CTLA4-Ig protects tacrolimus-induced oxidative stress via inhibiting the AKT/FOXO3 signaling pathway in rats

Background/Aims@#Although the conversion from tacrolimus (TAC) to cytotoxic T-lymphocyte-associated antigen 4-immunoglobulin (CTLA4-Ig) is effective in reducing TAC-induced nephrotoxicity, it remains unclear whether CTLA4-Ig has a direct effect on TAC-induced renal injury. In this study, we evaluated the effects of CTLA4-Ig on TAC-induced renal injury in terms of oxidative stress. @*Methods@#In vitro study was performed to assess the effect of CTLA4-Ig on TAC-induced cell death, reactive oxygen species (ROS), apoptosis, and the protein kinase B (AKT)/forkhead transcription factor (FOXO) 3 pathway in human kidney 2 cells. In the in vivo study, the effect of CTLA4-Ig on TAC-induced renal injury was evaluated using renal function, histopathology, markers of oxidative stress (8-hydroxy-2’-deoxyguanosine) and metabolites (4-hydroxy-2-hexenal, catalase, glutathione S-transferase, and glutathione reductase), and activation of the AKT/FOXO3 pathway with insulin-like growth factor 1 (IGF-1). @*Results@#CTLA4-Ig significantly decreased cell death, ROS, and apoptosis caused by TAC. TAC treatment increased apoptotic cell death and apoptosis-related proteins (increased Bcl-2-associated X protein and caspase-3 and decreased Bcl-2), but it was reversed by CTLA4-Ig treatment. The activation of p-AKT and p-FOXO3 by TAC decreased with CTLA4-Ig treatment. TAC-induced renal dysfunction and oxidative marker levels were significantly improved by CTLA4-Ig in vivo. Concomitant IGF-1 treatment abolished the effects of CTLA4-Ig. @*Conclusions@#CTLA4-Ig has a direct protective effect on TAC-induced renal injury via the inhibition of AKT/FOXO3 pathway.

Alleviation of renal ischemia/reperfusion injury by exosomes from induced pluripotent stem cell-derived mesenchymal stem cells

Background/Aims@#Renal ischemia followed by reperfusion (I/R) is a leading cause of acute kidney injury (AKI), which is closely associated with high morbidity and mortality. Studies have shown that induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) exert powerful therapeutic effects in renal ischemia. However, the efficacy of iMSC-derived exosomes (iExo) on I/R injuries remains largely unknown. @*Methods@#Human iPSCs were differentiated into iMSCs using a modified one-step method. Ultrafiltration, combined with purification, was used to isolate iExo from iMSCs. iExo was administered following I/R injury in a mouse model. The effect of iExo on I/R injury was assessed through changes in renal function, histology, and expression of oxidative stress, inflammation, and apoptosis markers. Further, we evaluated its association with the extracellular signal-regulated kinase (ERK) 1/2 signaling pathway. @*Results@#Mice subjected to I/R injury exhibited typical AKI patterns; serum creatinine level, tubular necrosis, apoptosis, inflammatory cytokine production, and oxidative stress were markedly increased compared to sham mice. However, treatment with iExo attenuated these changes, significantly improving renal function and tissue damage, similar to the renoprotective effects of iMSCs on I/R injury. Significant induction of activated ERK 1/2 signaling molecules was observed in mice treated with iExo compared to those in the I/R injury group. @*Conclusions@#The present study demonstrates that iExo administration ameliorated renal damage following I/R, suggesting that iMSC-derived exosomes may provide a novel therapeutic approach for AKI treatment.

Water-soluble coenzyme Q10 provides better protection than lipid-soluble coenzyme Q10 in a rat model of chronic tacrolimus nephropathy

Background/Aims@#Coenzyme Q10 (CoQ10), is a promising antioxidant; however, low bioavailability owing to lipid-solubility is a limiting factor. We developed water-soluble CoQ10 (CoQ10-W) and compared its effects with conventional lipid-soluble CoQ10 (CoQ10-L) in an experimental model of chronic tacrolimus (Tac) nephropathy. @*Methods@#CoQ10-W was developed from a glycyrrhizic-carnitine mixed layer CoQ10 micelle based on acyltransferases. Chronic nephropathy was induced in rats with 28-day Tac treatment; they were concomitantly treated with CoQ10-L or CoQ10-W. CoQ10 level in plasma and kidney were measured using liquid chromatography–mass spectrometry. CoQ10-W and CoQ10-L effects on Tac-induced nephropathy were assessed in terms of renal function, histopathology, oxidative stress, and apoptotic cell death. Their effects on cell viability and reactive oxygen species (ROS) production were assessed in cultured proximal tubular cells, human kidney 2 (HK-2) cells. @*Results@#The plasma CoQ10 level was significantly higher in the CoQ10-W group than in the CoQ10-L group. Tac treatment caused renal dysfunction, typical pathologic lesions, and oxidative stress markers. Serum creatinine was restored in the Tac + CoQ10-L or CoQ10-W groups compared with that in the Tac group. CoQ10-W administration reduced oxidative stress and apoptosis markers. Mitochondrial ultrastructure assessment revealed that the addition of CoQ10-L or CoQ10-W with Tac increased mitochondrial size and number than Tac treatment alone. In vitro investigations revealed that both CoQ10-L and CoQ10-W improved cell viability and reduced ROS production in the Tac-induced HK-2 cell injury. @*Conclusions@#CoQ10-W has a better therapeutic effect in Tac-induced renal injury than conventional CoQ10-L, possibly associated with improved CoQ10 bioavailability

Water-soluble coenzyme Q10 provides better protection than lipid-soluble coenzyme Q10 in a rat model of chronic tacrolimus nephropathy

Background/Aims@#Coenzyme Q10 (CoQ10), is a promising antioxidant; however, low bioavailability owing to lipid-solubility is a limiting factor. We developed water-soluble CoQ10 (CoQ10-W) and compared its effects with conventional lipid-soluble CoQ10 (CoQ10-L) in an experimental model of chronic tacrolimus (Tac) nephropathy. @*Methods@#CoQ10-W was developed from a glycyrrhizic-carnitine mixed layer CoQ10 micelle based on acyltransferases. Chronic nephropathy was induced in rats with 28-day Tac treatment; they were concomitantly treated with CoQ10-L or CoQ10-W. CoQ10 level in plasma and kidney were measured using liquid chromatography–mass spectrometry. CoQ10-W and CoQ10-L effects on Tac-induced nephropathy were assessed in terms of renal function, histopathology, oxidative stress, and apoptotic cell death. Their effects on cell viability and reactive oxygen species (ROS) production were assessed in cultured proximal tubular cells, human kidney 2 (HK-2) cells. @*Results@#The plasma CoQ10 level was significantly higher in the CoQ10-W group than in the CoQ10-L group. Tac treatment caused renal dysfunction, typical pathologic lesions, and oxidative stress markers. Serum creatinine was restored in the Tac + CoQ10-L or CoQ10-W groups compared with that in the Tac group. CoQ10-W administration reduced oxidative stress and apoptosis markers. Mitochondrial ultrastructure assessment revealed that the addition of CoQ10-L or CoQ10-W with Tac increased mitochondrial size and number than Tac treatment alone. In vitro investigations revealed that both CoQ10-L and CoQ10-W improved cell viability and reduced ROS production in the Tac-induced HK-2 cell injury. @*Conclusions@#CoQ10-W has a better therapeutic effect in Tac-induced renal injury than conventional CoQ10-L, possibly associated with improved CoQ10 bioavailability

Human kidney organoids model the tacrolimus nephrotoxicity and elucidate the role of autophagy

Background/Aims@#Tacrolimus has been used as an immunosuppressive agent in organ transplantation. Despite the therapeutic benefits, tacrolimus’s use is limited due to its nephrotoxicity. To reduce tacrolimus nephrotoxicity, effective humanized experimental models may be helpful. Here, we modeled tacrolimus nephrotoxicity using kidney organoids derived from human inducible pluripotent stem cells (iPSCs) in vitro. @*Methods@#Kidney organoids were differentiated from the CMC11 iPSC cell line, re-seeded in 96-well plates, and treated with tacrolimus at doses of 0, 30, or 60 μM for 24 hours. This in vitro model was compared to a mouse model of tacrolimus nephrotoxicity and the associated mechanisms were investigated. @*Results@#The size of the kidney organoids and cell viability decreased in dose-dependent manners after treatment with tacrolimus. The number of tubular cells decreased with a loss of polarity, similar to the effects seen in mouse tacrolimus nephrotoxicity. Ultrastructural analysis showed numerous vacuoles in the proximal tubular cells of the kidney organoids treated with tacrolimus. Tacrolimus treatment induced oxidative stress and mitochondrial dysfunction, and autophagic activity was enhanced in the kidney organoids. Rapamycin, an autophagy inducer, accelerated cell death in the kidney organoid model of tacrolimus nephrotoxicity, which was attenuated by treatment with 3-methyladenine, an autophagy inhibitor. These findings indicate that the augmentation of autophagy by rapamycin treatment accelerated tacrolimus nephrotoxicity. @*Conclusions@#Our data suggest that human kidney organoids are an effective in vitro model of tacrolimus nephrotoxicity and that autophagy plays a critical role in tacrolimus nephrotoxicity.

Modeling of endothelial cell dysfunction using human induced pluripotent stem cells derived from patients with end-stage renal disease

Background@#Endothelial cell (EC) dysfunction is a frequent feature in patients with end-stage renal disease (ESRD). The aim of this study was to generate human induced pluripotent stem cells, differentiate ECs (hiPSC-ECs) from patients with ESRD, and appraise the usefulness of hiPSC-ECs as a model to investigate EC dysfunction. @*Methods@#We generated hiPSCs using peripheral blood mononuclear cells (PBMCs) isolated from three patients with ESRD and three healthy controls (HCs). Next, we differentiated hiPSC-ECs using the generated hiPSCs and assessed the expression of endothelial markers by immunofluorescence. The differentiation efficacy, EC dysfunction, and molecular signatures of EC-related genes based on microarray analysis were compared between the ESRD and HC groups. @*Results@#In both groups, hiPSCs and hiPSC-ECs were successfully obtained based on induced pluripotent stem cell or EC marker expression in immunofluorescence and flow cytometry. However, the efficiency of differentiation of ECs from hiPSCs was lower in the ESRD-hiPSCs than in the HC-hiPSCs. In addition, unlike HC-hiPSC-ECs, ESRD-hiPSC-ECs failed to form interconnecting branching point networks in an in vitro tube formation assay. During microarray analysis, transcripts associated with oxidative stress and inflammation were upregulated and transcripts associated with vascular development and basement membrane extracellular matrix components were downregulated in ESRD-hiPSC-ECs relative to in HC-hiPSC-ECs. @*Conclusion@#ESRD-hiPSC-ECs showed a greater level of EC dysfunction than HC-hiPSC-ECs did based on functional assay results and molecular profiles. hiPSC-ECs may be used as a disease model to investigate the pathophysiology of EC dysfunction in ESRD.

The therapeutic efficacy of water-soluble coenzyme Q10 in an experimental model of tacrolimus-induced diabetes mellitus

Background/Aims@#Coenzyme Q10 (CoQ10) has antioxidant effects and is commercially available and marketed extensively. However, due to its low bioavailability, its effects are still controversial. We developed a water-soluble CoQ10-based micelle formulation (CoQ10-W) and tested it in an experimental model of tacrolimus (TAC)-induced diabetes mellitus (DM). @*Methods@#We developed CoQ10-W from a glycyrrhizic-carnitine mixed layer CoQ10 micelle preparation based on acyltransferases. TAC-induced DM rats were treated with either lipid-soluble CoQ10 (CoQ10-L) or CoQ10-W for 4 weeks. Their plasma and pancreatic CoQ10 concentrations were measured using liquid chromatography- tandem mass spectrometry. The therapeutic efficacies of CoQ10-W and CoQ10-L on TAC-induced DM were compared using functional and morphological parameters and their effects on cell viability and reactive oxygen species (ROS) production were also evaluated in cultured rat insulinoma cells. @*Results@#The plasma CoQ10 level was significantly increased in the CoQ10-W group compared to that in the CoQ10-L group. Intraperitoneal glucose tolerance tests and glucose-stimulated insulin secretion revealed that CoQ10-W controlled hyperglycemia and restored insulin secretion significantly better than CoQ10-L. The TAC-mediated decrease in pancreatic islet size was significantly attenuated by CoQ10-W but not by CoQ10-L. TAC-induced oxidative stress and apoptosis were significantly more reduced by CoQ10-W than CoQ10-L. Electron microscopy revealed that CoQ10-W restored TAC-induced attenuation in the number of insulin granules and the average mitochondrial area, unlike CoQ10-L. In vitro studies showed that CoQ10-L and CoQ10-W both improved cell viability and reduced ROS production in TAC-treated islet cells to a similar extent. @*Conclusions@#CoQ10-W has better therapeutic efficacy than CoQ10-L in TAC-induced DM.

Assessment of nephrotoxicity of herbal medicine containing aristolochic acid in mice

Background/Aims@#It is undetermined if herbal medicines (HM) containing aristolochic acid (AA)-containing have similar nephrotoxicity to AA itself. @*Methods@#We administered HM containing a high concentration of AA for 5 days (short-term study) or a low concentration of AA for 30 days (long-term study) to C57BL/6 mice; for comparison, same dose of AA compound was used as controls. @*Results@#The nephrotoxicity in the HM- and AA-treated mice was compared in terms of renal function, histopathology, oxidative stress, apoptotic cell death, and mitochondrial damage. Short-term HM treatment resulted in acute kidney injury (marked renal dysfunction, acute tubular necrosis, and neutrophil gelatinase-associated lipocalin [NGAL] expression) in which the severity of renal dysfunction and histopathology was comparable with that induced by the administration of AA alone. Long-term HM treatment resulted in features of chronic kidney disease (CKD, mild renal dysfunction and tubular atrophy and dilatation). No significant differences in these parameters were observed between the HM- and AA-treated mice. HM-induced oxidative stress (8-hydroxy-2’-deoxyguanosine and manganese- dependent superoxide dismutase expression) and apoptotic cell death (terminal deoxynucleotidyl transferase dUTP nick end labelling [TUNEL]-positive cells and active caspase-3 expression) were similar in HM- and AA-treated mice in the short-term and long-term studies. Mitochondrial injury, evaluated by electron microscopy, was also similar in HM- and AA-treated mice in the short-term and long-term studies. @*Conclusions@#The nephrotoxic potential of HM containing AA was similar to that of AA itself.

Shen-Kang protects against tacrolimus-induced renal injury

BACKGROUND/AIMS@#Evidence suggests that Shen-Kang (SK), a traditional Chinese herbal medicine, protects against various types of renal injury. In this study, we evaluated whether SK treatment confers renoprotection in a rat model of chronic tacrolimus (TAC) nephropathy.@*METHODS@#Rats were treated daily with TAC (1.5mg/kg, subcutaneously) and SK (450 mg/kg, intravenously) for 4 weeks. The effects of SK on TAC-induced renal injury were assessed by measuring renal function, urine albumin excretion, histopathology, inflammatory cell infiltration, expression of profibrotic (transforming growth factor β1 [TGF-β1] and TGF-β inducible gene-h3 [βig-h3]) and proinflammatory cytokines, oxidative stress, and apoptotic cell death.@*RESULTS@#Administration of SK preserved glomerular integrity (fractional mesangial area and Wilms tumor 1-positive glomeruli), attenuated tubulointerstitial fibrosis, and reduced the number of ectodermal dysplasia 1-positive cells, and this was paralleled by improved urine albumin excretion and renal dysfunction. At the molecular level, SK treatment suppressed expression of TGF-β1/Smad2/3, βig-h3, and proinflammatory cytokines. Oxidative stress and apoptotic cell death were significantly decreased with SK treatment, and apoptosis-related genes were regulated toward cell survival (active caspase-3 and the B-cell lymphoma-2/Bcl2-associated X [Bcl-2/Bax] ratio).@*CONCLUSIONS@#SK protects against TAC-induced renal injury.

The effects of addition of coenzyme Q10 to metformin on sirolimus-induced diabetes mellitus

BACKGROUND/AIMS@#This study was performed to determine whether adding coenzyme Q10 (CoQ(10)) to metformin (MET) has a beneficial effect as a treatment for sirolimus (SRL)-induced diabetes mellitus (DM).@*METHODS@#DM was induced in rats by daily treatment with SRL (0.3 mg/kg, subcutaneous) for 28 days, and animals were treated with CoQ(10) (20 mg/kg, oral) and MET (250 mg/kg, oral) alone or in combination for the latter 14 days of SRL treatment. The effects of CoQ(10) and MET on SRL-induced DM were assessed with the intraperitoneal glucose tolerance test (IPGTT) and by determining plasma insulin concentration and the homeostatic model assessment of insulin resistance (HOMA-R) index. We also evaluated the effect of CoQ(10) on pancreatic islet size, apoptosis, oxidative stress, and mitochondria morphology.@*RESULTS@#IPGTT revealed overt DM in SRL-treated rats. The addition of CoQ(10) to MET further improved hyperglycemia, decreased HOMA-R index, and increased plasma insulin concentration compared with the SRL group than MET alone therapy. While SRL treatment induced smaller islets with decreased insulin staining intensity, the combination of CoQ(10) and MET significantly improved insulin staining intensity, which was accompanied by a reduction in oxidative stress and apoptosis. In addition, co-treatment of CoQ(10) and MET significantly increased the levels of antiperoxidative enzymes in the pancreas islet cells compared with MET. At the subcellular level, addition of CoQ(10) to MET improved the average mitochondrial area and insulin granule number.@*CONCLUSIONS@#Addition of CoQ(10) to MET has a beneficial effect on SRL-induced DM compared to MET alone.

Effects of metformin on hyperglycemia in an experimental model of tacrolimus- and sirolimus-induced diabetic rats

BACKGROUND/AIMS: Metformin (MET) is a first-line drug for type 2 diabetes mellitus (DM); its effect on new-onset diabetes after transplantation caused by immunosuppressant therapy is unclear. We compared the effects of MET on DM caused by tacrolimus (TAC) or sirolimus (SRL). METHODS: DM was induced by injection of TAC (1.5 mg/kg) or SRL (0.3 mg/kg) for 2 weeks in rats, and MET (200 mg/kg) was injected for 2 more weeks. The effects of MET on DM caused by TAC or SRL were evaluated using an intraperitoneal glucose tolerance test (IPGTT) and by measuring plasma insulin concentration, islet size, and glucose-stimulated insulin secretion (GSIS). The effects of MET on the expression of adenosine monophosphate-activated protein kinase (AMPK), a pharmacological target of MET, were compared between TAC- and SRL-treated islets. RESULTS: IPGTT showed that both TAC and SRL induced hyperglycemia and reduced plasma insulin concentration compared with vehicle. These changes were reversed by addition of MET to SRL but not to TAC. Pancreatic islet cell size was decreased by TAC but not by SRL, but addition of MET did not affect pancreatic islet cell size in either group. MET significantly increased GSIS in SRL- but not in TAC-treated rats. AMPK expression was not affected by TAC but was significantly decreased in SRL-treated islets. Addition of MET restored AMPK expression in SRL-treated islets but not in TAC-treated islets. CONCLUSIONS: MET has different effects on hyperglycemia caused by TAC and SRL. The discrepancy between these drugs is related to their different mechanisms causing DM.

Role of dipeptidyl peptidase-4 inhibitors in new-onset diabetes after transplantation

Despite strict pre- and post-transplantation screening, the incidence of new-onset diabetes after transplantation (NODAT) remains as high as 60%. This complication affects the risk of cardiovascular events and patient and graft survival rates. Thus, reducing the impact of NODAT could improve overall transplant success. The pathogenesis of NODAT is multifactorial, and both modifiable and nonmodifiable risk factors have been implicated. Monitoring and controlling the blood glucose profile, implementing multidisciplinary care, performing lifestyle modifications, using a modified immunosuppressive regimen, administering anti-metabolite agents, and taking a conventional antidiabetic approach may diminish the incidence of NODAT. In addition to these preventive strategies, inhibition of dipeptidyl peptidase-4 (DPP4) by the gliptin family of drugs has recently gained considerable interest as therapy for type 2 diabetes mellitus and NODAT. This review focuses on the role of DPP4 inhibitors and discusses recent literature regarding management of NODAT.

Comparison of Early and Late Conversion of Sirolimus in Experimental Model of Chronic Cyclosporine Nephropathy

Sirolimus (SRL) is a promising drug for replacing calcineurin inhibitors. We performed this study to determine the optimal time of conversion from cyclosporine (CsA) to SRL in an experimental model of chronic CsA nephropathy. Three separate studies were performed. In the first study, SRL was given to rats with or without CsA for 4 weeks. In the second study, rats were treated initially with CsA for 1 week, and then switched to SRL (early conversion). In the third study, CsA was given for 4 weeks and then replaced by SRL for 4 weeks treatment of CsA (late conversion). The influence of SRL on CsA-induced renal injury was evaluated by assessing renal function, histopathology (interstitial inflammation and fibrosis), and apoptotic cell death. Combined CsA and SRL treatment significantly impaired renal function, increased apoptosis, and interstitial fibrosis and inflammation compared with CsA or SRL treatment alone. Early conversion to SRL did not change renal function, histopathology, or apoptosis compared with early CsA withdrawal. By contrast, late conversion to SRL significantly aggravated these parameters compared with late CsA withdrawal. In conclusion, early conversion from CsA to SRL is effective in preventing CsA-induced renal injury in a setting of CsA-induced renal injury.

Induction of Acute Cyclosporine Nephrotoxicity in Pig / 대한이식학회지

PURPOSE: Pigs are promising donor species for xenotransplantation. This study was performed to examine acute cyclosporine (CsA) nephrotoxicity in a pig model. METHODS: Adult pigs were treated daily for 1 week with vehicle (VH), or 7.5 mg/kg (CsA7.5), 15 mg/kg (CsA15), and 30 mg/kg (CsA30) CsA. The renal function, electrolyte levels, whole- blood CsA levels, and histopathological results (vacuolization and tubulointerstitial fibrosis) were compared among the different treatment groups. RESULTS: After 1 week of treatment, it was found that CsA induced characteristic lesions that were remarkably similar to those of chronic CsA nephropathy in humans. Compared with the results obtained for the VH group, CsA reduced renal function and yielded poor histopathological results. With an increase in the CsA concentration, the renal function and histological parameters worsened in a dose-dependent manner. CONCLUSION: The study showed that CsA dose-dependently induced renal injury in a pig model. These results provide basic data to estimate or prevent the adverse renal effects of immunosuppressants during porcine xenotransplantation in the future.

Induction of PPAR Gamma mRNA and Protein Expression by Rosiglitazone in Chronic Cyclosporine Nephropathy in the Rat

PURPOSE: We recently reported that rosiglitazone (RGTZ), a peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, has a protective effect against cyclosporine (CsA)- induced renal injury. Here we report the effect of RGTZ on peroxisome proliferator-activated receptor gamma (PPARgamma) expression in an experimental model of chronic cyclosporine (CsA) nephropathy. MATERIALS AND METHODS: Chronic CsA nephropathy was induced in Sprague-Dawley rats by administering CsA (15mg/kg per day) for 28 days, and control rats were treated with vehicle (VH group, olive oil 1mL/kg per day) for 28 days. RGTZ (3mg/kg) was concurrently administered via gavage to the CsA and VH groups. Expression of PPARgamma mRNA and protein was evaluated with RT-PCR, immunohistochemistry, and immunoblotting. RESULTS: PPARgamma mRNA expression was similar to the level of PPARgamma protein constitutively expressed in the kidneys of the VH treated rats, with expression in the glomerular epithelial, distal tubular cells, and collecting tubular cells. PPARgamma protein expression in CsA-treated rat kidneys was significantly less than in the VH group. However, concomitant administration of RGTZ restored PPARgamma protein expression in the kidneys of the CsA- reated rats. CONCLUSION: Exogenous administration of RGTZ treatment upregulates PPARgamma expression and that this mechanism may play a role in protecting against CsA-induced renal injury.

Activation of Intrarenal Complement System in Mouse Model for Chronic Cyclosporine Nephrotoxicity

PURPOSE: Local activation of the complement system plays a role in target organ damage. The aim of our study was to investigate the influence of cyclosporine (CsA)- induced renal injury on the complement system in the kidney. MATERIALS AND METHODS: Mice fed a low salt (0.01%) diet were treated with vehicle (VH, olive oil, 1mL/kg/day) or CsA (30mg/kg/day) for one or four weeks. Induction of chronic CsA nephrotoxicity was evaluated with renal function and histomorphology. Activation of the complement system was assessed through analysis of the expression of C3, C4d, and membrane attack complex (MAC), and the regulatory proteins, CD46 and CD55. CsA treatment induced renal dysfunction and typical morphology (tubulointerstitial inflammation and fibrosis) at four weeks. RESULTS: CsA-induced renal injury was associated with increased the expression of C3, C4d, and MAC (C9 and upregulation of complement regulatory proteins (CD 46 and CD55). Immunohistochemistry revealed that the activated complement components were mainly confined to the injured tubulointerstitium. CONCLUSION: CsA-induced renal injury is associated with activation of the intrarenal complement system.

Ischemia-Reperfusion Injury Enhances Dendritic Cell Infiltration and TLR2 Expression in Rat Kidneys / 대한신장학회잡지

BACKGROUND: It is well known that ischemia/reperfusion (I/R) injury enhances immunogenicity. But, its influence on innate immunity is still undetermined. This study was performed to evaluate whether I/R injury activates innate immunity in rat kidneys. METHODS: Sprague-Dawley rats were used. Ischemic injury was induced by clamping both renal arteries for 45 minutes. Sham operation was performed in a similar manner, except clamping the renal vessels. Rats were sacrificed on day 1, 3, 5, and 7 after I/R injury. Activation of innate immunity was evaluated in terms of toll-like receptor (TLR), dendritic cells and MHC class II antigen. TLR2 mRNA expression was detected by RT-PCR, and in situ hybridization. Dendritic cells and MHC class II antigens were detected with OX62 and OX6 monoclonal antibodies by immunohistochemistry. RESULTS: TLR2 mRNA was significantly increased in on day 3 and 5 after I/R injury (1 d: 120+/-2%, 3 d: 137+/-5%, 5 d: 173+/-5% 7 d: 120+/-8% vs. 100+/-11%, p<0.05 vs. sham), and in situ hybridization signal was observed on proximal, distal tubules, and interstitial cells. Compared to the sham-operated rat kidneys, number of dendritic cells significantly increased from day 1 to day 7 after I/R injury (1 d: 22.9+/-2.4, 3 d: 25.8+/-4.9, 5 d: 26.5+/-2.3, 7 d: 24.3+/-1.6 vs. 13.3+/-1.1, p<0.05 vs. sham) with peak value at day 5. Increased expression of MHC class II antigen was observed in the proximal tubules and interstitial cells in I/R injured rat kidney and there was maximal MHC class II protein level on day 3 after I/R injury. CONCLUSION: Ischemia-reperfusion injury itself can activate innate immunity on early period after injury.

Chronic Cyclosporine Nephrotoxicity: New Insights and Preventive Strategies

Cyclosporine (CsA) has improved patient and graft survival rates following solid-organ transplantation and has been increasingly applied with significant clinical benefits in the management of autoimmune diseases. However, the clinical use of CsA is often limited by acute and chronic nephrotoxicity, which remains a major problem. Acute nephrotoxicity depends on the dosage of CsA and seems to be caused by a reduction in renal blood flow related to afferent arteriolar vasoconstriction. However, the mechanisms underlying chronic CsA nephrotoxicity are not fully understood. Activation of the intrarenal renin-angiotensin system, increased release of endothelin-1, dysregulation of nitric oxide (NO) and NO synthase, upregulation of transforming growth factor-beta1, inappropriate apoptosis, stimulation of inflammatory mediators, and enhanced immunogenecity have all been implicated in the pathogenesis of chronic CsA nephrotoxicity. Reducing the CsA dose or withdrawing it and using combined nephroprotective drugs (mycophenolate mofetil, losartan, and pravastatin) may ameliorate chronic CsA-induced renal injury. This review discusses new insights and preventive strategies for this clinical dilemma.

Expression of alphasmooth Muscle Actin, Aquaporin 1 and Renin after Blocking AT1 Receptor in Developing Renal Arterial System of Rat / 대한신장학회잡지

BACKGROUND: Recent studies have demonstrated that renin, alphasmooth muscle actin(ASMA) and aquaporin-1(AQP1) participate in the development of renal arterial system. The components of the renin- angiotensin system have been shown to function as growth factors, apart from their classical roles in controlling blood volume and homeostasis. Interestingly, the vasoconstrictor angiotensin II(ANG II) appears to participate in the regulation of angiogenesis in various tissues. The present study examined the effect of ANG II type-1(AT1) receptor blocker losartan given during pregnancy or newborn rats on the expression of renin, ASMA and AQP1 in the developing renal arterial system. METHODS: Pregnant and newborn rats received losartan(10 mg/kg/day) or saline for 4 and 8 days from E14 to parturition, and for 4 and 9 days starting at day 1 after birth, respectively. Kidneys of 17-day-old fetuses and 1-, 4-, and 9-day- old pups were processed for immunohistochemistry using antibodies to renin(1 : 10,000), ASMA(1 : 1,000), and AQP1(1 : 1,000). RESULTS: In all pregnant groups, there were no differences in immunostaining for renin, ASMA, and AQP1 between losartan treated groups and saline treated groups. In all newborn groups, however, blockade of AT1 receptor with losartan found to increase expression of renin and ASMA but to have no effect on expression of AQP1 in the developing renal arterial system. CONCLUSION: These results suggest that AQP1 expression is not associated with renin or ASMA expression during development of renal arterial system.

Developmental Expression of eNOS in the Rat Renal Vasculature / 대한해부학회지

Nitric oxide (NO) has an important role in maintaining basal renal blood flow (RBF) and glomerular filtration rate (GFR) in the developing kidney. However, renal endothelial NO synthase (eNOS) has not been localized in the developing kidney. The purpose of this study was to examine the expression and localization of eNOS in the developing rat kidney using immunohistochemistry and western blotting. Kidneys from 14 (E14)-, 16 (E16)-, 18 (E18)- and 20-day-old (E20) fetuses, 1 (P1)-, 4 (P4)-, 7 (P7)-, 14 (P14)- and 21-day-old (P21) pups, and adult rats were extracted for immunohistochemistry, and western blot analysis. In the adult rat kidney, eNOS was expressed strongly in the endothelial cells of the arcuate artery and the vascular bundle in the medulla. Endothelial cells of the glomerulus and peritubular capillary network were weakly labeled for eNOS. There was no eNOS immunoreactivity in the uriniferous tubules, including the proximal tubules. In the developing rat kidney, eNOS appeared in the endothelial cells of the capillary network from E14. In the developing glomerular capillary, immunoreactivity for eNOS was observed in the S-shaped bodies (stage II glomeruli) and stage III glomeruli, whereas mature glomeruli (stage IV glomeruli) were faintly immunolabeled for eNOS. These eNOS-positive early-stage developing glomeruli were observed in the nephrogenic zone until seven days after birth. In the endothelial cells of the peritubular capillary network, eNOS was strongly expressed in the fetus and gradually decreased in intensity after birth. The endothelial cells of the arcuate artery were strongly immunoreactive for eNOS from E16 to the adult stages. In the renal medulla, eNOS was expressed in the endothelial cells of the capillary network surrounding the developing medullary collecting ducts of the fetal kidney. After birth, eNOS immunoreactivity gradually disappeared from the vasculature of the renal medulla and only remained in the vasa recta. In conclusion, the strong expression of eNOS in the early stages of the developing vasculature suggests that eNOS may contribute to angiogenesis and/or critically participate in the hemodynamics of the immature kidney.