Spexin-based galanin receptor 2 agonist improves renal injury in mice with type 2 diabetes.

The spexin-based GALR2 agonist (NS200) is a novel drug, which has shown antidepressant and anxiolytic action in a recent experimental study. In this study, we investigated the effects of NS200 on renal injury in an animal model of type 2 diabetes. Eight-week-old diabetic db/db mice were administered NS200 for 12 weeks. NS200 was intraperitoneally administered at a dose of 1.0 mg/kg/day. Metabolic parameters and structural and molecular changes in the kidneys were compared among the three groups: non-diabetic db/m control, db/db mice, and NS200-treated db/db mice. In db/db mice, NS200 administration did not impact the body weight, food and water intake, urinary volume, fasting blood glucose level, or HbA1c levels. Insulin and glucose tolerance were also unaffected by NS200 treatment. However, NS200 improved urinary albumin excretion and glomerulosclerosis in diabetic kidneys. Activation of TGFß1 and insulin signaling pathways, such as PI3 K /AKT/ERK, were inhibited by NS200. In conclusion, a spexin-based GALR2 agonist attenuated diabetic nephropathy by alleviating renal fibrosis in mice with type 2 diabetes. Spexin-based GALR2 agonists have considerable potential as novel treatment agents in diabetic nephropathy.

Pan-Nox inhibitor treatment improves renal function in aging murine diabetic kidneys.

Background: Aging is a risk factor for development of chronic kidney disease and diabetes mellitus with commonly shared features of chronic inflammation and increased oxidative stress. Here, we investigated the effect of pan-Nox-inhibitor, APX-115, on renal function in aging diabetic mice. Methods: Diabetes was induced by intraperitoneal injection of streptozotocin at 50 mg/kg/day for 5 days in 52-week-old C57BL/6J mice. APX-115 was administered by oral gavage at a dose of 60 mg/kg/day for 12 weeks in nondiabetic and diabetic aging mice. Results: APX-115 significantly improved insulin resistance in diabetic aging mice. Urinary level of 8-isoprostane was significantly increased in diabetic aging mice than nondiabetic aging mice, and APX-115 treatment reduced 8-isoprostane level. Urinary albumin and nephrin excretion were significantly higher in diabetic aging mice than nondiabetic aging mice. Although APX-115 did not significantly decrease albuminuria, APX-115 markedly improved mesangial expansion, macrophage infiltration, and expression of fibrosis molecules such as transforming growth factor beta 1 and plasminogen activator inhibitor 1. Interestingly, the expression of all Nox isoforms including Nox1, Nox2, and Nox4 was significantly increased in diabetic aging kidneys, and APX-115 treatment decreased Nox1, Nox2, and Nox4 protein expression in the kidney. Furthermore, Klotho expression was significantly decreased in diabetic aging kidneys, and APX-115 restored Klotho level. Conclusion: Our results provide evidence that pan-Nox inhibition may improve systemic insulin resistance and decrease oxidative stress, inflammation, and fibrosis in aging diabetic status and may have potential protective effects on aging diabetic kidney.

SH3YL1 Protein Predicts Renal Outcomes in Patients with Type 2 Diabetes.

NADPH oxidase (NOX)-derived oxidative stress is an important factor in renal progression, with NOX4 being the predominant NOX in the kidney. Recently, Src homology 3 (SH3) domain-containing YSC84-like 1 (SH3YL1) was reported to be a regulator of NOX4. In this study, we tested whether the SH3YL1 protein could predict 3-year renal outcomes in patients with type 2 diabetes. A total of 131 patients with type 2 diabetes were enrolled in this study. Renal events were defined as a 15% decline in the estimated glomerular filtration rate (eGFR) from the baseline, the initiation of renal replacement therapy, or death during the 3 years. The levels of the urinary SH3YL1-to-creatinine ratio (USCR) were significantly different among the five stages of chronic kidney disease (CKD) and the three groups, based on albuminuria levels. The USCR levels showed a significant negative correlation with eGFR and a positive correlation with the urinary albumin-to-creatinine ratio (UACR). Plasma SH3YL1 levels were significantly correlated with UACR. The highest tertile group of USCR and plasma SH3YL1 had a significantly lower probability of renal event-free survival. Furthermore, the highest tertile group of USCR showed a significant association with the incidence of renal events after full adjustment: adjusted hazard ratio (4.636: 95% confidence interval, 1.416-15.181, p = 0.011). This study suggests that SH3YL1 is a new diagnostic biomarker for renal outcomes in patients with type 2 diabetes.

The Role of V-Set Ig Domain-Containing 4 in Chronic Kidney Disease Models.

V-set Ig domain-containing 4 (VSIG4) regulates an inflammatory response and is involved in various diseases. However, the role of VSIG4 in kidney diseases is still unclear. Here, we investigated VSIG4 expression in unilateral ureteral obstruction (UUO), doxorubicin-induced kidney injury mouse, and doxorubicin-induced podocyte injury models. The levels of urinary VSIG4 protein significantly increased in the UUO mice compared with that in the control. The expression of VSIG4 mRNA and protein in the UUO mice was significantly upregulated compared with that in the control. In the doxorubicin-induced kidney injury model, the levels of urinary albumin and VSIG4 for 24 h were significantly higher than those in the control mice. Notably, a significant correlation was observed between urinary levels of VSIG4 and albumin (r = 0.912, p < 0.001). Intrarenal VSIG4 mRNA and protein expression were also significantly higher in the doxorubicin-induced mice than in the control. In cultured podocytes, VSIG4 mRNA and protein expressions were significantly higher in the doxorubicin-treated groups (1.0 and 3.0 µg/mL) than in the controls at 12 and 24 h. In conclusion, VSIG4 expression was upregulated in the UUO and doxorubicin-induced kidney injury models. VSIG4 may be involved in pathogenesis and disease progression in chronic kidney disease models.

Role of NADPH Oxidases in Renal Aging.

INTRODUCTION: Aging of the kidney is associated with complex molecular, histological, and functional changes. Although the aging process itself does not induce renal damage, underlying disease such as diabetes mellitus can aggravate kidney injury during aging. Although oxidative stress is considered an important mediator in age-related renal fibrosis, it is unclear how oxidative stress increases during normal and diabetic aging. METHODS: In this study, we investigated molecular changes in the kidney in normal and diabetic aging mice. C57BL/6 mice were studied at 2, 12, and 24 months of age, and leptin receptor-deficient db/db mice were studied at 8, 12, 16, 20, 24, and 38 weeks of age. We measured renal functional parameters, fibrotic and inflammatory markers, and oxidative stress markers at all the above time points. RESULTS: Both nondiabetic and diabetic mice exhibited progressive microalbuminuria during their lifespan. Interestingly, both diabetic aging and normal aging mice showed progressive increases in oxidative stress markers such as plasma and urinary 8-isoprostane, as well as renal lipid hydroperoxide content. In renal tissues, proinflammatory and profibrotic molecules were significantly upregulated in an age-dependent manner. Expression of three NADPH oxidase (Nox) isoforms, namely, Nox1, Nox2, and Nox4, was significantly increased during aging. Compared with normal aging mice, diabetic db/db mice demonstrated more dramatic changes during aging process. CONCLUSIONS: Our findings suggest that NADPH oxidases play an important role in the aging kidney under both normal and diabetic conditions. Targeting of these oxidases might be a new promising therapy to treat issues associated with aging kidneys.

Upregulation of VSIG4 in Type 2 Diabetic Kidney Disease.

Fibrosis is the final common finding in patients with advanced diabetic kidney disease. V-set Ig domain containing 4 (VSIG4) is related to fibrosis in several diseases. It also contributes to fibrosis under high-glucose conditions in renal tubule cells. To determine the role of VSIG4 in type 2 diabetes, we examined VSIG4 expression in a type 2 diabetic animal model and podocyte. Urinary excretion of albumin and VSIG4 was significantly higher in db/db mice than in the control group. Urine VSIGs levels for 6 h were about three-fold higher in db/db mice than in db/m mice at 20 weeks of age: 55.2 ± 37.8 vs. 153.1 ± 74.3 ng, p = 0.04. Furthermore, urinary VSIG4 levels were significantly correlated with urinary albumin levels (r = 0.77, p < 0.01). Intrarenal VSIG4 mRNA expression was significantly higher in db/db mice than in control mice (1.00 ± 0.35 vs. 1.69 ± 0.77, p = 0.04). Further, VSIG4 expression was almost twice as high in db/db mice at 20 weeks of age. Intrarenal VSIG immunoreactivity in db/db mice was also significantly higher than that in control mice. In cultured podocytes, both high glucose and angiotensin II significantly upregulated the expression of VSIG4 mRNA and protein. In conclusion, VSIG4 was upregulated in an animal model of type 2 diabetes and was related to albuminuria and pro-fibrotic markers. Considering these relationships, VSIG4 may be an important mediator of diabetic nephropathy progression.

A Novel Dipeptidyl Peptidase-4 Inhibitor DA-1229 Ameliorates Tubulointerstitial Fibrosis in Cyclosporine Nephrotoxicity in Mice.

Cyclosporine A (CyA) is an immunosuppressive agent that induces nephrotoxicity with long-term treatment. The roles of DPP-4 and its inhibitors in cyclosporine nephrotoxicity are not fully understood. Therefore, we investigated the effects of a novel DPP-4 inhibitor, DA-1229, on the progression of renal disease in an experimental cyclosporine nephrotoxicity model. Chronic cyclosporine nephrotoxicity was induced in six-week-old male ICR mice by subcutaneous injections of CyA at a dose of 30 mg/kg for four weeks. Animals were treated with DA-1229 at a dose of 300 mg/kg per day in food for four weeks. Although DPP-4 activity did not increase in the kidneys of mice with induced cyclosporine nephrotoxicity, DA-1229 treatment significantly suppressed DPP-4 activity in both plasma and renal tissues. DPP-4 inhibition by DA-1229 led to significantly decreased albuminuria and urinary excretion of 8-isoprosatane. DPP-4 inhibition also substantially suppressed pro-inflammatory effects, profibrotic molecules, and macrophage infiltration, and led to the improvement in renal structural changes. Our results suggest that DPP-4 inhibition by DA-1229 provides renoprotective effects in an animal model of cyclosporine nephrotoxicity via antioxidant, anti-inflammatory, and anti-fibrotic mechanisms. DPP-4 inhibition may be a useful new therapeutic approach for the management of progressive renal disease in cyclosporine nephrotoxicity.

LPS-Induced Acute Kidney Injury Is Mediated by Nox4-SH3YL1.

Cytosolic proteins are required for regulation of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase (Nox) isozymes. Here we show that Src homology 3 (SH3) domain-containing YSC84-like 1 (SH3YL1), as a Nox4 cytosolic regulator, mediates lipopolysaccharide (LPS)-induced H2O2 generation, leading to acute kidney injury. The SH3YL1, Ysc84p/Lsb4p, Lsb3p, and plant FYVE proteins (SYLF) region and SH3 domain of SH3YL1 contribute to formation of a complex with Nox4-p22phox. Interaction of p22phox with SH3YL1 is triggered by LPS, and the complex induces H2O2 generation and pro-inflammatory cytokine expression in mouse tubular epithelial cells. After LPS injection, SH3YL1 knockout mice show lower levels of acute kidney injury biomarkers, decreased secretion of pro-inflammatory cytokines, decreased infiltration of macrophages, and reduced tubular damage compared with wild-type (WT) mice. The results strongly suggest that SH3YL1 is involved in renal failure in LPS-induced acute kidney injury (AKI) mice. We demonstrate that formation of a ternary complex of p22phox-SH3YL1-Nox4, leading to H2O2 generation, induces severe renal failure in the LPS-induced AKI model.

Inhibition of Renal Stellate Cell Activation Reduces Renal Fibrosis.

Interstitial fibrosis is a common feature of chronic kidney disease, and platelet-derived growth factor receptor-ß (PDGFR-ß)-positive mesenchymal cells are reportedly the major source of scar-producing myofibroblasts. We had previously demonstrated that albumin and its derivative R-III (a retinol-binding protein-albumin domain III fusion protein) inhibited the transdifferentiation/activation of hepatic stellate cells (HSCs) to myofibroblasts and that R-III administration reduced liver fibrosis. In this study, we isolated cells (referred to as renal stellate cells, RSCs) from rat kidney tissues using the HSC isolation protocol and compared their morphological and biochemical characteristics with those of HSCs. RSCs shared many characteristics with HSCs, such as storage of vitamin A-containing lipid droplets and expression of HSC markers as well as pericyte markers. RSCs underwent spontaneous transdifferentiation into myofibroblasts in in vitro culture, which was inhibited by albumin expression or R-III treatment. We also evaluated the therapeutic effects of R-III in unilateral ureteral obstruction (UUO)-induced renal fibrosis in mice. Injected R-III localized predominantly in cytoglobin/stellate cell activation-associated protein (Cygb/STAP)-positive cells in the kidney and reduced renal fibrosis. These findings suggest that RSCs can be recognized as the renal counterparts of HSCs and that RSCs represent an attractive therapeutic target for anti-fibrotic therapy.

APX-115, a first-in-class pan-NADPH oxidase (Nox) inhibitor, protects db/db mice from renal injury.

Recent studies have suggested that renal Nox is important in the progression of diabetic nephropathy. Therefore, we investigated the effect of a novel pan-NOX-inhibitor, APX-115, on diabetic nephropathy in type 2 diabetic mice. Eight- week-old db/m and db/db mice were treated with APX-115 for 12 weeks. APX-115 was administered by oral gavage at a dose of 60 mg/kg per day. To compare the effects of APX-115 with a dual Nox1/Nox4 inhibitor, db/db mice were treated with GKT137831 according to the same protocol. APX-115 significantly improved insulin resistance in diabetic mice, similar to GKT137831. Oxidative stress as measured by plasma 8-isoprostane level was decreased in the APX-115 group compared with diabetic controls. All lipid profiles, both in plasma and tissues improved with Nox inhibition. APX-115 treatment decreased Nox1, Nox2, and Nox4 protein expression in the kidney. APX-115 decreased urinary albumin excretion and preserved creatinine level. In diabetic kidneys, APX-115 significantly improved mesangial expansion, but GKT137831 did not. In addition, F4/80 infiltration in the adipose tissue and kidney decreased with APX-115 treatment. We also found that TGF-ß stimulated ROS generation in primary mouse mesangial cells (pMMCs) from wild-type, Nox1 KO, and Duox1 KO mice, but did not induce Nox activity in pMMCs from Nox2 knockout (KO), Nox4 KO, or Duox2 KO mice. These results indicate that activating Nox2, Nox4, or Duox2 in pMMCs is essential for TGF-ß-mediated ROS generation. Our findings suggest that APX-115 may be as effective or may provide better protection than the dual Nox1/Nox4 inhibitor, and pan-Nox inhibition with APX-115 might be a promising therapy for diabetic nephropathy.

Renoprotective Effects of a Highly Selective A3 Adenosine Receptor Antagonist in a Mouse Model of Adriamycin-induced Nephropathy.

The concentration of adenosine in the normal kidney increases markedly during renal hypoxia, ischemia, and inflammation. A recent study reported that an A3 adenosine receptor (A3AR) antagonist attenuated the progression of renal fibrosis. The adriamycin (ADX)-induced nephropathy model induces podocyte injury, which results in severe proteinuria and progressive glomerulosclerosis. In this study, we investigated the preventive effect of a highly selective A3AR antagonist (LJ1888) in ADX-induced nephropathy. Three groups of six-week-old Balb/c mice were treated with ADX (11 mg/kg) for four weeks and LJ1888 (10 mg/kg) for two weeks as following: 1) control; 2) ADX; and 3) ADX + LJ1888. ADX treatment decreased body weight without a change in water and food intake, but this was ameliorated by LJ1888 treatment. Interestingly, LJ1888 lowered plasma creatinine level, proteinuria, and albuminuria, which had increased during ADX treatment. Furthermore, LJ1888 inhibited urinary nephrin excretion as a podocyte injury marker, and urine 8-isoprostane and kidney lipid peroxide concentration, which are markers of oxidative stress, increased after injection of ADX. ADX also induced the activation of proinflammatory and profibrotic molecules such as TGF-ß1, MCP-1, PAI-1, type IV collagen, NF-κB, NOX4, TLR4, TNFα, IL-1ß, and IFN-γ, but they were remarkably suppressed after LJ1888 treatment. In conclusion, our results suggest that LJ1888 has a renoprotective effect in ADX-induced nephropathy, which might be associated with podocyte injury through oxidative stress. Therefore, LJ1888, a selective A3AR antagonist, could be considered as a potential therapeutic agent in renal glomerular diseases which include podocyte injury and proteinuria.

Chronic Administration of Visfatin Ameliorated Diabetic Nephropathy in Type 2 Diabetic Mice.

BACKGROUND/AIMS: Visfatin is a known adipokine which may improve insulin resistance in obesity and have an anti-diabetic effect via the insulin receptor. We studied the effects of visfatin on diabetic nephropathy in type 2 diabetic mice. METHODS: Diabetic male db/db mice were treated with intraperitoneal injections of visfatin. Basal parameters were measured in all mice and glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed in diabetic mice. The histopathological and molecular changes were evaluated in diabetic nephropathy. RESULTS: Visfatin treatment had no effect on body weight, water and food intake, urinary volume, blood glucose, and HbA1c level. However, visfatin improved HOMA-IR, GTT, ITT and decreased plasma insulin and visfatin level, but not adiponectin level. Plasma cholesterol and triglyceride level were also improved by visfatin treatment. Significantly, visfatin decreased albuminuria in diabetic mice. Glomerulosclerotic change and mesangial expansion in the kidneys were significantly reduced. In addition, visfatin inhibited the expression of proinflammatory and profibrotic cytokines such as MCP-1, TGFß1, type IV collagen, and PAI-1. The enzymes related to lipid metabolism in the kidney, HMG-CoAR was suppressed by visfatin treatment, whereas FXR and ABCA1 were significantly elevated by treatment. CONCLUSION: Visfatin might have a protective effect in diabetic nephropathy without the hypoglycemic effect.

Enhanced conjugation stability and blood circulation time of macromolecular gadolinium-DTPA contrast agent.

In this study, we prepared macromolecular MR T1 contrast agent: pullulan-conjugated Gd diethylene triamine pentaacetate (Gd-DTPA-Pullulan) and estimated residual free Gd(3+), chelation stability in competition with metal ions, plasma and tissue pharmacokinetics, and abdominal MR contrast on rats. Residual free Gd(3+) in Gd-DTPA-Pullulan was measured using colorimetric spectroscopy. The transmetalation of Gd(3+) incubated with Ca(2+) was performed by using a dialysis membrane (MWCO 100-500 Da) and investigated by ICP-OES. The plasma concentration profiles of Gd-DTPA-Pullulan were estimated after intravenous injection at a dose 0.1 mmol/kg of Gd. The coronal-plane abdominal images of normal rats were observed by MR imaging. The content of free Gd(3+), the toxic residual form, was less than 0.01%. Chelation stability of Gd-DTPA-Pullulan was estimated, and only 0.2% and 0.00045% of Gd(3+) were released from Gd-DTPA-Pullulan after 2h incubation with Ca(2+) and Fe(2+), respectively. Gd-DTPA-Pullulan displayed the extended plasma half-life (t1/2,α=0.43 h, t1/2,ß=2.32 h), much longer than 0.11h and 0.79 h of Gd-EOB-DTPA. Abdominal MR imaging showed Gd-DTPA-Pullulan maintained initial MR contrast for 30 min. The extended plasma half-life of Gd-DTPA-Pullulan probably allows the prolonged MR acquisition time in clinic with enhanced MR contrast.

DA-1229, a dipeptidyl peptidase IV inhibitor, protects against renal injury by preventing podocyte damage in an animal model of progressive renal injury.

Although dipeptidyl peptidase IV (DPPIV) inhibitors are known to have renoprotective effects, the mechanism underlying these effects has remained elusive. Here we investigated the effects of DA-1229, a novel DPPIV inhibitor, in two animal models of renal injury including db/db mice and the adriamycin nephropathy rodent model of chronic renal disease characterized by podocyte injury. For both models, DA-1229 was administered at 300 mg/kg/day. DPPIV activity in the kidney was significantly higher in diabetic mice compared with their nondiabetic controls. Although DA-1229 did not affect glycemic control or insulin resistance, DA-1229 did improve lipid profiles, albuminuria and renal fibrosis. Moreover, DA-1229 treatment resulted in decreased urinary excretion of nephrin, decreased circulating and kidney DPPIV activity, and decreased macrophage infiltration in the kidney. In adriamycin-treated mice, DPPIV activity in the kidney and urinary nephrin loss were both increased, whereas glucagon-like peptide-1 concentrations were unchanged. Moreover, DA-1229 treatment significantly improved proteinuria, renal fibrosis and inflammation associated with decreased urinary nephrin loss, and kidney DPP4 activity. In cultured podocytes, DA-1229 restored the high glucose/angiotensin II-induced increase of DPPIV activity and preserved the nephrin levels in podocytes. These findings suggest that activation of DPPIV in the kidney has a role in the progression of renal disease, and that DA-1229 may exert its renoprotective effects by preventing podocyte injury.

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.

Comparison of adverse drug reaction profiles of two tacrolimus formulations in rats.

Tacrobell(®) (TB) is a generic tacrolimus which showed the comparable efficacy to original product, Prograf(®) (PG) in renal transplantation, but toxicity between two drugs is unclear. The aim of this study was to compare the toxicity between these two formulations. TB and PG (0.5, 1 and 2 mg/kg/day) was administered to rats for 4 weeks. The rat survival rate, kidney, liver and pancreas injury was investigated. The survival rate was similar between TB- and PG-treated rats. TB and PG induced renal dysfunction in a dose-dependent manner. Compared to PG treatment in equal dose, TB treatment reduced urinary creatinine clearance in a less degree and renal interstitial fibrosis was comparable between two regimens. The r-glutamyl transpeptidase was aggravated by tacrolimus treatment, and this was not different between TB and PG treatment. In the intraperitoneal glucose tolerance test, a significant diabetogenic effect was observed in all tacrolimus treated-rats. The glucose tolerance of TB-treated rats was similar to those of PG-treated rats in each dose. The decrement in pancreatic ß-cell mass by tacrolimus showed the dose-dependent response and it was comparable between TB and PG treatment. In conclusion, TB is similar to PG in terms of nephrotoxicity, hepatoxicity and diabetogenic effect.

Angiotensin II blockade upregulates the expression of Klotho, the anti-ageing gene, in an experimental model of chronic cyclosporine nephropathy.

BACKGROUND: The Klotho gene plays a role in suppressing ageing-related disorders. It is suggested that activation of renin-angiotensin system (RAS) or oxidative stress suppresses Klotho in the kidney. This study evaluated the association between Klotho expression and RAS in cyclosporine (CsA)-induced renal injury. METHODS: Chronic CsA nephropathy was induced by administering CsA (30 mg/kg) to mice on a low-salt diet (LSD) for 4 weeks. A normal-salt diet (NSD) was used as the control. Reverse transcription-polymerase chain reaction, western blot and immunohistochemistry were performed for Klotho and intrarenal RAS activity was measured using immunohistochemistry for angiotensinogen and renin. Oxidative stress was measured with urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG). RESULTS: CsA treatment decreased Klotho mRNA and protein in mouse kidney in a dose-dependent and time-dependent manner, but a concurrent treatment with losartan, an angiotensin II type 1 (AT1) receptor blocker, reversed the decrease in Klotho expression with histological improvement. This finding was more marked in the LSD than the NSD. Klotho expression was correlated with angiotensinogen and renin expression, tubulointerstitial fibrosis score and urinary 8-OHdG excretion. CONCLUSIONS: Angiotensin II may play a pivotal role in regulating Klotho expression in CsA-induced renal injury. AT1 receptor blocker may inhibit the ageing process by decreasing oxidative stress caused by CsA.

Early and delayed effects of AST-120 on chronic cyclosporine nephropathy.

BACKGROUND: Removal of uraemic toxins by AST-120 (Kremezin(®)) decreases the progression of chronic kidney disease by reducing oxidative stress. We performed this study to evaluate whether AST-120 has a similar effect on progression of cyclosporine (CsA)-induced renal injury. METHODS: Two separate studies were performed in adult Sprague-Dawley rats. First, AST-120 was administered with CsA (15 mg/kg) for 4 weeks (early treatment). Second, AST-120 was administered to the rats for 3 weeks after treatment with CsA for 3 weeks (delayed treatment). Uraemic toxin and oxidative stress were evaluated with plasma indoxyl sulphate (IS) levels and urinary 8-OHdG excretion. The effects of AST-120 on CsA-induced renal injury were evaluated in terms of renal function, interstitial fibrosis, inflammation, and apoptotic cell death. RESULTS: CsA treatment for 4 weeks showed 2-fold increase in plasma IS and urinary 8-OHdG levels compared with the VH group. Early treatment with AST-120 significantly decreased both parameters, and this was accompanied by improved renal function and decreased interstitial inflammation, fibrosis, and apoptotic cell death compared with those of rats that received CsA alone. Delayed treatment with AST-120 also decreased the plasma IS and urinary 8-OHdG levels, and reduced the progression of chronic CsA nephropathy. Furthermore, delayed AST-120 treatment decreased the epithelial-mesenchymal transition in chronic CsA nephropathy. CONCLUSIONS: Removal of uraemic toxins with AST-120 treatment is effective in decreasing the progression of CsA-induced renal injury by reducing oxidative stress.

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.

Upregulation of hyaluronan and its binding receptors in an experimental model of chronic cyclosporine nephropathy.

AIM: Hyaluronan (HA) is an important extracellular matrix (ECM) proteoglycan. The localization of HA and its binding receptors, CD44 and LYVE-1, was evaluated in an experimental model of chronic cyclosporine A (CsA)-induced nephropathy. METHODS: Sprague-Dawley rats maintained on a low-salt diet (0.05% sodium) received an s.c. injection of vehicle (1 mL/kg per day olive oil; VH groups) or CsA (15 mg/kg per day; CsA groups) for 1 or 4 weeks. Induction of chronic CsA nephropathy was evaluated according to renal function and pathology and expression of HA, CD44, LYVE-1, ED-1 and alpha-smooth muscle actin (alpha-SMA). RESULTS: CsA treatment for 4 weeks caused renal dysfunction, which was accompanied by typical striped interstitial fibrosis. In the VHroup, HA immunoreactivity was observed only in the inner medulla. However, the area of HA immunoreactivity increased with the duration of CsA treatment: CsA treatment for 1 week extended HA immunoreactivity to the outer medulla, and CsA treatment for 4 weeks caused a further extension of HA immunoreactivity to the cortex, which was vulnerable to CsA-induced renal injury. HA binding receptor, CD44 and LYVE-1 expression were also upregulated in the CsA groups, and were localized to the area of fibrosis and the peritubular capillaries of the cortex. In the CsA groups, ED-1 and alpha-SMA were predominantly expressed in fibrotic areas in which HA had accumulated. CONCLUSION: These findings suggest that upregulation of HA and its binding receptors are involved in interstitial fibrosis in chronic CsA-induced renal injury.