No Association Between Single Nucleotide Polymorphisms and the Development of Nephrotoxicity After Orthotopic Heart Transplantation

Survival for heart transplantation (HTx) patients is limited by nephrotoxicity of the calcineurin inhibitors cyclosporine and tacrolimus. To determine whether genetic factors are involved in the development of renal dysfunction under immunosuppressive therapy, we screened various genes for sequence variations. In a case-control study we analyzed in parallel polymorphisms within the transforming growth factor-beta1 gene (TGF-beta1; L10P, R25P), the multidrug resistance gene MDR 1 (A893T/S) and the CYP3A5 gene (CYP3A5*1/*3 allele). In total, we included 53 cardiac allograft recipients with renal insufficiency (serum creatinine >or=1.8 mg/dl and glomerular filtration rate <50 ml/min/1.73 m(2)) and 53 patients with normal renal function as controls. The controls were matched with patients for age, gender and post-HTx time. The polymorphisms were assessed by denaturing high-performance liquid chromatography (dHPLC) and direct sequencing. We performed univariate and multivariate logistic regression analysis to assess the association between different gene variants and renal dysfunction. No significant (p > 0.05) relationship was found between the polymorphisms investigated and the susceptibility of renal insufficiency under immunosuppressive therapy. Our data do not justify genotyping of the investigated single nucleotide polymorphisms (SNPs) to assess the development of renal dysfunction post-HTx.