Renal Cortical Slices Research Articles

Cladophora glomerata Kützing extract exhibits antioxidant, anti-inflammation, and anti-nitrosative stress against impairment of renal organic anion transport in an in vivo study.

Cladophora glomerata extract (CGE), rich in polyphenols, was reported to exhibit antidiabetic and renoprotective effects by modulating the functions of protein kinases-mediated organic anion transporter 1 (Oat1) and 3 (Oat3) in rats with type 2 diabetes mellitus (T2DM). Nevertheless, the antioxidant effects of CGE on such renoprotection have not been investigated. This study examined the mechanisms involved in the antioxidant effects of CGE on renal organic anion transport function in an in vivo study. Diabetes was induced in the rats through a high-fat diet combined with a single dose of 40 mg/kg body weight (BW) streptozotocin. Subsequently, normal-diet rats were supplemented with a vehicle or 1,000 mg/kg BW of CGE, while T2DM rats were supplemented with a vehicle, CGE, or 200 mg/kg BW of vitamin C for 12 weeks. The study evaluated the general characteristics of T2DM and renal oxidative stress markers. The renal organic transport function was assessed by measuring the para-aminohippurate (PAH) uptake using renal cortical slices and renal inflammatory cytokine expression in the normal diet (ND) and ND + CGE treated groups. CGE supplementation significantly reduced hyperglycemia, hypertriglyceridemia, insulin resistance, and renal lipid peroxidation in T2DM rats. This was accompanied by the normalization of high expressions of renal glutathione peroxidase and nuclear factor kappa B by CGE and vitamin C. The renal anti-inflammation of CGE was evidenced by the reduction of tumor necrosis factor-1α and interleukin-1β. CGE directly blunted sodium nitroprusside-induced renal oxidative/nitrosative stresses and mediated the PAH uptake in the normally treated CGE in rats was particularly noteworthy. These data also correlated with reduced nitric oxide production, highlighting the potential of CGE as a therapeutic agent for managing T2DM-related renal complications. These findings suggest that CGE has antidiabetic effects and directly prevents diabetic nephropathy through oxidative/nitrosative stress pathways.

Inhibitory effects of indoxyl sulfate and creatinine on the renal transport of meropenem and biapenem in rats

Carbapenem antibiotics are excreted preferentially in the urine after intravenous administration, with organic anion transporters (OATs) known to be involved in the renal tubular secretion of carbapenem antibiotics. Various uremic toxins (UTs) accumulate in the blood of patients with end-stage renal failure, and some UTs such as indoxyl sulfate (IS) and creatinine (Cr) are excreted in the urine via OATs. However, information about the possible interactions between these UTs and carbapenems in the renal secretion remains limited. In this study, we investigated the effects of IS and Cr on the renal transport of anionic meropenem and zwitterionic biapenem by using rat renal cortical slices. The uptake of meropenem and biapenem in the renal cortical slices was significantly decreased in the presence of 0.1 mM IS or 1 mM Cr. When biapenem and Cr were co-administered to rats intravenously, biapenem clearance from the plasma was clearly retarded, reflecting the current in vitro results. However, IS and Cr exerted no inhibitory effect on the uptake of metformin, a substrate of renal organic cation transporter (OCT) 2, in the renal cortical slices. Thus, our findings indicate that IS and Cr interfere with the renal secretion of carbapenem antibiotics by preferentially inhibiting OATs.

Abstract 16703: Regulation of Cell Proliferation in Renal Injury by Dopamine D2 Receptors

Introduction: Genetic factors contribute to the susceptibility to chronic renal disease associated with essential hypertension. The renal dopamine D2 receptor (D2R) has a significant role in blood pressure (BP) control and in negative regulation of the development of inflammation and injury. Several common single nucleotide polymorphisms of the human DRD2 gene are associated with decreased D2R expression and function. Impaired D2R function results in renal inflammation and end-organ damage. Injury to the kidney triggers a cell proliferation response that can be either adaptive, leading to repair of the tubular epithelium, or maladaptive, leading to fibrosis and chronic kidney disease. Hypothesis: We tested the role of renal D2Rs in the cell proliferation response Methods: We used models of renal injury in mice and human renal proximal tubule cells (hRPTCs) Results: Renal selective silencing of the D2R in mice resulted in an increase in the mRNA expression of the proliferation marker Ki-67 (2.8±0.8 vs 1.0±0.1 fold; qRT-PCR; P<0.05; n=4-5), along with an increase in the number of proliferating cells in renal cortical slices determined by nuclear Ki-67 staining (36±3 vs 6±1; positive cells/field; P<0.05), in comparison with mice with no silencing. Renal tubule-selective rescue of D2R function in these mice by the ureteral infusion of AAV carrying D2R reduced the expression of cortical Ki-67 mRNA (42±3 vs 100±5 %; P<0.05; n=4) and the number of positive cells/field (16±2 vs 32±5; P<0.05), in comparison with mice treated with a control AAV. In mice subjected to renal ischemia reperfusion Ki-67 mRNA expression was higher (1.5±0.03 vs 1.0±0.05 fold; P<0.05; n=3-4) than in mice with sham operation. Renal tubule treatment with AAV carrying D2R after ischemic injury reduced Ki-67 expression (0.65±0.05 vs 1±0.1; P<0.05) and the number of Ki-67 positive cells in kidney sections. Rescue of function or renal tubule-selective D2R overexpression reduced tissue damage, fibrosis and the increase in BP in these models. Treatment of hRPTCs with the nephrotoxic, aristolochic acid (5 μg/ml, 24h), increased mRNA Ki-67 (2.8±0.2 fold; P<0.05; n=4-5) in comparison with cells treated with vehicle while the presence of a D2R agonist, quinpirole (1μM, 24h), in the medium reduced the increase by 42% and also reduced the increase in fibronectin 1 and collagen 1a1 elicited by aristolochic acid Conclusions: Our data suggest that regulation of cell proliferation is one of the mechanisms involved in the protective effect of D2R function on renal inflammation and injury.

Exhaustive exercise decreases renal organic anion transporter 3 function.

This study aimed to investigate the effects of various types of exercise on organic anion transporter 3 (Oat3) function, a major transporter that plays a role in the secretion of a variety of drugs and endogenous compounds. Male Wistar rats were randomly allocated to non-exercise, exhaustive, acute and training exercise groups. The function of Oat3 was assessed by the uptake of [3H]-estrone sulfate ([3H]-ES) into rat renal cortical slices. Acute and training exercises had no effect on [3H]-ES uptake whereas a marked reduction in [3H]-ES uptake occurred immediately after exhaustive exercise. However, the reduction in Oat3 function was gradually recovered at 6 and 24h after the exercise session. Importantly, the impairment of Oat3 function was associated with a decrease in renal Oat3 protein expression. Our results indicate that exhaustive exercise produces a significant impact on renal organic anion transport function, which in turn could alter the plasma level of drugs and compounds in the body.

Abstract 052: Role of Dopamine D2 Receptors in The Regulation of Cell Proliferation in Renal Injury

The dopamine D2 receptor (D2R) in the kidney has a direct and significant role in blood pressure (BP) control and in negatively regulating the mechanisms involved in the development of inflammation and injury. Impaired D2R function results in renal inflammation and end organ damage. Injury to the kidney triggers a cell proliferation response that can be adaptive, leading to repair of the tubular epithelium, or maladaptive, leading to fibrosis and chronic kidney disease. We investigated the role of renal D2Rs in the cell proliferation response in models of renal injury in mice and human renal proximal tubule cells (hRPTCs). Renal selective silencing of the D2R in mice resulted in an increase in the mRNA expression of the proliferation marker Ki-67 (2.8±0.8 vs 1.0±0.1 fold; qRT-PCR; P<0.05; n=4-5) along with an increase in the number of proliferating cells in renal cortical slices determined by nuclear Ki-67 staining (36±3 vs 6±1; positive cells/ field; P<0.05) in comparison with mice with no silencing. Rescue of D2R function in these mice by the ureteral infusion of AAV carrying a D2R vector reduced the expression of cortical Ki-67 mRNA (42±3 vs 100±5 %; P<0.05; n=4) and the number of positive cells/field (16±2 vs 32±5; P<0.05) in comparison with mice treated with a control AAV. In mice subjected to renal ischemia reperfusion KI-67 mRNA expression was higher (1.5±0.03 vs 1.0±0.05 fold; P<0.05; n=3-4) than in mice with sham operation. Treatment with AAV carrying D2R after ischemic injury reduced Ki-67 expression (0.65±0.05 vs 1±0.1; P<0.05) and the number of Ki-67 positive cells in kidney sections. Rescue of function or D2R overexpression reduced tissue damage, fibrosis and the increase in BP in these models. Treatment of hRPTCs with the nephrotoxic, aristolochic acid (5 μg/ml, 24h), increased mRNA Ki-67 expression (2.8±0.2 fold; P<0.05) in comparison with cells treated with vehicle while the presence in the medium of a D2R agonist, quinpirole (1μM, 24h), reduced the increase by 42%. The D2R agonist also reduced the increase in fibronectin 1 and collagen 1a1 elicited by aristolochic acid. Our data suggest that regulation of cell proliferation is one of the mechanisms involved in the protective effect of D2R function on renal inflammation and injury.

The urinary excretion of metformin, ceftizoxime and ofloxacin in high serum creatinine rats: Can creatinine predict renal tubular elimination?

The renal excretion of creatinine and most drugs are the net result of glomerular filtration and tubular secretion, and their tubular secretions are mediated by individual transporters. Thus, we hypothesized that the increase of serum creatinine (SCr) levels attributing to inhibiting tubular transporters but not glomerular filtration rate (GFR) could be used to evaluate the tubular excretion of drugs mediated by identical or partial overlap transporter with creatinine. In this work, we firstly developed the creatinine excretion inhibition model with normal GFR by competitively inhibiting tubular transporters, and investigated the renal excretion of metformin, ceftizoxime and ofloxacin in vivo and in vitro. The results showed that the 24-hour urinary excretion of metformin and ceftizoxime in model rats were decreased by 25% and 17% compared to that in control rats, respectively. The uptake amount and urinary excretion of metformin and ceftizoxime could be inhibited by creatinine in renal cortical slices and isolated kidney perfusion. However, the urinary excretion of ofloxacin was not affected by high SCr. These results showed that the inhibition of tubular creatinine transporters by high SCr resulted to the decrease of urinary excretion of metformin and ceftizoxime, but not ofloxacin, which implied that the increase of SCr could also be used to evaluate the tubular excretion of drugs mediated by identical or partial overlap transporter with creatinine in normal GFR rats.

Erratum to: Activation of liver X receptor inhibits OCT2-mediated organic cation transport in renal proximal tubular cells.

Liver X receptor (LXR) is transcriptional factor that plays an important role in the regulation of energy metabolism such as cholesterol, lipid, and glucose metabolism as well as membrane transporters and channels. Using both in vitro and in vivo models, LXR regulation of the expression and function of renal organic cation transporter 2 (OCT2) was observed. Synthetic LXR agonist (GW3965) and endogenous LXR agonist (22R-hydroxycholesterol) significantly reduced the uptake of 3H–MPP+, a prototypic substrate of OCT2, in both OCT2- Chinese hamster ovary K1 and human renal proximal tubular cells (RPTEC/TERT1). GW3965 decreased transport activity of OCT2 via a reduction of the maximal transport rate of MPP+ without affecting transporter affinity. The inhibitory effect of GW3965 was attenuated by co-treatment with LXR antagonist (fenofibrate) indicating the inhibition was LXR-dependent mechanism. In addition, co-treatment with a retinoic X receptor (RXR) ligand, 9-cis retinoic acid enhanced the inhibitory effect of GW3965, indicating negative regulation of OCT2 transport activity by the LXR/RXR complex. Treatment RPTEC/TERT1 cells with GW3965 significantly reduced OCT2 protein expression without changing mRNA expression. In parallel, the effect of LXR activation on OCT2 function was investigated in intact mouse kidney. Treating mice with 50 mg/kg BW T0901317 for 14 days significantly decreased 3H–MPP+ uptake into renal cortical slices, correlating with decreased OCT2 protein expression in renal cortex without changes in mRNA expression levels. Taken together, LXR/RXR activation downregulates the protein expression and function of OCT2 in renal proximal tubule, suggesting LXR might affect the total profile of renal excretion of cationic compounds.

Activation of liver X receptor inhibits OCT2-mediated organic cation transport in renal proximal tubular cells.

Liver X receptor (LXR) is transcriptional factor that plays an important role in the regulation of energy metabolism such as cholesterol, lipid, and glucose metabolism as well as membrane transporters and channels. Using both in vitro and in vivo models, LXR regulation of the expression and function of renal organic cation transporter 2 (OCT2) was observed. Synthetic LXR agonist (GW3965) and endogenous LXR agonist (22R-hydroxycholesterol) significantly reduced the uptake of 3H-MPP+, a prototypic substrate of OCT2, in both OCT2- Chinese hamster ovary K1 and human renal proximal tubular cells (RPTEC/TERT1). GW3965 decreased transport activity of OCT2 via a reduction of the maximal transport rate of MPP+ without affecting transporter affinity. The inhibitory effect of GW3965 was attenuated by co-treatment with LXR antagonist (fenofibrate) indicating the inhibition was LXR-dependent mechanism. In addition, co-treatment with a retinoic X receptor (RXR) ligand, 9-cis retinoic acid enhanced the inhibitory effect of GW3965, indicating negative regulation of OCT2 transport activity by the LXR/RXR complex. Treatment RPTEC/TERT1 cells with GW3965 significantly reduced OCT2 protein expression without changing mRNA expression. In parallel, the effect of LXR activation on OCT2 function was investigated in intact mouse kidney. Treating mice with 50mg/kg BW T0901317 for 14days significantly decreased 3H-MPP+ uptake into renal cortical slices, correlating with decreased OCT2 protein expression in renal cortex without changes in mRNA expression levels. Taken together, LXR/RXR activation downregulates the protein expression and function of OCT2 in renal proximal tubule, suggesting LXR might affect the total profile of renal excretion of cationic compounds.

Gastrin stimulates renal dopamine production by increasing the renal tubular uptake of l-DOPA.

Gastrin is a peptide hormone that is involved in the regulation of sodium balance and blood pressure. Dopamine, which is also involved in the regulation of sodium balance and blood pressure, directly or indirectly interacts with other blood pressure-regulating hormones, including gastrin. This study aimed to determine the mechanisms of the interaction between gastrin and dopamine and tested the hypothesis that gastrin produced in the kidney increases renal dopamine production to keep blood pressure within the normal range. We show that in human and mouse renal proximal tubule cells (hRPTCs and mRPTCs, respectively), gastrin stimulates renal dopamine production by increasing the cellular uptake of l-DOPA via the l-type amino acid transporter (LAT) at the plasma membrane. The uptake of l-DOPA in RPTCs from C57Bl/6J mice is lower than in RPTCs from normotensive humans. l-DOPA uptake in renal cortical slices is also lower in salt-sensitive C57Bl/6J than in salt-resistant BALB/c mice. The deficient renal cortical uptake of l-DOPA in C57Bl/6J mice may be due to decreased LAT-1 activity that is related to its decreased expression at the plasma membrane, relative to BALB/c mice. We also show that renal-selective silencing of Gast by the renal subcapsular injection of Gast siRNA in BALB/c mice decreases renal dopamine production and increases blood pressure. These results highlight the importance of renal gastrin in stimulating renal dopamine production, which may give a new perspective in the prevention and treatment of hypertension.

Protocols and Applications of Cellular Metabolomics in Safety Studies Using Precision-Cut Tissue Slices and Carbon 13 NMR.

Numerous xenobiotics are toxic to human and animal cells by interacting with their metabolism, but the precise metabolic step affected and the biochemical mechanism behind such a toxicity remain often unknown. In an attempt to reduce the ignorance in this field, we have developed a new approach called cellular metabolomics. This approach, developed in vitro, provides a panoramic view not only of the pathways involved in the metabolism of physiological substrates of any normal or pathological human or animal cell but also of the beneficial and adverse effects of xenobiotics on these metabolic pathways. Unlike many cell lines, precision-cut tissue slices, for which there is a renewed interest, remain metabolically differentiated for at least 24-48h and allow to study the effect of xenobiotics during short-term and long-term incubations. Cellular metabolomics (or metabolic flux analysis), which combines enzymatic and carbon 13 NMR measurements with mathematical modeling of metabolic pathways, is illustrated in this brief chapter for studying the effect of insulin on glucose metabolism in rat liver precision-cut slices and of valproate on glutamine metabolism in human renal cortical precision-cut slices. The use of very small amounts of test compounds allows to predict their toxic effect and eventually their beneficial effects very early in the research and development processes. Cellular metabolomics is complementary to other omics approaches, but, unlike them, provides functional, mechanistic, and dynamic pieces of information by measuring enzymatic fluxes.

Riceberry bran extract prevents renal dysfunction and impaired renal organic anion transporter 3 (Oat3) function by modulating the PKC/Nrf2 pathway in gentamicin-induced nephrotoxicity in rats

PurposeThis study investigated the protective effects of Riceberry bran extract (RBBE) on renal function, and the function and expression of renal organic anion transporter 3 (Oat3) in gentamicin-induced nephrotoxicity in rats and explored the mechanisms for its protective effects. Material and methodsMale Sprague Dawley rats (n= 42) were divided into six groups to receive normal saline, gentamicin (100mg/kg), co-treatment of gentamicin and RBBE (at dose of 250, 500 and 1000mg/kg), and RBBE (at dose of 1000mg/kg) only, for consecutive fifteen days. Renal function, oxidative and antioxidative markers, the function and expression of Oat3 and histological changes in the kidney were evaluated. ResultsElevation of BUN, serum creatinine levels and reduction in urine creatinine and creatinine clearance indicated decreased renal function in the gentamicin-treated rats. The decrease of [3H]ES uptake in the renal cortical slices of these rats, reflecting the attenuation of Oat3 transport function that was accompanied by decreased expression of Oat3. Moreover, increased MDA level and reduced superoxide dismutase (SOD) and glutathione (GSH) activities were found in gentamicin-treated rats compared to the control group. These changes were associated with the upregulated PKCα, Nrf-2, Keap 1, NQO-1 and HO-1 expressions in kidneys. RBBE treatment improved the renal function and Oat3 transport function and expression in gentamicin-treated rats. The oxidative status was also restored by RBBE treatment. ConclusionRBBE protects kidney injury by its antioxidant effect, subsequently leading to modulation of the PKC/Nrf2 antioxidant defense pathway.

3,5-dichloronitrobenzene induced nephrotoxicity in vitro.

Dichloroanilines (DCAs) and dichloronitrobenzenes (DCNBs) are important chemical intermediates in the manufacture of a wide variety of industrial, agricultural, and pharmaceutical agents and have been found to induce nephrotoxicity in vivo and/or in vitro. CAs may also be metabolized to CNBs and vice versa. Among the DCNBs, 3,5-dichloronitrobenzene (3,5-DCNB) was the most potent nephrotoxicant in renal cortical slices. The current study was designed to explore the nephrotoxicity of 3,5-DCNB and its possible renal biotransformation using isolated renal cortical cells (IRCC) as the in vitro model. Briefly, IRCC (~4.0 million cells/ml, 3 ml) from male Fisher 344 rats were treated with DMSO (vehicle control) or 3,5-DCNB (0.5 or 1.0 mM), and cytotoxicity determined over 90 min by measuring lactate dehydrogenase release. In some experiments, a pretreatment was added before 3,5-DCNB (1.0 mM) followed by 60 min incubations. The pretreatments were antioxidants, cytochrome P450 (CYP) inhibitors, a cyclooxygenase inhibitor, a peroxidase inhibitor or flavin monooxygenase (FMO) inhibitors. All antioxidants and metabolizing enzyme inhibitor pretreatments completely or partially attenuated 3,5-DCNB cytotoxicity. These results suggest that the bioactivation of 3,5-DCNB is complex, as inhibition of enzyme systems as different as CYPs, FMOs, cyclooxygenase and peroxidases can provide some cytoprotection. It can also be concluded that free radicals play a role in 3,5-DCNB nephrotoxicity either via participating in the in vitro renal biotransformation of 3,5-DCNB or via being produced as a consequence of 3,5-DCNB biotransformation. Additional studies are needed to ascertain which pathways are critical for 3,5-DCNB nephrotoxicity. Supported by NIH Grant P20GM103434.

Atorvastatin improves renal organic anion transporter 3 and renal function in gentamicin-induced nephrotoxicity in rats.

What is the central question of this study? This study was designed to determine the renoprotective effects of atorvastatin treatment in an experimental model of gentamicin-induced nephrotoxicity through modulating the Nrf2 pathway by decreasing the oxidative stress. What is the main finding and its importance? Atorvastatin exerts a nephroprotective effect by attenuating oxidative stress, protecting renal function and renal organic anion transporter 3 function from the effects of gentamicin. Atorvastatin might protect the tissues via its antioxidant property and by modulating the antioxidant enzymes through the Nrf2 signalling pathway, which may be the underlying mechanisms of these protective effects. Recent evidence demonstrates that statins, 3-hydroxy-3-methylglutaryl coenzymeA reductase inhibitors, exert not only lipid-lowering effects but also antioxidant, anti-inflammatory and anti-apoptotic effects. Nephrotoxicity, a serious side-effect of gentamicin, is related to an increase in reactive oxygen species in the kidney. This study was designed to determine the renoprotective effects of atorvastatin treatment in an experimental model of gentamicin-induced nephrotoxicity. Male Sprague-Dawley rats were used. Nephrotocixity was induced by i.p. injection of gentamicin, 100mgkg(-1) day(-1) , for 15days. Atorvastatin, 10mgkg(-1) day(-1) , was administered by gavage 30min before gentamicin injection (pretreatment) for 15days or only on days 10-15 (post-treatment). Renal function and renal organic anion transporter 3 (Oat3) function and expression were examined. Gentamicin-treated rats demonstrated impaired renal function by attenuation of creatinine clearance and increased oxidative stress. Gentamicin treatment also decreased renal Oat3 function and expression as shown by decreased [(3) H]estrone sulfate uptake into renal cortical slices and decreased expression. The protein expressions of protein kinaseC, Nrf2, NAD(P)H:quinone oxidoreductase1, haeme oxygenase 1 and glutamate-cysteine ligase were markedly increased in gentamicin-treated rats, indicating the increase in oxidative stress. Administration of atorvastatin improved renal function and alleviated oxidative stress, and atorvastatin pretreatment had a greater ability to decrease oxidative stress than atorvastatin post-treatment. These effects helped to preserve renal function and Oat3 function and expression. These results indicate that atorvastatin has a renoprotective effect against gentamicin-induced nephrotoxicity by decreasing overoxidation in the kidney, and might be used in conjunction with gentamicin to protect against renal damage.

Interaction of green tea catechins with renal organic cation transporter 2

1. Green tea extract (GTE) and EGCG have previously shown to increase the uptake of MPP+ into Caco-2 cells. However, whether GTE and its derivatives interact with renal basolateral organic cation transporter 2 (Oct2) which plays a crucial role for cationic clearance remains unknown. Thus, this study assessed the potential of drug-green tea (GT) catechins and its derivatives interactions with rat Oct2 using renal cortical slices and S2 stably expressing rat Oct2 (S2rOct2).2. Both GTE and ECG inhibited MPP+ uptake in renal slices in a concentration-dependent manner (IC50 = 2.71 ± 0.360 mg/ml and 0.87 ± 0.151 mM), and this inhibitory effect was reversible. Inhibition of [3H]MPP+ transport in S2rOct2 by either GTE or ECG (IC50 = 1.90 ± 0.087 mg/ml and 1.67 ± 0.088 mM) was also observed.3. The weak and reversible interactions of GTE and ECG with rOct2 indicate that consumption of GT beverages could not interfere with cationic drugs secreted via renal OCT2 in humans. However, the rise of therapeutic use of GTE and ECG might have to take into account the significant possibility of adverse drug–green tea catechins interactions which could alter renal organic cation drug clearance.

16. Controlling cryoprotectant toxicity and chilling injury

Cryoprotectant toxicity and chilling injury are the central problems blocking the full deployment of vitrification, including its use for organ cryopreservation. Controlling these sources of injury is handicapped by the fact that little information is available about the nature of either cryoprotectant toxicity or chilling injury. To date, toxicity has been controlled by following 14 largely empirical rules of toxicity suppression in vitrification, but recently both theory and empirical experiment have begun to point the way to a coming ability to transcend cryoprotectant toxicity and with it, chilling injury as well. One interpretation of a recent gene expression profiling experiment on rat liver slices is that cryoprotectant toxicity in advanced vitrification solutions is related to protein denaturation. This is consistent with independent evidence that toxicity is correlated with stronger water hydrogen bonding by the permeating cryoprotectants in such solutions, which might destabilize the protein hydration layer and thus induce denaturation. Individual cryoprotectants apparently injure cells by specific mechanisms that don’t include denaturation, but when individual agents are diluted and combined to form vitrification solutions of high total concentration, injury may be primarily non-specific and mediated by denaturation linked to reduced water availability. Although worrisome at first, cells have evolved many ways of coping with protein denaturation, which may be augmented to achieve a therapeutic effect. In addition, chilling injury of vitrifiable liver slices was found to mimic cryoprotectant toxicity based on additional gene expression profiling, suggesting that chilling injury during vitrification may arise from the superimposition of cold denaturation on pre-existing protein destabilization by cryoprotectants. This implies that reducing cryoprotectant toxicity will also reduce chilling injury, but other interventions for chilling injury may also be helpful, including interventions that have been aimed at preventing membrane lipid phase transitions in other systems. Interestingly, those interventions include using antifreeze proteins to stabilize the cell membrane, which raises the question of whether other ice-active agents that might be more practically incorporated into vitrification solutions would have similar protective effects in addition to their intended role of interfering with ice formation. Another factor in controlling chilling injury has been tonicity adjustment. It has been reported that setting the nominal osmolality of non-penetrating species in a vitrification solution to 1.2–1.5 times isotonic minimizes chilling injury in rabbit renal cortical slices at all temperatures from −20 °C to −80 °C, below which chilling injury no longer increases. While helpful, this intervention places constraints on the amount of impermeant that can be used in vitrification solutions for the kidney. The mechanism of this effect is unknown, although it has been speculated that hypertonicity prevents injury otherwise caused by thermal contraction of cell membranes.

Antidiabetic and Renoprotective Effects ofCladophora glomerataKützing Extract in Experimental Type 2 Diabetic Rats: A Potential Nutraceutical Product for Diabetic Nephropathy

Cladophora glomerata extract (CGE) has been shown to exhibit antigastric ulcer, anti-inflammatory, analgesic, hypotensive, and antioxidant activities. The present study investigated antidiabetic and renoprotective effects of CGE in type 2 diabetes mellitus (T2DM) rats. The rats were induced by high-fat diet and streptozotocin and supplemented daily with 1 g/kg BW of CGE for 12 weeks. The renal transport function was assessed by the uptake of para-aminohippurate mediated organic anion transporters 1 (Oat1) and 3 (Oat3), using renal cortical slices. These two transporters were known to be upregulated by insulin and PKCζ while they were downregulated by PKCα activation. Compared to T2DM, CGE supplemented rats had significantly improved hyperglycaemia, hypertriglyceridemia, insulin resistance, and renal morphology. The baseline uptake of para-aminohippurate was not different among experimental groups and was correlated with Oat1 and 3 mRNA expressions. Nevertheless, while insulin-stimulated Oat1 and 3 functions in renal slices were blunted in T2DM rats, they were improved by CGE supplementation. The mechanism of CGE-restored insulin-stimulated Oat1 and 3 functions was clearly shown to be associated with upregulated PKCζ and downregulated PKCα expressions and activations. These findings indicate that CGE has antidiabetic effect and suggest it may prevent diabetic nephropathy through PKCs in a T2DM rat model.

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.

Decreased Renal Organic Anion Transporter 3 Expression in Type 1 Diabetic Rats

BackgroundOrganic anion transporter 3 (Oat3) plays an essential role in the renal excretion of organic anions. Reduced renal Oat3 expression potentially contributes to an impaired anion clearance and causes abnormal kidney function. This study examined the effects of diabetes on the expression and function of rat renal Oat3. MethodsExperimental diabetes was induced by the administration of streptozotocin. Diabetic rats were randomly assigned to the treatment group or no treatment group with insulin for 4 weeks. The expression of renal Oat3, protein kinase Cα (PKCα), phospho-PKCα and nuclear factor kappa B (NF-κB) p65 were determined by immunoblotting. Estrone sulfate (Es) uptake into renal cortical slices was used as an indicator of renal Oat3 function. ResultsThe reduced expression of renal Oat3 was related to the decrease in [3H]Es uptake in a renal cortical slice of the diabetic rat. Insulin treatment restored the impairment of renal Oat3 function and expression. These may be because of the hyperglycemia-induced oxidative stress effectively activating the PKCα and NF-κB. Insulin treatment abolished these processes. ConclusionsThese data are the first to show that the decreased function and expression of renal Oat3 in diabetes was associated with an increase in reactive oxygen species production coinciding with the activation of PKC and NF-κB signaling pathway. These events may affect the transporter protein translocation and/or expression.

Resveratrol attenuates cisplatin renal cortical cytotoxicity by modifying oxidative stress

Cisplatin, a cancer chemotherapy drug, is nephrotoxic. The aim of this study was to investigate whether resveratrol (RES) reduced cisplatin cytotoxicity and oxidative stress. Rat renal cortical slices were pre-incubated 30min with 0 (VEH, ethanol) or 30μg/ml RES followed by 60, 90 or 120min co-incubation with 0, 75, or 150μg/ml cisplatin. Lactate dehydrogenase (LDH) leakage was unchanged at 60 and 90min by cisplatin. Cisplatin increased (p<0.05) LDH leakage at 120min which was protected by RES. Cisplatin induced oxidative stress prior to LDH leakage as cisplatin depressed glutathione peroxidase and superoxide dismutase (SOD) activity, increased lipid peroxidation, protein carbonyls and 4-hydroxynonenal (4-HNE) adducted proteins within 60min. RES failed to reverse glutathione (GSH) depression by cisplatin. In order to eliminated an extracellular interaction between RES and cisplatin, additional studies (RINSE studies) allowed a 30min RES uptake into slices, transfer of slices to buffer lacking RES, followed by 120min cisplatin incubation. RES in the RINSE studies prevented LDH leakage by cisplatin indicating that RES protection was not via a physical interaction with cisplatin in the media. These findings indicate that RES diminished cisplatin in vitro renal toxicity and prevented the development of oxidative stress.

Human organic anion transporters function as a high-capacity transporter for p-cresyl sulfate, a uremic toxin

Recent clinical studies have shown that increased serum levels of p-cresyl sulfate (PCS), a uremic toxin, are associated with the progression of chronic kidney disease (CKD) and cardiovascular outcomes. Using rat renal cortical slices, we previously reported that the rat organic anion transporter (OAT) could play a key role in the renal tubular secretion of PCS. However, no information is currently available regarding the transport of PCS via human OAT (hOAT) isoforms, hOAT1 and hOAT3. Uptake experiments of PCS were performed using HEK293 cells, which stably express hOAT1 or hOAT3. PCS was taken up by hOAT1/HEK293 and hOAT3/HEK293 cells in a time- and concentration-dependent manner. The apparent K m for the hOAT1-mediated transport of PCS was 128 μM, whereas in hOAT3/HEK293, saturation was not observed at the highest tested PCS concentration of 5 mM. Probenecid, an OAT inhibitor, inhibited PCS transport by hOAT1 and hOAT3. The uptake of p-aminohippurate by hOAT1 and estron-3-sulfate by hOAT3 was decreased with increasing PCS concentration. The apparent 50 % inhibitory concentrations for PCS were 690 and 485 μM for hOAT1 and hOAT3, respectively. PCS is a substrate for hOAT1 and hOAT3, and hOAT1 and hOAT3 appear to play a physiological role as a high-capacity PCS transporter. Since hOATs are expressed not only in the kidneys, but also in blood vessels and osteoblasts, etc., these findings are of great significance in terms of elucidating the renal clearance, tissue disposition of PCS and the mechanism of its toxicity in CKD.