Shang Guo Piao

Chronic cyclosporine nephropathy is characterized by excessive autophagosome formation and decreased autophagic clearance.

Background The study was performed to investigate the influence of cyclosporine A (CsA)–induced renal injury on autophagy in an experimental model of chronic CsA nephropathy. Methods Three dosages of CsA (7.5, 15, and 30 mg/kg/day) were administered to mice for 4 weeks. The formation of autophagosomes was measured with microtubule-associated protein 1 light chain 3 phospholipid-conjugated form (LC3-II) and beclin-1, and the ability of autophagic clearance was examined with sequestosome-1 (p62). Autophagic vacuoles were visualized and counted using electron microscopy. Double immunolabeling of LC3-II and active caspase-3 was performed to evaluate the association between autophagy and apoptosis. Oxidative stress was evaluated by measuring urinary 8-hydroxy-2′-deoxyguanosine excretion, demonstrating oxidative DNA damage. Antioxidative drugs, pravastatin and N-acetylcysteine, were used to evaluate the role of CsA-induced oxidative stress on autophagy. Results CsA treatment increased the expressions of LC3-II and beclin-1 in the kidney in a dose-dependent manner. The number of p62-positive cells was also significantly increased in a CsA dose–dependent manner. Electron microscopy revealed excessive autophagic vacuoles in the CsA group compared with the vehicle group. Expression of active caspase-3 was increased in a CsA dose–dependent manner and was colocalized with LC3-II in the injured area of CsA-treated kidneys. Concurrent pravastatin or N-acetylcysteine treatment reduced urinary excretion of 8-hydroxy-2′-deoxyguanosine and subsequently decreased LC3-II expression and the number of p62-positive cells compared with the CsA group. Conclusions Chronic CsA nephropathy is a state of excessive autophagic vacuoles and decreased autophagic clearance. Oxidative stress may play an importation role in the induction of autophagy.

Effect of Sirolimus on Calcineurin Inhibitor-Induced Nephrotoxicity Using Renal Expression of KLOTHO, an Antiaging Gene

Background. The aim of this study was to observe the effect of sirolimus (SRL) on calcineurin inhibitor (CNI)-induced nephrotoxicity in the aging process by using renal expression of KLOTHO, an antiaging gene. Methods. Mice were treated with vehicle (VH; 1 mL/kg/day of olive oil), cyclosporine A (CsA; 30 mg/kg/day), or tacrolimus (FK; 1 mg/kg/day) with or without SRL (0.3 mg/kg/day) for 2 weeks. KLOTHO expression was evaluated by using reverse-transcriptase polymerase chain reaction, immunoblotting, and immunohistochemistry. Oxidative stress was evaluated by using immunohistochemistry and urinary excretion of 8-hydroxy-2′-deoxyguanosine (8-OHdG). The calcium metabolism was evaluated by using renal ectopic calcification, serum intact parathyroid hormone level, and renal fibroblast factor 23 (FGF23) expression. Results. Treatment with CsA or FK alone significantly decreased KLOTHO expression and increased urinary 8-OHdG excretion compared with VH treatment but SRL treatment did not. Treatment SRL+CsA or SRL+FK further decreased KLOTHO expression and increased urinary 8-OHdG excretion compared with treatment of CsA or FK alone. There was a strong correlation between KLOTHO expression and urinary 8-OHdG excretion (r=−0.893; P<0.001). Treatment of CsA or FK alone increased renal ectopic calcification and serum intact parathyroid hormone level and decreased renal FGF23 expression compared with VH treatment (P<0.05) but SRL treatment did not. Treatment with SRL+CNI aggravated these parameters compared with CNI alone. Conclusions. SRL accelerates the CNI-induced oxidative process by down-regulating the renal antioxidant KLOTHO expression in the kidney.

Dysregulation of Th17 Cells during the Early Post-Transplant Period in Patients under Calcineurin Inhibitor Based Immunosuppression

Accumulating evidence suggests that Th17 cells play a role in the development of chronic allograft injury in transplantation of various organs. However, the influence of current immunosuppressants on Th17-associated immune responses has not been fully investigated. We prospectively investigated the changes in Th17 cells in peripheral blood mononuclear cells (PBMCs) collected before and 1 and 3 months after KT in 26 patients and we investigated the suppressive effect of tacrolimus on Th17 in vitro. In the early posttransplant period, the percentage of Th17 cells and the proportion of IL-17-producing cells in the effector memory T cells (TEM) were significantly increased at 3 months after transplantation compared with before transplantation (P<0.05), whereas Th1/Th2 cells and TEM cells were significantly decreased. The degree of increase in Th17 during the early posttransplant period was significantly associated with allograft function at 1 year after transplantation (r = 0.4, P<0.05). In vitro, tacrolimus suppressed Th1 and Th2 cells in a concentration-dependent manner, but did not suppress Th17 cells even at high concentration. This suggests that current immunosuppression based on tacrolimus is inadequate to suppress Th17 cells in KTRs, and dysregulation of Th17 may be associated with the progression of CAD.

Ginseng Treatment Attenuates Chronic Cyclosporine Nephropathy via Reducing Oxidative Stress in an Experimental Mouse Model

Background: This study was performed to investigate whether ginseng extract has a protective effect in an experimental mouse model of chronic cyclosporine (CsA) nephropathy. Methods: Mice were treated with CsA (30 mg/kg/day, subcutaneously) with or without Korean red ginseng extract (KRG) (0.2, 0.4 g/kg/day, orally) on a 0.01% salt diet for 4 weeks. The effect of KRG on CsA-induced renal injury was evaluated by assessing renal function and pathology, mediators of inflammation, tubulointerstitial fibrosis and apoptotic cell death. Using an in vitro model, we also examined the effect of KRG on CsA-treated proximal tubular cells (HK-2). Oxidative stress was measured by assessing 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels in 24-hour urine, tissue sections, and culture media. Results: Four weeks of CsA treatment caused renal dysfunction, typical pathologic lesions and apoptotic cell death. KRG treatment reduced serum creatinine and blood urea nitrogen and histopathology and increased creatinine clearance. Proinflammatory and profibrotic molecules such as induced nitric oxide synthase, cytokines, transforming growth factor (TGF)-β1 and TGF-β1-inducible gene h3 and apoptotic cell death, also decreased with KRG treatment. Consistent with these results, in vitro studies showed that addition of KRG protected against CsA-induced morphological changes, cytotoxicity, inflammation, and apoptotic cell death as demonstrated by annexin V binding. These changes were accompanied by decrease in the level of 8-OHdG in urine and culture supernatant after KRG treatment. Conclusion: The results of our in vivo and in vitro studies demonstrate that KRG has a protective effect in CsA-induced renal injury via reducing oxidative stress.

Statin Upregulates the Expression of Klotho, an Anti-Aging Gene, in Experimental Cyclosporine Nephropathy

Background: We recently reported that long-term cyclosporine (CsA)-induced oxidative stress is associated with decreased expression of klotho, an anti-aging gene. This study evaluated whether the antioxidant effect of statin might upregulate klotho expression in CsA-induced renal injury. Methods: Two separate experiments were performed. First, the dose-dependent effect of statin on klotho expression was evaluated in normal mouse kidneys. Second, the effect of statin on klotho expression was evaluated in experimental chronic CsA nephropathy in mice. We performed immunohistochemistry and immunoblotting for klotho, Forkhead box O transcription factors [FoxOs; phosphorylated FoxO1 (p-FoxO1) and FoxO3a (p-FoxO3a)] and their target molecules, manganese superoxide dismutase (MnSOD), Bim and hemeoxygenase-1. Results: Statin treatment upregulated klotho expression in a dose-dependent manner in the normal mouse kidney and alleviated the decrease in klotho expression in kidneys exhibiting CsA nephropathy. CsA administration increased p-FoxO1 expression and decreased p-FoxO3a expression, whereas concurrent statin treatment reversed these changes, increased the expression of the antioxidant enzymes MnSOD and hemeoxygenase-1 and decreased the expression of the pro-apoptotic protein Bim. Conclusion: Statin-mediated upregulation of klotho expression and differential regulation of FoxO expression promote resistance to CsA-induced oxidative stress.

Clinical significance of the ratio between FOXP3 positive regulatory T cell and interleukin-17 secreting cell in renal allograft biopsies with acute T-cell-mediated rejection.

The aim of this study is to investigate the clinical significance of the ratio between interleukin‐17 (IL‐17) secreting cell and FOXP3‐positive regulatory T cell (FOXP3+ Treg) infiltration in renal allograft tissues with acute T‐cell‐mediated rejection (ATCMR). Fifty‐six patients with biopsy‐proven ATCMR were included. Infiltration of FOXP3+ Treg and IL‐17‐secreting cells was evaluated with immunostaining for FOXP3 or IL‐17 on the biopsy specimens, and the patients were divided into the FOXP3 high group (Log FOXP3/IL‐17 > 0·45) or the IL‐17 high group (Log FOXP3/IL‐17 < 0·45). We compared the allograft function, severity of tissue injury, and clinical outcome between the two groups. In the IL‐17 high group, allograft function was significantly decreased compared with the FOXP3 high group (P < 0·05). The severity of interstitial and tubular injury in the IL‐17 high group was higher than the FOXP3 high group (P < 0·05). The proportions of steroid‐resistant rejection, incomplete recovery and recurrent ATCMR were higher in the IL‐17 high group than in the FOXP3 high group (all indicators, P < 0·05). The IL‐17 high group showed lower 1‐year (54% versus 90%, P < 0·05) and 5‐year (38% versus 85%, P < 0·05) allograft survival rates compared with the FOXP3 high group. Multivariate analysis revealed that the FOXP3/IL‐17 ratio was a significant predictor for allograft outcome. The FOXP3/IL‐17 ratio is a useful indicator for representing the severity of tissue injury, allograft dysfunction and for predicting the clinical outcome of ATCMR.

Dipeptidyl Peptidase IV Inhibitor MK-0626 Attenuates Pancreatic Islet Injury in Tacrolimus-Induced Diabetic Rats

Background Tacrolimus (TAC)-induced pancreatic islet injury is one of the important causes of new-onset diabetes in transplant recipients. This study was performed to evaluate whether a dipeptidyl peptidase IV (DPP IV) inhibitor is effective in improving TAC-induced diabetes mellitus by reducing pancreatic islet injury. Methods Rats were treated with TAC (1.5 mg/kg, subcutaneously) and the DPP IV inhibitor MK-0626 (10 or 20 mg/kg, oral gavage) for 4 weeks. The effect of MK-0626 on TAC-induced diabetes was evaluated by assessing pancreatic islet function, histopathology. TAC-induced incretin dysfunction was also examined based on active glucagon-like peptide-1 (GLP-1) levels in the serum after glucose loading. The protective effect of MK-0626 was evaluated by measuring markers of oxidative stress, oxidative resistance, and apoptosis. To determine whether enhanced GLP-1 signaling is associated with these protective effects, we measured the expression of the GLP-1 receptor (GLP-1R) and the effect of the GLP-1 analog exendin-4 on cell viability and oxidative stress in isolated islets. Results MK-0626 treatment attenuated TAC-induced pancreatic islet dysfunction and islet morphology. TAC treatment led to a defect in active GLP-1 secretion; however, MK-0626 reversed these effects. TAC treatment increased the level of 8-hydroxy-2′-deoxyguanosine (8-OHdG), the number of apoptotic death, and the level of active caspase-3, and decreased the level of manganese superoxide dismutase and heme oxygenase-1; MK-0626 treatment reversed these changes. MK-0626 treatment restored the expression of GLP-1R, and direct administration of exendin-4 to isolated islets reduced TAC-induced cell death and 8-OHdG expression. Conclusions The DPP IV inhibitor MK-0626wasan effective antidiabetic agent that exerted antioxidative and antiapoptotic effects via enhanced GLP-1 signaling in TAC-induced diabetics.

Drug interaction between cyclosporine and mTOR inhibitors in experimental model of chronic cyclosporine nephrotoxicity and pancreatic islet dysfunction.

Background. It is known that sirolimus (SRL) aggravates cyclosporine A (CsA)-induced nephrotoxicity and pancreatic injury, but the influence of everolimus (EVR) on CsA-induced organ injury is undetermined. Methods. Rats were treated with CsA (15 mg/kg) and EVR or SRL (0.3 mg/kg) subcutaneously for 4 weeks. The influences of EVR or SRL on CsA-induced nephrotoxicity and pancreatic islet dysfunction were compared, and drug interactions between CsA and EVR or SRL were evaluated at blood and tissue levels. Results. Treatment with EVR or SRL alone did not cause severe pancreatic dysfunction and renal injury, but when combined with CsA they aggravated CsA-induced pancreatic and renal injury. Drug interactions between CsA and EVR or SRL differed at the tissue level. Combined treatment with CsA and SRL significantly increased the CsA or SRL levels in kidney and pancreas compared with CsA or SRL alone. However, combined treatment with CsA and EVR did not increase CsA or EVR levels in kidney and pancreas. Conclusions. Both EVR and SRL aggravate CsA-induced organ injury, but the pharmacologic interaction between EVR and CsA at the tissue level is less than that between CsA and SRL. This finding provides better understanding of the difference between EVR and SRL when combined with CsA treatment.

Higher infiltration by Th17 cells compared with regulatory T cells is associated with severe acute T-cell-mediated graft rejection

The aim of this study was to evaluate whether the Th17 and Treg cell infiltration into allograft tissue is associated with the severity of allograft dysfunction and tissue injury in acute T cell-mediated rejection (ATCMR). Seventy-one allograft tissues with biopsy-proven ATCMR were included. The biopsy specimens were immunostained for FOXP3 and IL-17. The allograft function was assessed at biopsy by measuring serum creatinine (Scr) concentration, and by applying the modified diet in renal disease (MDRD) formula, which provides the estimated glomerular filtration rate (eGFR). The severity of allograft tissue injury was assessed by calculating tissue injury scores using the Banff classification. The average numbers of infiltrating Treg and Th17 cells were 11.6 ± 12.2 cells/mm2 and 5.6 ± 8.0 cells/mm2, respectively. The average Treg/Th17 ratio was 5.6 ± 8.2. The Treg/Th17 ratio was significantly associated with allograft function (Scr and MDRD eGFR) and with the severity of interstitial injury and tubular injury (P < 0.05, all parameters). In separate analyses of the number of infiltrating Treg and Th17 cells, Th17 cell infiltration was significantly associated with allograft function and the severity of tissue injury. By contrast, Treg cell infiltration was not significantly associated with allograft dysfunction or the severity of tissue injury. The results of this study show that higher infiltration of Th17 cell compared with Treg cell is significantly associated with the severity of allograft dysfunction and tissue injury.

Inhibition of dipeptidyl peptidase IV protects tacrolimus-induced kidney injury.

Accumulating evidence shows that a gut-released hormone, the glucagon-like peptide-1 (GLP-1), has not only a glucose-lowering effect but also a renoprotective effect against kidney injury. In this study, we investigated whether a dipeptidyl peptidase (DPP) IV inhibitor has a protective effect against tacrolimus-induced renal injury. Rats were treated with tacrolimus (1.5 mg/kg, subcutaneously) and the DPP IV inhibitor MK0626 (10 or 20 mg/kg, oral gavage) for 4 weeks. MK0626 treatment attenuated tacrolimus-induced renal dysfunction, tubulointerstitial fibrosis, and arteriolopathy. Moreover, these improvements were accompanied by a reduction in oxidative stress and apoptosis. MK0626 treatment increased the blood level of GLP-1 and the level of its receptor in tissue sections but did not alter the levels of other DPP IV substrates, such as neuropeptide Y and the stromal cell-derived factor-1. These data suggest that DPP IV inhibition has an important role in the renoprotection against tacrolimus-induced nephrotoxicity via antioxidative and antiapoptotic effects and preservation of the GLP-1 system.