Expression Of Renal Transporters Research Articles

Cold Ischemia, Renal Transplantation and Tacrolimus Significantly Alter the Gene Expression of Renal Transporters in Rats.

Cold Ischemia, Renal Transplantation and Tacrolimus Significantly Alter the Gene Expression of Renal Transporters in Rats.

Effect of early life Ca and P restriction on renal transporters gene expression in post weaning piglets

The objective of this study was to evaluate the effect of Ca and P restriction during the post‐weaning period in pigs. Piglets (n=5/treatment) were assigned to one of 4 treatments: Control (100% NRC requirements), or 7‐d, 14‐d or 21‐d postweaning Ca and P restriction (33% of NRC requirements). After restriction periods, piglets were fed 100% of NRC requirements. Performance was measured until d 35, at which time kidney gene expression of renal transporters was measured using qPCR relative to the control group. Performance did not differ between treatments. However, during d 21–35 the gain‐to‐feed ratio was greater (P < 0.05) in piglets restricted for 21 d (0.692 G:F) or 14 d (0.672 G:F) compared with 7 d (0.583 G:F). Relative mRNA level of the solute carrier family 34 member 1(SLC34A1) was up‐regulated (P < 0.05) in piglets restricted for 21 d (1.16‐fold) or 14 d (1.18‐fold), consistent with improved gain‐to‐feed ratio and indicating improved active phosphate transport into cells. The solute carrier family 8 member 1 (SLC8A1) was down‐regulated (P < 0.05) in piglets restricted for 7 d (−1.78‐fold), demonstrating a long‐term effect of early life nutrition on Ca metabolism. No differences were observed in ATP2B1, SLC22A and SLC34A2 mRNA levels. These results suggest performance and renal capacity benefits in response to 14 to 21 d post‐weaning Ca and P restriction.Grant Funding Source: Alltech, Inc.

WNK-OSR1/SPAK-NCC signal cascade has circadian rhythm dependent on aldosterone

Blood pressure and renal salt excretion show circadian rhythms. Recently, it has been clarified that clock genes regulate circadian rhythms of renal transporter expression in the kidney. Since we discovered the WNK-OSR1/SPAK-NaCl cotransporter (NCC) signal cascade, which is important for regulating salt balance and blood pressure, we have sought to determine whether NCC protein expression or phosphorylation shows diurnal rhythms in the mouse kidneys. Male C57BL/6J mice were sacrificed every 4h (at 20:00, 0:00, 4:00, 8:00, 12:00, and 16:00), and the expression and phosphorylation of WNK4, OSR1, SPAK, and NCC were determined by immunoblot. (Lights were turned on at 8:00, which was the start of the rest period, and turned off at 20:00, which was the start of the active period, since mice are nocturnal.) Although expression levels of each protein did not show diurnal rhythm, the phosphorylation levels of OSR1, SPAK, and NCC were increased around the start of the active period and decreased around the start of the rest period. Oral administration of eplerenone (10mg/day) attenuated the phosphorylation levels of these proteins and also diminished the diurnal rhythm of NCC phosphorylation. Thus, the activity of the WNK4-OSR1/SPAK-NCC cascade was shown to have a diurnal rhythm in the kidney that may be governed by aldosterone.

Endocrine and metabolic regulation of renal drug transporters

Renal xenobiotic transporters are important determinants of urinary secretion and reabsorption of chemicals. In addition to glomerular filtration, these processes are key to the overall renal clearance of a diverse array of drugs and toxins. Alterations in kidney transporter levels and function can influence the efficacy and toxicity of chemicals. Studies in experimental animals have revealed distinct patterns of renal transporter expression in response to sex hormones, pregnancy, and growth hormone. Likewise, a number of disease states including diabetes, obesity, and cholestasis alter the expression of kidney transporters. The goal of this review is to provide an overview of the major xenobiotic transporters expressed in the kidneys and an understanding of metabolic conditions and hormonal factors that regulate their expression and function.

Severe diabetes and leptin resistance cause differential hepatic and renal transporter expression in mice

BackgroundType-2 Diabetes is a major health concern in the United States and other Westernized countries, with prevalence increasing yearly. There is a need to better model and predict adverse drug reactions, drug-induced liver injury, and drug efficacy in this population. Because transporters significantly contribute to drug clearance and disposition, it is highly significant to determine whether a severe diabetes phenotype alters drug transporter expression, and whether diabetic mouse models have altered disposition of acetaminophen (APAP) metabolites.ResultsTransporter mRNA and protein expression were quantified in livers and kidneys of adult C57BKS and db/db mice, which have a severe diabetes phenotype due to a lack of a functional leptin receptor. The urinary excretion of acetaminophen-glucuronide, a substrate for multidrug resistance-associated proteins transporters was also determined. The mRNA expression of major uptake transporters, such as organic anion transporting polypeptide Slco1a1 in liver and kidney, 1a4 in liver, and Slc22a7 in kidney was decreased in db/db mice. In contrast, Abcc3 and 4 mRNA and protein expression was more than 2 fold higher in db/db male mouse livers as compared to C57BKS controls. Urine levels of APAP-glucuronide, -sulfate, and N-acetyl cysteine metabolites were higher in db/db mice.ConclusionA severe diabetes phenotype/presentation significantly altered drug transporter expression in liver and kidney, which corresponded with urinary APAP metabolite levels.

Renal efflux transporter expression in pregnant mice with Type I diabetes

Prior research suggests that sex hormones and metabolic changes, such as obesity and hyperglycemia, can alter renal transporter expression in rodents. The purpose of this study was to characterize the expression of kidney efflux transporters and regulatory transcription factors in response to Type I diabetes and pregnancy. Female C57BL/6 mice were treated with multiple low doses of streptozotocin (STZ) to induce hyperglycemia and then mated with normoglycemic male mice. Transporter mRNA and protein expression were quantified in kidneys from vehicle- and STZ-treated non-pregnant and pregnant mice on gestation day 14. Pregnancy decreased the expression of Mdr1b, Mrp4, and 5 proteins and increased the mRNA and protein expression of Mrp3 by 50–60%. STZ treatment elevated Mrp1, 2, 4, and 5 and reduced Mrp3, 6, and Mdr1b mRNA and/or protein in non-pregnant mice. Pregnancy had little effect on STZ-mediated changes in renal efflux transporter expression. Transcriptional profiles of Hnf1α, PXR, AhR, and Nrf2 were altered in patterns similar to some efflux transporters suggesting potential involvement in their regulation. Taken together, these results suggest that renal drug efflux transporters and regulatory signaling pathways are altered by endocrine and metabolic changes that occur during pregnancy and Type I diabetes.

Renal drug transporter expression in pregnant mice with type 1 diabetes

Sex hormones and metabolic changes (such as obesity and hyperglycemia) can alter renal transporter expression in rodents. The purpose of this study was to investigate renal transporter expression in response to pregnancy and type 1 diabetes. Female mice were treated with vehicle or the diabetogenic drug, streptozotocin (STZ) 2 weeks prior to mating. Renal transporter mRNA and protein expression were evaluated in vehicle‐ and STZ‐treated pregnant and virgin mice. The majority of renal transporters were unaffected by pregnancy in vehicle‐treated mice, with only modest 40–80% increases in Mrp3, Oatp2b1, Pept1, and Cnt1 mRNA. In contrast, renal transporters were markedly changed in STZ‐treated virgin mice with decreased Oat2, 5, and Mrp3 mRNA and elevated Oatp2b1, 4c1, Mrp2, 4, 5, and Pept1 mRNA. In combination, STZ treatment and pregnancy downregulated Oatp4c1, Mdr1b, and Mrp3 mRNA. Transcriptional profiling suggests Hnf1α, Nrf2, PXR, AhR, and PPARα may be involved in regulating renal transporter expression. In conclusion, renal drug transport pathways are altered by hormone changes that occur during pregnancy and diabetes. Supported by ES‐007148, ES‐005022, DK‐080774, ES‐020522.

Alteration of Hepatic but Not Renal Transporter Expression in Diet-Induced Obese Mice

Drug pharmacokinetics can be altered in obese and diabetic subjects. In consideration of the prevalence of obesity and diabetes, characterization of transporter expression in mouse models of diabetes and obesity may be a useful tool to aid in prediction of altered drug pharmacokinetics or adverse drug reactions. It has been reported that ob/ob mice, which display a severe obesity and diabetes phenotype, exhibit multiple changes in drug transporter expression in liver and kidney. In the present study, the mRNA and protein expression of major drug transporters was determined in livers and kidneys of diet-induced obese (DIO) C57BL/6J male mice. The mice were fed a high-fat diet (HFD) (60% fat) from 6 weeks of age and display obesity, fatty liver, and mild hyperglycemia. The HFD diet increased expression of multidrug resistance-associated proteins Abcc3 and 4 mRNA and protein in liver by 3.4- and 1.4-fold, respectively, compared with that detected in control mice fed a low-fat diet (LFD). In contrast, Abcc1 mRNA and protein decreased by 50% in livers of DIO mice compared with those in livers to lean mice. The HFD did not alter transporter expression in kidney compared with the LFD. In summary, unlike ob/ob and db/db mice, DIO mice exhibited a selective induction of efflux transporter expression in liver (i.e., Abcc3 and 4). In addition, diet-induced obesity affects transporter expression in liver but not kidney in the C57BL/6J mouse model. These data indicate that hepatic transporter expression is only slightly altered in a model of mild diabetes and nonalcoholic fatty liver disease and obesity.

Renal Function of Rats with Isoproternol‐induced Cardiac Hypertrophy

AimAvaliate the effect of Isoproterenol‐induced cardiac hypertrophy on renal function and proteic expression in rats.MethodsMale Wistar rats were submitted to subcutaneous injection of isoproterenol (ISO) (0.3 mg.kg(‐1).day(‐1) sc) or vehicle for 7 days. Renal plasmatic flow (RPF) and glomerular filtration rate (GFR) were determined by para aminohypurate and inulin clearance. The excretion of ions was also analyzed. Cardiac hypertrophy and organs congestion were evaluated by respective organs weights. The proteic expression of many renal transporters and Angiotensin II receptors will be analysed by Western Blot technique.Results and DiscussionChronic β‐adrenergic stimulation does not affect hemodynamic parameters, despite cardiac hypertrophy. Our results showed that ISO‐treatment does not affect RPF (control: 4,19 ± 0,45; n = 12 versus ISO: 4,17 ± 0,36; n = 11), which is consistent with a normal cardiac output found in this model. However, there was a significant decrease of GFR (control: 2,24 ± 0,22; n = 12 versus ISO: 1,49 ± 0,13; n = 11 * p< 0.01), which could be related to inflammatory process, leading to alteration in ultratiltration coefficient (Kf). Urinary Na+ and K+ were significantly reduced and enhanced, respectively. These changes in excretion of ions could be related to alterations in expression of renal transporters and are under investigation.Research Support: Fapesp

The role of kidney on sodium balance in AngiotensinII‐induced hypertension.

AimInvestigate the renal function and modulation of epithelial transporters of rats with angiotensin‐induced hypertension.MethodsNormal 8‐week‐old male Wistar rats were divided in two groups: control (sham) or treated with 200 ng/kg/min of angiotensin II (ANG II) for 28 days, using osmotic minipumps. Blood pressure (BP) was monitored weekly by tail cuff plethysmography. Renal plasmatic flow (RPF) and glomerular filtration rate (GFR) were determined by para‐aminohippurate sodium and inulin clearance, respectively. The excretion of ions was analyzed and proteic expression of renal transporters or ANG II receptors will be analyzed by Western Blot technique.Results and DiscussionOur preliminary results animals showed that the dose of ANG II used is sufficient to induce hypertension [110 ± 1,6 (sham) n = 4 versus 158 ± 1,7 (ANG II) mmHg n = 4 *p<0,001]. Urinary Na+ and K+ were reduced in hypertensive rats. RPF and GFR were also reduced [RPF = 13,29 ± 0,8 (sham) n = 14 versus 6,48 ± 0,44 (ANG II) ml/min/kg n = 14 *p<0,001] and [GFR = 8,4 ± 0,52 (sham) n=14 versus 4,73 ± 0,52 (ANG II) ml/min/Kg n = 14 *p<0,001], suggesting changes in renal hemodynamics and tubular function. However, epithelial transporters will be analyzed in the renal cortex and medulla. Financial Support: CNPq and FAPESP

Hemodynamic Parameters During Normal and Hypertensive Pregnancy in Rats: Evaluation of Renal Salt and Water Transporters

Objective: To determine whether alterations in extracellular volume expansion observed during normal and hypertensive pregnancy run in parallel to changes in the mRNA expression of renal transporters. Methods: Wistar rats were divided into four groups: control (C, n = 5); pregnancy (P, n = 5); Nω-nitro-l-arginine methyl ester (L-NAME; 50 mg/kg/d)-treated control (H, n = 6); and pregnant rats (HP, n = 6). Hemodynamic studies were performed on day 14 of pregnancy, at which time we also analyzed of the sodium transporters (NHE3, Na/K/2Cl and Na/Cl), potassium channel (ROMK2) and water channel (AQP2). Results: As expected, P rats presented high cardiac output (CO) and normal blood pressure (BP), whereas H rats presented lower CO and elevated BP. A significant (threefold) increase in total vascular resistance and a decrease in stroke volume were observed in the HP group. Hypertension resulting from nitric oxide (NO) synthesis inhibition blunted systemic hemodynamic adaptations during pregnancy. Compared with C rats, mRNA expression of ROMK2 in P rats was lower, whereas that of AQP2 was higher. Expression of AQP2 was significantly higher in H than in C or HP groups. Expression of BSC and NHE3 was lower in the HP than in the P group. The NO inhibition also provoked renal transporter alterations in HP. Conclusions: Our results suggest that tubule transporter variants may mediate the hemodynamic adaptations seen during pregnancy, although we cannot rule out the hypothesis that other factors are also mediating hemodynamic changes.

Reversal of renal tubule transporter down-regulation during severe leptospirosis with antimicrobial therapy

Tubular dysfunction is a hallmark of severe leptospirosis. Antimicrobial therapy is thought to interfere on renal involvement. We evaluated the expression of a proximal tubule type- 3N a + /H + exchanger (NHE3) and a thick ascending limb Na + -K + -2Clco-transporter (NKCC2) in controls and treated hamsters. Animals infected by a serovar Copenhageni isolate, were treated or not with ampicillin (AMP) and/or N-acetylcysteine (NAC). Leptospiral antigen(s) and expression of renal transporters were evaluated by immunohistochemistry, and serum thiobarbituric acid (TBARS) was quantified. Infected hamsters had high amounts of detectable leptospiral antigen(s) in target tissues while renal expression of NHE3 and NKCC2 decreased. Ampicillin treatment was associated with minimal or no detection of leptospiral antigens, normal expression of NHE3 and NKCC2 transporters, and reduced levels of TBARS. NAC effect was restricted to lowering TBARS. Early and late AMP treatment rescued tubular defects in severe leptospirosis disease, and there was no evidence of benefit from antioxidant therapy.

Reversal of Renal Tubule Transporter Downregulation during Severe Leptospirosis with Antimicrobial Therapy

Tubular dysfunction is a hallmark of severe leptospirosis. Antimicrobial therapy is thought to interfere on renal involvement. We evaluated the expression of a proximal tubule type-3 Na+/H+ exchanger (NHE3) and a thick ascending limb Na+-K+-2Cl(-) cotransporter (NKCC2) in controls and treated hamsters. Animals infected by a serovar Copenhageni isolate, were treated or not with ampicillin (AMP) and/or N-acetylcysteine (NAC). Leptospiral antigen(s) and expression of renal transporters were evaluated by immunohistochemistry, and serum thiobarbituric acid (TBARS) was quantified. Infected hamsters had high amounts of detectable leptospiral antigen(s) in target tissues while renal expression of NHE3 and NKCC2 decreased. Ampicillin treatment was associated with minimal or no detection of leptospiral antigens, normal expression of NHE3 and NKCC2 transporters, and reduced levels of TBARS. NAC effect was restricted to lowering TBARS. Early and late AMP treatment rescued tubular defects in severe leptospirosis disease, and there was no evidence of benefit from antioxidant therapy.

The Na +/PO 4 cotransporter SLC20A1 gene labels distinct restricted subdomains of the developing pronephros in Xenopus and zebrafish embryos

The embryonic pronephric kidneys of Xenopus and zebrafish serve as models to study vertebrate nephrogenesis. Recently, multiple subdomains within the Xenopus pronephros have been defined based on the expression of several transport proteins. In contrast, very few studies on the expression of renal transporters have been conducted in zebrafish. We have recently shown that the anterior and posterior segments of the zebrafish pronephric duct may correspond to the proximal tubule and distal tubule/duct compartments of the Xenopus and higher vertebrate pronephros, respectively. Here, we report the embryonic expression pattern of the Na +/PO 4 cotransporter SLC20A1 ( PiT1/Glvr-1) gene encoding a type III sodium-dependent phosphate cotransporter in Xenopus and zebrafish. In Xenopus, SLC20A1 mRNA is expressed in the somitic mesoderm and lower level of expression is detected in the neural tube, eye, and neural crest cells. From stage 25, SLC20A1 is also detectable in the developing pronephros where expression is restricted to the late portion of the distal pronephric tubules. In zebrafish, SLC20A1 is transcribed from mid-somitogenesis in the anterior part of the pronephros where its expression corresponds to the rostral portion of the expression of other proximal tubule-specific markers. Outside the pronephros, lower level of SLC20A1 expression is also observed in the posterior cardinal and caudal veins. Based on the SLC20A1 expression domain and that of other transporters, four segments have been defined within the zebrafish pronephros. Together, our data reveal that the zebrafish and Xenopus pronephros have non-identical proximo-distal organizations.

Expression of rat thick limb Na/H exchangers in potassium depletion and chronic metabolic acidosis

Regulation of renal transporter expression has been shown to support adaptation of transporter activities in several chronic situations. Basolateral and apical Na/H exchangers (NHE) in medullary thick ascending limb (MTAL) are involved in NH4+ and HCO3+ absorption, respectively. The NH4+ absorption rate in Henle's loop is increased in chronic metabolic acidosis (CMA) and potassium depletion (KD), which may be secondary to the increased NH4+ concentration in luminal fluid and/or to an increased NH4+ absorptive capacity of MTAL. HCO3- absorptive capacity in Henle's loop is increased in CMA and decreased in metabolic alkalosis, but is unchanged in KD despite the presence of metabolic alkalosis. The present study compared the effects of NH4Cl-induced CMA and KD on the expression of basolateral NHE-1 and the effect of KD on the expression of apical NHE-3 in MTAL. NHE-1 and NHE-3 mRNAs and proteins were assessed by a competitive reverse transcription-polymerase chain reaction (RT-PCR) method and semiquantitative immunoblots, respectively, in MTAL-purified suspensions from rats with CMA and KD. NHE-1 protein abundance was similarly increased (approximately 90%) at two and five weeks of KD, while NHE-1 mRNA amount in MTAL cells was increased at two weeks of KD and returned to normal values by five weeks of KD. In contrast, NHE-1 mRNA and protein abundance did not change in CMA. NHE-3 protein abundance remained unchanged in both two and five weeks of KD, while NHE-3 mRNA was unchanged by two weeks of KD and reduced by approximately 50% at five weeks of KD. The results suggest the following: (1) in KD, where the increased NH4+ concentration of luminal fluid that favors NH4+ absorption is counterbalanced by a decrease in BSC1 expression and activity, the increased NHE-1 expression may support an increased MTAL NH4+ absorptive capacity in CMA, NHE-1 expression is not specifically regulated and remains unchanged, suggesting that the increase in NH4+ concentration in luminal fluid is the main determinant of increased NH4+ absorption in MTAL. (2) In KD, NHE-3 expression did not decrease despite the presence of metabolic alkalosis, in agreement with the unchanged HCO3- absorptive capacity of Henle's loop.