Impaired insulin signaling affects renal organic anion transporter 3 (Oat3) function in streptozotocin-induced diabetic rats.

Organic anion transporter 3 (Oat3) is a major renal Oats expressed in the basolateral membrane of renal proximal tubule cells. We have recently reported decreases in renal Oat3 function and expression in diabetic rats and these changes were recovered after insulin treatment for four weeks. However, the mechanisms by which insulin restored these changes have not been elucidated. In this study, we hypothesized that insulin signaling mediators might play a crucial role in the regulation of renal Oat3 function. Experimental diabetic rats were induced by a single intraperitoneal injection of streptozotocin (65 mg/kg). One week after injection, animals showing blood glucose above 250 mg/dL were considered to be diabetic and used for the experiment in which insulin-treated diabetic rats were subcutaneously injected daily with insulin for four weeks. Estrone sulfate (ES) uptake into renal cortical slices was examined to reflect the renal Oat3 function. The results showed that pre-incubation with insulin for 30 min (short term) stimulated [3H]ES uptake into the renal cortical slices of normal control rats. In the untreated diabetic rats, pre-incubation with insulin for 30 min failed to stimulate renal Oat3 activity. The unresponsiveness of renal Oat3 activity to insulin in the untreated diabetic rats suggests the impairment of insulin signaling. Indeed, pre-incubation with phosphoinositide 3-kinase (PI3K) and protein kinase C zeta (PKCζ) inhibitors inhibited insulin-stimulated renal Oat3 activity. In addition, the expressions of PI3K, Akt and PKCζ in the renal cortex of diabetic rats were markedly decreased. Prolonged insulin treatment in diabetic rats restored these alterations toward normal levels. Our data suggest that the decreases in both function and expression of renal Oat3 in diabetes are associated with an impairment of renal insulin-induced Akt/PKB activation through PI3K/PKCζ/Akt/PKB signaling pathway

Pinocembrin Attenuates Gentamicin-induced Nephrotoxicity in Rats

Oxidative stress-mediated apoptosis of renal tubular cells is a major pathology of gentamicin-induced nephrotoxicity which is one of the prevailing causes of acute renal failure. Pinocembrin is a major flavonoid found in rhizomes of fingerroot (Boesenbergia pandurata). It has pharmacological and biological activities including antimicrobial, anti-inflammatory, and antioxidant effects. Preclinical studies have suggested that pinocembrin protects rat brain and heart against oxidation and apoptosis induced by ischemia-reperfusion model. The aim of the current study was to investigate the mechanisms of renoprotection elicited by pinocembrin in gentamicin-induced nephrotoxicity. Nephrotoxic rats were induced by intraperitoneal injection (i.p) of gentamicin and pinocembrin was administered via i.p. 30 min before gentamicin treatment for 10 days. Gentamicin-induced nephrotoxicity was indicated by the reduced renal function and renal Oat3 function and expression. Gentamicin treatment also stimulated Nrf2, HO-1, and NQO1 and the pro-apoptotic protein, Bax and caspase-3, concomitant with the attenuation of Bcl-XL expressions in the renal cortical tissues. Pinocembrin pretreatment improved renal function, renal Oat3 function, reduced oxidative stress and apoptotic conditions. These findings indicate that pinocembrin has a protective effect against gentamicin-induced nephrotoxicity which may be due in part to its antioxidant and anti-apoptotic effects, subsequently leading to improved renal function.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Amelioration of Renal Inflammation, Endoplasmic Reticulum Stress and Apoptosis Underlies the Protective Effect of Low Dosage of Atorvastatin in Gentamicin-Induced Nephrotoxicity.

Gentamicin is a commonly used aminoglycoside antibiotic. However, its therapeutic use is limited by its nephrotoxicity. The mechanisms of gentamicin-induced nephrotoxicity are principally from renal inflammation and oxidative stress. Since atorvastatin, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, exerts lipid-lowering effects, antioxidant, anti-inflammatory as well as anti-apoptotic effects, this study aimed to investigate the protective effects of atorvastatin against gentamicin-induced nephrotoxicity. Male Sprague Dawley rats were used and nephrotoxicity was induced by intraperitoneal injection of gentamicin, 100 mg/kg/day, for 15 days. Atorvastatin, 10 mg/kg/day, was administered by orally gavage 30 min before gentamicin injection on day 1 to 15 (pretreatment) or on day 10 to15 (delayed treatment). For only atorvastatin treatment group, it was given on day 1 to 15. At the end of the experiment, kidney weight, blood urea nitrogen and serum creatinine as well as renal inflammation (NF-κB, TNFαR1, IL-6 and iNOS), renal fibrosis (TGFβ1), ER stress (calpain, GRP78, CHOP, and caspase 12) and apoptotic markers (cleaved caspase-3, Bax, and Bcl-2) as well as TUNEL assay were determined. Gentamicin-induced nephrotoxicity was confirmed by marked elevations in serum urea and creatinine, kidney hypertrophy, renal inflammation, fibrosis, ER stress and apoptosis and attenuation of creatinine clearance. Atorvastatin pre and delayed treatment significantly improved renal function and decreased renal NF-κB, TNFαR1, IL-6, iNOS and TGFβ1 expressions. They also attenuated calpain, GRP78, CHOP, caspase 12, Bax, and increased Bcl-2 expressions in gentamicin-treated rat. These results indicate that atorvastatin treatment could attenuate gentamicin-induced nephrotoxicity in rats, substantiated by the reduction of inflammation, ER stress and apoptosis. The effect of atorvastatin in protecting from renal damage induced by gentamicin seems to be more effective when it beginning given along with gentamicin or pretreatment

Effect of PKCζ inhibitor on ES uptake in renal cortical slices.

<p>Tissue slices prepared from rat renal cortex were pre-incubated under different experimental conditions as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096236#s2" target="_blank">Materials and Methods</a> section. Following pre-incubation, the slices were incubated for 30 min in 50 nM [³H]ES. Values are expressed as means ± SEM from five animals per group (6 slices/animal). **p<0.01, compared to control; ^††p<0.01, compared to insulin alone.</p

Effects of diabetes on basal and short term insulin-stimulated ES uptake in renal cortical slices.

<p>Renal cortical slices from control, diabetic (DM) and diabetic with insulin-treated rats (DM-treated) were pre-incubated for 30 min in buffer alone (C), or buffer containing 30 µg/mL insulin (In) at room temperature. After pre-incubation, renal cortical slices were incubated in buffer containing 50 nM [³H]ES for 30 min. Values are expressed as means of T/M ± SEM. from five animals (5 slices/experimental group/animal). ^††<i>p</i><0.01, compared to the corresponding control; **<i>p</i><0.01, compared to the control (non DM rats); ^##<i>p</i><0.01, compared to the diabetic rats.</p

Effects of diabetes on Akt and phospho-Akt expressions in the renal cortex.

<p>(A) and (C); Western blot analysis for Akt and phospho-Akt in the membrane and cytosolic fractions of renal cortical tissues in control, diabetic (DM) and diabetic with insulin-treated (DM-treated) rats, respectively. (B) and (D); The signal intensity of Akt and phospho-Akt in the membrane and cytosolic fractions normalized to β<i>-</i>actin, respectively. Na⁺-K⁺-ATPase and β-actin expressions were used as a membrane marker and loading control, respectively. Bar graphs indicate means ± SEM from five independent experiments. **<i>p</i><0.01, compared to control; ^##<i>p</i><0.01, compared to diabetes.</p

Effect of diabetes on Oat3 expression in the renal cortex.

<p>(A) and (C); Western blot analysis for Oat3 in the membrane and whole cell lysate fractions of renal cortical tissues in control, diabetic (DM) and diabetic with insulin-treated (DM-treated) rats, respectively. (B) and (D); The signal intensity of Oat3 in membrane and whole cell lysate fractions normalized to β-actin, respectively. Na⁺-K⁺-ATPase and β-actin expressions were used as a membrane marker and loading control, respectively. Bar graphs indicate means ± SEM from five independent experiments. **<i>p</i><0.01, compared to control; ^##<i>p</i><0.01, compared to diabetes.</p

Effects of diabetes on PKCζ and phospho-PKCζ expressions in the renal cortex.

<p>(A) and (C); Western blot analysis for PKCζ and phospho-PKCζ in the membrane and cytosolic fractions of renal cortical tissues in control, diabetic (DM) and diabetic with insulin-treated (DM-treated) rats, respectively. (B) and (D); The signal intensity of PKCζ and phospho-PKCζ in the membrane and cytosolic fractions normalized to β<i>-</i>actin, respectively. Na⁺-K⁺-ATPase and β-actin expressions were used as a membrane marker and loading control, respectively. β<i>-</i>actin for the membrane fractions of PKCζ (A) and phospho-PKCζ (C) were obtained from the same membrane/loading protein. Bar graphs indicate means ± SEM from five independent experiments. **<i>p</i><0.01, compared to control; ^##<i>p</i><0.01, compared to diabetes.</p

Effects of diabetes and insulin treatment on physiological and biochemical parameters.

<p>BW: body weight; KW: kidney weight; KW/BW: kidney weight to body weight; PG: plasma glucose; TG: serum triglyceride; MDA: malondialdehyde; DM: diabetic rat; DM-treated: insulin-treated diabetic rat. Values are expressed as means ± SEM of 10 rats/group.</p><p>**<i>p</i><0.01, compared to control; ^##<i>p</i><0.01, compared to diabetic (DM).</p

Effect of Wortmannin on ES uptake in renal cortical slices.

<p>Tissue slices prepared from rat renal cortex were pre-incubated under different experimental conditions as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096236#s2" target="_blank">Materials and Methods</a> section. Following pre-incubation, the slices were incubated for 30 min with 50 nM [³H]ES. Values are expressed as means ± SEM from five animals per group (6 slices/animal). **<i>p</i><0.01, compared to control; ^††<i>p</i><0.01, compared to insulin alone.</p