Nicorandil protects podocytes via modulation of antioxidative capacity in acute puromycin aminonucleoside-induced nephrosis in rats.

Nephrotic syndrome, characterized by proteinuria and hypoalbuminemia, results from the dysregulation of glomerular podocytes and is a significant cause of end-stage kidney disease. Patients with idiopathic nephrotic syndrome are generally treated with immunosuppressive agents; however, these agents produce various adverse effects. Previously, we reported the renoprotective effects of a stimulator of the mitochondrial ATP-dependent K+ channel (MitKATP), nicorandil, in a remnant kidney model. Nonetheless, the cellular targets of these effects remain unknown. Here, we examined the effect of nicorandil on puromycin aminonucleoside-induced nephrosis (PAN) rats, a well-established model of podocyte injury and human nephrotic syndrome. PAN was induced using a single intraperitoneal injection. Nicorandil was administered orally at 30 mg/kg/day. We found that proteinuria and hypoalbuminemia in PAN rats were significantly ameliorated following nicorandil treatment. Immunostaining and ultrastructural analysis under electron microscopy demonstrated that podocyte injury in PAN rats showed a significant partial attenuation following nicorandil treatment. Nicorandil ameliorated the increase in the oxidative stress markers nitrotyrosine and 8-hydroxy-2-deoxyguanosine in glomeruli. Conversely, nicorandil prevented the decrease in levels of the antioxidant enzyme manganese superoxide dismutase in PAN rats. We found that mitochondrial Ca2+ uniporter levels in glomeruli were higher in PAN rats than in control rats, and this increase was significantly attenuated by nicorandil. We conclude that stimulation of MitKATP by nicorandil reduces proteinuria by attenuating podocyte injury in PAN nephrosis, which restores mitochondrial antioxidative capacity, possibly through mitochondrial Ca2+ uniporter modulation. These data indicate that MitKATP may represent a novel target for podocyte injury and nephrotic syndrome.NEW & NOTEWORTHY Our findings suggest that the mitochondrial Ca2+ uniporter may be an upstream regulator of manganese superoxide dismutase and indicate a biochemical basis for the interaction between the ATP-sensitive K+ channel and Ca2+ signaling. We believe that our study makes a significant contribution to the literature because our results indicate that the ATP-sensitive K+ channel may be a potential therapeutic target for podocyte injury and nephrotic syndrome.

Characterization of pendrin in urinary extracellular vesicles in a rat model of aldosterone excess and in human primary aldosteronism.

Pendrin is a Cl-/HCO3- exchanger selectively present in the intercalated cells of the kidney. Although experimental studies have demonstrated that pendrin regulates blood pressure downstream of the renin-angiotensin-aldosterone system, its role in human hypertension remains unclear. Here, we analyzed the quantitative changes in pendrin in urinary extracellular vesicles (uEVs) isolated from a total of 30 patients with primary aldosteronism (PA) and from a rat model of aldosterone excess. Western blot analysis revealed that pendrin is present in dimeric and monomeric forms in uEVs in humans and rats. In a rodent model that received continuous infusion of aldosterone with or without concomitant administration of the selective mineralocorticoid receptor (MR) antagonist esaxerenone, pendrin levels in uEVs, as well as those of epithelial Na+ channel (ENaC) and Na-Cl-cotransporter (NCC), were highly correlated with renal abundance. In patients with PA, pendrin levels in uEVs were reduced by 49% from baseline by adrenalectomy or pharmacological MR blockade. Correlation analysis revealed that the magnitude of pendrin reduction after treatment significantly correlated with the baseline aldosterone-renin ratio (ARR). Finally, a cross-sectional analysis of patients with PA confirmed a significant correlation between the ARR and pendrin levels in uEVs. These data are consistent with experimental studies showing the role of pendrin in aldosterone excess and suggest that pendrin abundance is attenuated by therapeutic interventions in human PA. Our study also indicates that pendrin analysis in uEVs, along with other proteins, can be useful to understand the pathophysiology of hypertensive disorders.

Cardio-renal protective effect of the xanthine oxidase inhibitor febuxostat in the 5/6 nephrectomy model with hyperuricemia.

Although hyperuricemia has been shown to be associated with the progression of cardiovascular disorder and chronic kidney disease (CKD), there is conflicting evidence as to whether xanthine oxidase (XO) inhibitors confer organ protection besides lowering serum urate levels. In this study, we addressed the cardio-renal effects of XO inhibition in rodent CKD model with hyperuricemia. Sprague-Dawley rats underwent 5/6 nephrectomy and received a uricase inhibitor oxonic acid for 8 weeks (RK + HUA rats). In some rats, a XO inhibitor febuxostat was administered orally. Compared with control group, RK + HUA group showed a significant increase in albuminuria and renal injury. Febuxostat reduced serum uric acid as well as urinary albumin levels. Histological and immunohistochemical analysis of the kidney revealed that febuxostat alleviated glomerular, tubulointerstitial, and arteriolar injury in RK + HUA rats. Moreover, in the heart, RK + HUA showed individual myofiber hypertrophy and cardiac fibrosis, which was significantly attenuated by febuxostat. We found that renal injury and the indices of cardiac changes were well correlated, confirming the cardio-renal interaction in this model. Finally, NF-E2-related factor 2 (Nrf2) and the downstream target heme oxygenase-1 (HO-1) protein levels were increased both in the heart and in the kidney in RK + HUA rats, and these changes were alleviated by febuxostat, suggesting that tissue oxidative stress burden was attenuated by the treatment. These data demonstrate that febuxostat protects against cardiac and renal injury in RK + HUA rats, and underscore the pathological importance of XO in the cardio-renal interaction.

ABCG2 expression and uric acid metabolism of the intestine in hyperuricemia model rat.

To elucidate roles of the intestine in uric acid (UA) metabolism, we examined ABCG2 expression, tissue UA content and xanthine oxidoreductase (XOR) activity in different intestinal segments. Male SD rats were assigned to control group or oxonic acid-induced hyperuricemia (HUA) group. In control rats, ABCG2 was present both in villi and crypts in each segment. Tissue UA content and XOR activity were relatively high in duodenum and jejunum. However, in HUA rats, tissue UA content was significantly elevated in the ileum, whereas it remained unaltered in other segments. Moreover, ABCG2 expression in the HUA group was upregulated both in the villi and crypts of the ileum. These data indicate that the ileum may play an important role in the extra-renal UA excretion.

Insulin stimulates uric acid reabsorption via regulating urate transporter 1 and ATP-binding cassette subfamily G member 2.

Accumulating data indicate that renal uric acid (UA) handling is altered in diabetes and by hypoglycemic agents. In addition, hyperinsulinemia is associated with hyperuricemia and hypouricosuria. However, the underlying mechanisms remain unclear. In this study, we aimed to investigate how diabetes and hypoglycemic agents alter the levels of renal urate transporters. In insulin-depleted diabetic rats with streptozotocin treatment, both UA excretion and fractional excretion of UA were increased, suggesting that tubular handling of UA is altered in this model. In the membrane fraction of the kidney, the expression of urate transporter 1 (URAT1) was significantly decreased, whereas that of ATP-binding cassette subfamily G member 2 (ABCG2) was increased, consistent with the increased renal UA clearance. Administration of insulin to the diabetic rats decreased UA excretion and alleviated UA transporter-level changes, while sodium glucose cotransporter 2 inhibitor (SGLT2i) ipragliflozin did not change renal UA handling in this model. To confirm the contribution of insulin in the regulation of urate transporters, normal rats received insulin and separately, ipragliflozin. Insulin significantly increased URAT1 and decreased ABCG2 levels, resulting in increased UA reabsorption. In contrast, the SGLT2i did not alter URAT1 or ABCG2 levels, although blood glucose levels were similarly reduced. Furthermore, we found that insulin significantly increased endogenous URAT1 levels in the membrane fraction of NRK-52E cells, the kidney epithelial cell line, demonstrating the direct effects of insulin on renal UA transport mechanisms. These results suggest a previously unrecognized mechanism for the anti-uricosuric effects of insulin and provide novel insights into the renal UA handling in the diabetic state.

Proteomics Approach Identifies Factors Associated With the Response to Low-Density Lipoprotein Apheresis Therapy in Patients With Steroid-Resistant Nephrotic Syndrome.

Low-density lipoprotein apheresis (LDL-A) has been shown to reduce proteinuria in a subgroup of nephrotic syndrome patients refractory to immunosuppressive therapy. Factors influencing the efficacy of LDL-A in nephrotic syndrome are completely unknown. Using a proteomics approach, we aimed to identify biological markers that predict the response to LDL-A in patients with steroid-resistant nephrotic syndrome (SRNS). Identification of plasma proteins bound to the dextran-sulfate column at the first session of LDL-A was determined by mass spectrometry. To investigate biological factors associated with the response to LDL-A, we compared profiles of column-bound proteins between responders (defined by more than 50% reduction of proteinuria after the treatment) and non-responders by 2-dimensional gel electrophoresis (2-DE) coupled to mass spectrometry in seven patients with SRNS. Evaluation of proteins adsorbed to LDL-A column in patients with SRNS revealed the identity of 62 proteins, which included apolipoproteins, complement components, and serum amyloid P-component (SAP). Comparative analysis of the column-bound proteins between responders and non-responders by 2-DE demonstrated that apolipoprotein E (APOE) and SAP levels were increased in non-responders as compared with responders. These results were confirmed by western blotting. Moreover, serum levels of APOE and SAP were significantly higher in the non-responder group than in the responder group by ELISA. Our data provide comprehensive analysis of proteins adsorbed by LDL-A in SRNS, and demonstrate that the serum levels of APOE and SAP may be used to predict the response to LDL-A in these patients.