Rat Proximal Tubule Research Articles

Protective Effect of Vitamin E on Formaldehyde-Induced Injuries in the Rat Kidney

Results: Comparison of the control and formaldehyde groups with the vitamin E group revealed that there were significant differences in lumen size and nuclear color of proximal tubules in rats treated by vitamin E (P < 0.05). Cell degeneration, lumen size, number and size of nucleus of distal tubules were statistically different among the three groups. In the vitamin E treated group observed histological changes were improved compared to the formaldehyde group. Conclusions: According to the results of this study, vitamin E probably improves the harmful effects of formaldehyde on rat kidney tissue.

Abstract 518: Loss of Caveolin-1 in Mouse, Rat and Human Proximal Tubule Cells Increases GRK4 Activity and Decreases D1-Like Receptor Cell Surface Levels

Normal dopaminergic signaling in the renal proximal tubule under conditions of sodium loading leads to natriuresis without the need to increase blood pressure. Low caveolin-1 (CAV1) expression in the kidney is associated with dopaminergic signaling defects and salt sensitive hypertension in both mice and rats. We and others have shown that SNPs in human CAV1 are associated with hypertension. We hypothesize that low CAV1 expression causes increased GRK4 kinase activity due to lack of physical interaction and inhibition of GRK4; thus overactive GRK4 would inactivate the dopamine-1 receptor (D 1 R). We have previously shown that CAV1 reduction, knockdown or knockout in mouse, rat and human renal proximal tubule cells (RPTC) leads to decreased dopamine D 1 -like receptor binding at the proximal tubule cell surface and that re-expression of CAV1 alpha isoform is necessary to rescue this dopaminergic defect. Here we extend those studies by showing that defects in D 1 R cell surface expression due to low CAV1 expression can also be rescued by inhibiting GRK4 in human RPTC using GRK4 siRNA or by introducing heparin (1 μmol/L, 24 hrs) in mouse and rat RPTC. GRK4 siRNA lowered GRK4 expression by greater than 80% in all four human RPTC cell lines and partially restored fluorescent D 1 -like receptor antagonist bodipy630 SKF83566 cell surface binding (scrambled siRNA 66,231±7,286 RFU, GRK4 siRNA 96,206±11,035 RFU, P&lt;0.05, N=28). In RPTC from CAV1 knockout mice, electroporation of heparin restored bodipy630 SKF83566 cell surface binding (vehicle 21,346±3,219 RFU, heparin 58,380±8,032 RFU, P&lt;0.05, N=7). In rat SHR RPTC, CAV1 expression is very low, and the stable transfection of CAV1 partially restored their D 1 -like receptor surface expression. Electroporation of heparin in these CAV1 transfected cells (SHR CAV1) restored bodipy630 SKF83566 cell surface binding back to levels found in WKY RPTC (SHR CAV1 35,241±4,280 RFU, SHR CAV1 heparin 54,218±6,564 RFU, WKY 56,821± 3,546 RFU, P&lt;0.05, N=7). In summary, we show that dopamine D 1 -like receptor defects caused by loss of CAV1 are likely due to increased GRK4 activity and can be restored by either increasing CAV1 expression or decreasing GRK4 expression.

A histological study of the effect of exogenous melatonin on gentamicin induced structural alterations of proximal tubules in rats

The aim of this research was to assess the reactive changes of rat proximal tubules caused by gentamicin and the effect of relatively low doses of melatonin. 48 adult male Wistar rats were distributed into six groups of equal size which all received one of the following daily intraperitoneal injections: vehicle (5% ethanol in Ringer solution) during 11 days (C); gentamicin (80 mg/kg) during 8 days (G), two groups which concomitantly received gentamicin (80 mg/kg) during 8 days and melatonin in two different test doses (5 or 20 mg/kg) during 11 days (GM1, GM2) and two groups treated only with melatonin in two different doses (5 or 20 mg/kg) during 11 days (M1, M2). Histological analysis included qualitative and semi-quantitative light microscopy analysis of proximal tubules. Exogenous melatonin had no significant effect on the microstructure, independently of dosis. The changes of proximal tubules microstructure induced by gentamicin were expressed in the form of granulovacuolar degeneration, necrosis and desquamation. The grade of proximal tubular changes was smaller in animals who besides gentamicin received melatonin. Melatonin has a dose dependent protective effect on the structural alterations of proximal tubules of the kidney induced by gentamicin.

A mathematical model of rat proximal tubule and loop of Henle (1137.9)

PT and LOH function are coupled, with proximal transport determining loop fluid composition, and loop transport modulating glomerular filtration via tubuloglomerular feedback (TGF). To examine this interaction, we began with published models of superficial rat proximal convoluted tubule (PCT) (including flow‐dependent transport in a compliant tubule), and rat thick ascending Henle limb (AHL). This PCT was scaled down to represent outer medullary proximal straight tubule (PST), which was connected to thick AHL via a short descending limb. Superficial PT was scaled up to a juxtamedullary nephron, and connected to thick AHL via outer and inner medullary descending limbs, and inner medullary thin AHL. Medullary interstitial solute concentrations were specified. End‐tubule hydrostatic pressure was required to conform to a distal nephron flow resistance. The TGF signal was represented as a linear function of end‐AHL cytosolic Cl concentration. These two distal conditions required iterative solution of the model. Calculations revealed Na, K, and HCO3 reabsorption in all segments, with thin limb transport driven by the increases in lumen concentrations produced by osmotic water reabsorption. PST was a critical locus for NH4 secretion, due to high interstitial NH4, and cellular uptake via the Na,K‐ATPase. Late AHL cytosolic Cl was a sigmoidal function of flow, and appears suitable to represent the TGF signal.Grant Funding Source: Supported by NIH RO1‐DK29857

Modulation of Na+/H+exchanger 3 trafficking and activity in rat proximal tubule

Modulation of Na⁺/H⁺exchanger 3 trafficking and activity in rat proximal tubule

Fructose Stimulates Na/H Exchange Activity and Sensitizes the Proximal Tubule to Angiotensin II

The proximal nephron reabsorbs 60% to 70% of the fluid and sodium and most of the filtered bicarbonate via Na/H exchanger 3. Enhanced proximal nephron transport is implicated in hypertension. Our findings show that a fructose-enriched diet causes salt sensitivity. We hypothesized that fructose stimulates luminal Na/H exchange activity and sensitizes the proximal tubule to angiotensin II. Na/H exchange was measured in rat proximal tubules as the rate of intracellular pH (pHi) recovery in fluorescent units/s. Replacing 5 mmol/L glucose with 5 mmol/L fructose increased the rate of pHi recovery (1.8±0.6 fluorescent units/s; P<0.02; n=8). Staurosporine, a protein kinase C inhibitor, blocked this effect. We studied whether this effect was because of the addition of fructose or removal of glucose. The basal rate of pHi recovery was first tested in the presence of a 0.6-mmol/L glucose and 1, 3, or 5 mmol/L fructose added in a second period. The rate of pHi recovery did not change with 1 mmol/L but it increased with 3 and 5 mmol/L of fructose. Adding 5 mmol/L glucose caused no change. Removal of luminal sodium blocked pHi recovery. With 5.5 mmol/L glucose, angiotensin II (1 pmol/L) did not affect the rate of pHi recovery (change, -1.1±0.5 fluorescent units/s; n=9) but it increased the rate of pHi recovery with 0.6 mmol/L glucose/5 mmol/L fructose (change, 4.0±2.2 fluorescent units/s; P<0.02; n=6). We conclude that fructose stimulates Na/H exchange activity and sensitizes the proximal tubule to angiotensin II. This mechanism is likely dependent on protein kinase C. These results may partially explain the mechanism by which a fructose diet induces hypertension.

Proteomic analysis of rat proximal tubule cells following stretch-induced apoptosis in an in vitro model of kidney obstruction

Urinary tract obstruction (UTO) is a commonly noted disorder on prenatal ultrasound that has the potential to lead to permanent loss of renal function. To study the molecular processes of the disease, an in vitro model has been developed which involves mechanical stretch of proximal tubule cells grown on flexible plates which mimics the physiological conditions during UTO. This study employs a one dimensional SDS-PAGE fractionation procedure, followed by in-gel digest and LC-MS/MS analysis in a semi-quantitative experiment using spectral counting to relatively quantify changes in protein expression following the established model of UTO. Quantitative analysis shows 317 of the 1630 identified proteins express altered abundance, with 135 increased and 182 decreased in abundance as a result of stretch. Gene ontology (GO) and KEGG annotations implicate a number of physiological processes that have been previously shown in addition to some potentially novel processes in UTO. The quantitative proteomic analysis we performed here provides a more complete characterization of changes in protein abundance as a result of stretch than previous studies, and provides a number of previously undescribed proteins in proximal tubule cells that may play a role in UTO. Urinary tract obstruction (UTO) is a commonly noted abnormality on prenatal ultrasound that can either resolve spontaneously or require surgical intervention to prevent permanent renal damage or loss of function. While targeted studies of UTO have shown a number of pathological responses in proximal tubule cells, there are currently no large-scale quantitative studies that aim to elucidate a global cellular response. This study uses a semi-quantitative approach and applies a well characterized model of UTO to determine a number of cellular processes affected by UTO simulation and identifies a number of proteins with altered abundance that have not been noted previously in UTO. This article is part of a Special Issue entitled: Can Proteomics Fill the Gap Between Genomics and Phenotypes?

Abstract 626: Caveolin-1 (CAV1) Levels Determine the Cell Surface Expression of the Dopamine D1-like Receptors

In human renal proximal tubule cells, the dopamine-1 receptor (D1R) increases cAMP production and negatively regulates sodium transport. Defects in D1R signaling have been associated with SNPs in G-protein coupled receptor kinase type 4 (GRK4), which hyperphosphorylates and decreases D1R function, leading to sodium retention. We have previously shown that knockdown of CAV1 in proximal tubule cells impairs sodium transport. In this report we show that CAV1 reduction, knockdown or knockout leads to decreased dopamine D1-like receptor binding at the proximal tubule cell surface. Dopamine D1-like brush border and cell surface binding was measured using Bodipy-FL conjugated SKF83566 . WT and Caveolin 1 knockout (CAV1KO) kidney cortex brush border membrane isolation was performed by magnetic lotus tetragonolobus agglutinin affinity purification and fluorescent SKF83566 binding by microplate fluorometry. CAV1KO had a 35.8±3.3% decrease in binding vs WT (p&lt;0.01, n=6 per group, WT level 18,492 RFU above background). In hTERT immortalized human renal proximal tubule cell lines, lentiviral shRNAi CAV1 stable knockdown cell lines had a 76.4±2.5%, p&lt;0.001 n=4, reduction in CAV1 expression by western blot. Cell surface D1-like receptor binding was reduced by 34.0±1.3% (p&lt;0.001, n=4) in the CAV1 knockdown cell lines (scrambled control levels 117193 RFU). We previously have shown that CAV1 expression is dramatically reduced in the hypertensive SHR proximal tubule cell line vs the normotensive WKY proximal tubule cell line. The SHR cell line had a 57.5±1.2% (p&lt;0.001, n=7) reduction in D1-like receptor binding (WKY 116114 RFU). Re-expression of rat CAV1 alpha or CAV1 beta isoform in the SHR cell line by stable lentiviral infection increased binding by 51.6±2.1%, and 63.3±1.6% respectively (p&lt;0.01, n=14). Sodium transport was decreased by CAV1 alpha and CAV1 beta expression in SHR cell lines. The average number of domes per 10x objective fields of view was 9.5±1.6 in SHR cell, 4.5±0.3 in SHR CAV1alpha (p&lt;0.05, n=4 vs SHR) and in SHR CAV1beta 5.0±0.3 (p&lt;0.05, n=4 vs SHR). In mouse, rat, and human renal proximal tubule cells, CAV1 is necessary for dopamine D1-like receptor cell surface localization and for normal dopaminergic inhibition of sodium transport.

Serum-free culture of rat proximal tubule cells with enhanced function on chitosan

The proximal tubule performs a variety of important renal functions and is the major site for nutrient reabsorption. The purpose of this study is to culture rat renal proximal tubule cells (PTCs) on chitosan without serum to maintain a transcellular pathway to transport water and ions effectively without loss of highly differentiated cell function. The effect of chitosan, which is structurally similar to glycosaminoglycans, in the absence of serum on the primary cultured PTCs was compared that of collagen with or without serum. Two days after seeding, more tubule fragments and higher PTC viability were observed on chitosan than on collagen with or without serum. Proliferation marker Ki-67 immunostaining and phosphorylated extracellular-regulated kinase (ERK) expression results displayed similar proliferation capability of PTCs established on chitosan without serum and collagen with 2% fetal bovine serum after 4days of incubation. When grown to confluence, PTCs formed a monolayer with well-organized tight junctions and formation of domes on chitosan without serum. Moreover, evaluation of the transepithelial electrical resistance showed that both chitosan and serum were involved in the modification of water and ion transport in confluent cells. By showing the direct suppression of PTC growth and dome formation treated with heparinase, we demonstrated that the interaction between cell surface heparin sulfate proteoglycan and chitosan played an important role in PTC proliferation and differentiation. A successful primary culture of PTCs has now been produced on chitosan in serum-free culture condition, which offers potential applications for chitosan in renal tissue engineering.

Dose-dependent effects of angiotensin-(1–7) on the NHE3 exchanger and [Ca2+]iin in vivo proximal tubules

The acute direct action of angiotensin-(1–7) [ANG-(1–7)] on bicarbonate reabsorption ( JHCO3−) was evaluated by stationary microperfusions on in vivo middle proximal tubules in rats using H ion-sensitive microelectrodes. The control JHCO3−is 2.82 ± 0.078 nmol·cm−2·s−1(50). ANG-(1–7) (10−12or 10−9M) in luminally perfused tubules decreases JHCO3−(36 or 60%, respectively), but ANG-(1–7) (10−6M) increases it (80%). A779 increases JHCO3−(30%) and prevents both the inhibitory and the stimulatory effects of ANG-(1–7) on it. S3226 decreases JHCO3−(45%) and changes the stimulatory effect of ANG-(1–7) to an inhibitory effect (30%) but does not affect the inhibitory effect of ANG-(1–7). Our results indicate that in the basal condition endogenous ANG-(1–7) inhibits JHCO3−and that the biphasic dose-dependent effect of ANG-(1–7) on JHCO3−is mediated by the Mas receptors via the Na+/H+exchanger 3 (NHE3). The control value of intracellular Ca2+concentration ([Ca2+]i), as monitored using fura-2 AM, is 101 ± 2 nM ( 6 ), and ANG-(1–7) (10−12, 10−9, or 10−6M) transiently (3 min) increases it (by 151, 102, or 52%, respectively). A779 increases the [Ca2+]i(25%) but impairs the stimulatory effect of all doses of ANG-(1–7) on it. The use of BAPTA or thapsigargin suggests a correlation between the ANG-(1–7) dose-dependent effects on [Ca2+]iand JHCO3−. Therefore, the interaction of the opposing dose-dependent effects of ANG II and ANG-(1–7) on [Ca2+]iand JHCO3−may represent an physiological regulatory mechanism of extracellular volume and/or pH changes. However, whether [Ca2+]imodification is an important direct mechanism for NHE3 activation by these peptides or is a side effect of other signaling pathways will require additional studies.

Anti-Angiogenic and Anti-Metastatic Activity of Synthetic Phosphoethanolamine

BackgroundRenal cell carcinoma (RCC) is the most common type of kidney cancer, and represents the third most common urological malignancy. Despite the advent of targeted therapies for RCC and the improvement of the lifespan of patients, its cost-effectiveness restricted the therapeutic efficacy. In a recent report, we showed that synthetic phosphoethanolamine (Pho-s) has a broad antitumor activity on a variety of tumor cells and showed potent inhibitor effects on tumor progress in vivo.Methodology/Principal FindingsWe show that murine renal carcinoma (Renca) is more sensitive to Pho-s when compared to normal immortalized rat proximal tubule cells (IRPTC) and human umbilical vein endothelial cells (HUVEC). In vitro anti-angiogenic activity assays show that Pho-s inhibits endothelial cell proliferation, migration and tube formation. In addition, Pho-s has anti-proliferative effects on HUVEC by inducing a cell cycle arrest at the G2/M phase. It causes a decrease in cyclin D1 mRNA, VEGFR1 gene transcription and VEGFR1 receptor expression. Pho-s also induces nuclear fragmentation and affects the organization of the cytoskeleton through the disruption of actin filaments. Additionally, Pho-s induces apoptosis through the mitochondrial pathway. The putative therapeutic potential of Pho-s was validated in a renal carcinoma model, on which our remarkable in vivo results show that Pho-s potentially inhibits lung metastasis in nude mice, with a superior efficacy when compared to Sunitinib.Conclusions/SignificanceTaken together, our findings provide evidence that Pho-s is a compound that potently inhibits lung metastasis, suggesting that it is a promising novel candidate drug for future developments.

Intracellular calcium release modulates polycystin-2 trafficking

BackgroundPolycystin-2 (PC2), encoded by the gene that is mutated in autosomal dominant polycystic kidney disease (ADPKD), functions as a calcium (Ca2+) permeable ion channel. Considerable controversy remains regarding the subcellular localization and signaling function of PC2 in kidney cells.MethodsWe investigated the subcellular PC2 localization by immunocytochemistry and confocal microscopy in primary cultures of human and rat proximal tubule cells after stimulating cytosolic Ca2+ signaling. Plasma membrane (PM) Ca2+ permeability was evaluated by Fura-2 manganese quenching using time-lapse fluorescence microscopy.ResultsWe demonstrated that PC2 exhibits a dynamic subcellular localization pattern. In unstimulated human or rat proximal tubule cells, PC2 exhibited a cytosolic/reticular distribution. Treatments with agents that in various ways affect the Ca2+ signaling machinery, those being ATP, bradykinin, ionomycin, CPA or thapsigargin, resulted in increased PC2 immunostaining in the PM. Exposing cells to the steroid hormone ouabain, known to trigger Ca2+ oscillations in kidney cells, caused increased PC2 in the PM and increased PM Ca2+ permeability. Intracellular Ca2+ buffering with BAPTA, inositol 1,4,5-trisphosphate receptor (InsP3R) inhibition with 2-aminoethoxydiphenyl borate (2-APB) or Ca2+/Calmodulin-dependent kinase inhibition with KN-93 completely abolished ouabain-stimulated PC2 translocation to the PM.ConclusionsThese novel findings demonstrate intracellular Ca2+-dependent PC2 trafficking in human and rat kidney cells, which may provide new insight into cyst formations in ADPKD.

Retention of fetuin-A in renal tubular lumen protects the kidney from nephrocalcinosis in rats

The serum glycoprotein fetuin-A is an important inhibitor of extraosseous calcification. The importance of fetuin-A has been confirmed in fetuin-A null mice, which develop widespread extraosseous calcification including the kidney. However, the mechanism how fetuin-A protects kidneys from nephrocalcinosis remains uncertain. Here, we demonstrate that intratubular fetuin-A plays a role in the prevention of nephrocalcinosis in the proximal tubules. Although normal rat kidney did not express mRNA for fetuin-A, we found punctate immunohistochemical staining of fetuin-A mainly in the S1 segment of the proximal tubules. The staining pattern suggested that fetuin-A passed through the slit diaphragm, traveled in the proximal tubular lumen, and was introduced into proximal tubular cells by megalin-mediated endocytosis. To test this hypothesis, we inhibited the function of megalin by intravenous injection of histidine-tagged soluble receptor-associated protein (His-sRAP), a megalin inhibitor. His-sRAP injection diminished fetuin-A staining in the proximal tubules and led to urinary excretion of fetuin-A. We further analyzed the role of fetuin-A in nephrocalcinosis. Continuous injection of parathyroid hormone (PTH) 1-34 induced nephrocalcinosis mainly in the proximal tubules in rats. His-sRAP retained fetuin-A in renal tubular lumen and thereby protected the kidneys of PTH-treated rats from calcification. Our findings suggest that tubular luminal fetuin-A works as a natural inhibitor against calcification in the proximal tubules under PTH-loaded condition.

Urinary chemokine (C-C motif) ligand 2 (monocyte chemotactic protein-1) as a tubular injury marker for early detection of cisplatin-induced nephrotoxicity

Because of the difficulty in detecting segment-specific response in the kidney, we investigated the molecular events underlying acute kidney injury in the proximal tubules of rats with cisplatin (cis-diamminedichloroplatinum II)-induced nephrotoxicity. Microarray analysis revealed that mRNA levels of several cytokines and chemokines, such as interleukin-1beta, chemokine (C-C motif) ligand (CCL) 2, CCL20, chemokine (C-X-C motif) ligand (CXCL) 1, and CXCL10 were significantly increased after cisplatin treatment in both isolated proximal tubules and whole kidney. Interestingly, tubular CCL2 mRNA levels increased soon after cisplatin administration, whereas CCL2 mRNA levels in whole kidney first decreased and then increased. Levels of both CCL2 and kidney injury molecule-1 (KIM-1) in the whole kidney increased after cisplatin administration. Immunofluorescence analysis revealed CCL2 changes in the proximal tubular cells initially and then in the medullary interstitium. Urinary CCL2 excretion significantly increased approximately 3-fold compared with controls the day after cisplatin administration (5mg/kg), when no changes were observed plasma creatinine and blood urea nitrogen levels. Urinary levels of KIM-1 also increased 3-fold after cisplatin administration. In addition, urinary CCL2 rather than KIM-1 increased in chronic renal failure rats after administration of low-dose cisplatin (2mg/kg), suggesting that urinary CCL2 was selective for cisplatin-induced nephrotoxicity in renal impairment. These results indicated that the increase in cytokine and chemokine expression in renal epithelial cells might be responsible for kidney deterioration in cisplatin-induced nephrotoxicity, and that urinary CCL2 is associated with tubular injury and serves as a sensitive and noninvasive marker for the early detection of cisplatin-induced tubular injury.

Divergent localization of angiotensinogen mRNA and protein in proximal tubule segments of normal rat kidney

Angiotensinogen in the kidneys is formed primarily in the proximal tubule cells and is secreted into the tubular fluid. Structurally, proximal tubules can be divided into three segments. The first segment, segment 1 (S1) is mainly confined to the pars convoluta, the second segment, segment 2 (S2) comprises the end of pars convoluta, and the third segment, segment 3 (S3) includes the major part of the pars recta. There are some reports describing angiotensinogen localization in kidneys; however, it remains uncertain which proximal tubule segments express angiotensinogen. To determine the detailed localization of angiotensinogen in the three proximal tubule segments, we established multistaining methods using segment-specific protein markers. Using kidneys from Wistar-Kyoto rats, we performed immunohistochemistry and double or triple staining by fluorescence in-situ hybridization and/or immunofluorescence. Our results show that angiotensinogen mRNA and protein are expressed in the cortex and outer medulla of the normal rat kidney. Angiotensinogen mRNA was hardly detected in S1, detected weakly in S2 and strongly in S3 segments. In contrast, angiotensinogen protein was detected in S1 at high levels and less in S2 and S3 segments. These data indicate divergence of angiotensinogen mRNA transcription and angiotensinogen protein synthesis and metabolism in different segments of the normal rat proximal tubules.

Effects of cadmium on the sub-cellular localization of β-catenin and β-catenin-regulated gene expression in NRK-52E cells

The E-cadherin/β-catenin complex is a structural component of adherens-type junctions in epithelial cells. Moreover, β-catenin acts as an intracellular signaling molecule that can influence the expression of a variety of genes that regulate apoptosis and cell cycle control. Cadmium (Cd) is an environmental toxicant that causes renal dysfunction and disrupts cadherin-dependent cell-cell adhesion in various types of epithelial cells. In this study, we examined the effects of Cd on the subcellular localization of β-catenin, the cadherin/β-catenin complex and β-catenin-mediated gene transcription in rat proximal tubule NRK-52E cells. Exposure to 5-10μM Cd for 4h caused the NRK cells to separate from each other without killing the cells or causing them to detach from the growing surface. This effect was associated with the loss of β-catenin and E-cadherin from the cell-cell contacts and apparent changes in the accumulation of β-catenin in the nuclear cell subfraction. The expression of the β-catenin-sensitive gene, c-jun was significantly increased in cells exposed to 5μM Cd. However, there was no change in the expression of several other β-catenin-regulated genes including: c-myc, cyclin D1 and matrilysin. Additional studies utilizing the TOPFLASH β-catenin reporter gene construct showed that Cd caused a 2-3 fold increase in the expression of the luciferase reporter gene. Overall, these results indicate that Cd disrupts the cadherin/β-catenin complex in NRK-52E cells, but this effect leads to only partial activation of β-catenin-mediated gene transcription.

MiR-127 Protects Proximal Tubule Cells against Ischemia/Reperfusion: Identification of Kinesin Family Member 3B as miR-127 Target

Ischemia/reperfusion (I/R) is at the basis of renal transplantation and acute kidney injury. Molecular mechanisms underlying proximal tubule response to I/R will allow the identification of new therapeutic targets for both clinical settings. microRNAs have emerged as crucial and tight regulators of the cellular response to insults including hypoxia. Here, we have identified several miRNAs involved in the response of the proximal tubule cell to I/R. Microarrays and RT-PCR analysis of proximal tubule cells submitted to I/R mimicking conditions in vitro demonstrated that miR-127 is induced during ischemia and also during reperfusion. miR-127 is also modulated in a rat model of renal I/R. Interference approaches demonstrated that ischemic induction of miR-127 is mediated by Hypoxia Inducible Factor-1alpha (HIF-1α) stabilization. Moreover, miR-127 is involved in cell-matrix and cell-cell adhesion maintenance, since overexpression of miR-127 maintains focal adhesion complex assembly and the integrity of tight junctions. miR-127 also regulates intracellular trafficking since miR-127 interference promotes dextran-FITC uptake. In fact, we have identified the Kinesin Family Member 3B (KIF3B), involved in cell trafficking, as a target of miR-127 in rat proximal tubule cells. In summary, we have described a novel role of miR-127 in cell adhesion and its regulation by HIF-1α. We also identified for the first time KIF3B as a miR-127 target. Both, miR-127 and KIF3B appear as key mediators of proximal epithelial tubule cell response to I/R with potential al application in renal ischemic damage management.

Cadmium induced oxidative stress in rat proximal tubules: To die or not to die

Cadmium induced oxidative stress in rat proximal tubules: To die or not to die

Male-Dominant Activation of Rat Renal Organic Anion Transporter 1 (Oat1) and 3 (Oat3) Expression by Transcription Factor BCL6

BackgroundOrganic anion transporters 1 (Oat1) and 3 (Oat3) mediate the transport of organic anions, including frequently prescribed drugs, across cell membranes in kidney proximal tubule cells. In rats, these transporters are known to be male-dominant and testosterone-dependently expressed. The molecular mechanisms that are involved in the sex-dependent expression are unknown. Our aim was to identify genes that show a sex-dependent expression and could be involved in male-dominant regulation of Oat1 and Oat3.Methodology/Principal FindingsPromoter activities of Oat1 and Oat3 were analyzed using luciferase assays. Expression profiling was done using a SurePrint G3 rat GE 8×60K microarray. RNA was isolated from renal cortical slices of four adult rats per sex. To filter the achieved microarray data for genes expressed in proximal tubule cells, transcription database alignment was carried out. We demonstrate that predicted androgen response elements in the promoters of Oat1 and Oat3 are not functional when the promoters were expressed in OK cells. Using microarray analyses we analyzed 17,406 different genes. Out of these genes, 56 exhibit a sex-dependent expression in rat proximal tubule cells. As genes potentially involved in the regulation of Oat1 and Oat3 expression, we identified, amongst others, the male-dominant hydroxysteroid (17-beta) dehydrogenase 1 (Hsd17b1), B-cell CLL/lymphoma 6 (BCL6), and polymerase (RNA) III (DNA directed) polypeptide G (Polr3g). Moreover, our results revealed that the transcription factor BCL6 activates promoter constructs of Oat1 and Oat3.ConclusionThe results indicate that the male-dominant expression of both transporters, Oat1 and Oat3, is possibly not directly regulated by the classical androgen receptor mediated transcriptional pathway but appears to be regulated by the transcription factor BCL6.

Insulin regulates hSGLT2 expression via PKA and PKC activation

Recent evidence suggest that in the rat proximal tubule SGLT2 (Sodium dependent Glucose Transporter 2) is phosphorylated. To test if human SGLT2 is regulated by posphorylation we analyze the effect of PKA and PKC activation on HEK‐293T cells expressing hSGLT2 using patch clamp and radio tracer uptake. PKA activation was induced by incubation of cells in 8‐bromoadenosine 3′‐5′‐cyclic monophosphate (Br‐cAMP) and sn‐1,2‐dioctanoylglycerol (DOG) was use as activator of PKC. After 1 h incubation in Br‐cAMP we observed a 2.5 fold increase in [14 C]α‐MDG uptake confirmed by a similar increase in glucose induced current. Incubation in DOG resulted in a 50 % increase in [14C]α‐MDG uptake and glucose induced current. In both cases increase is caused by an increase in Imax and not in the change in glucose affinity. These effects were completely suppressed by mutation of the SGLT2 phosphorylation site (S625A). In order to determine the initial event triggering the phosphorylation, we measured the effect of insulin on [14C]α‐MDG uptake. Incubating cells expressing hSGLT2 wt or S625A mutant with 400 pM insulin induced a 3 fold increase in glucose transport in wild type hSGLT2 but not in the mutant. In conclusion our results indicate that insulin via PKA and PKC activation regulates hSGLT2 expression in a fast time scale by an increasing trafficking of hSGLT2 to the plasma membrane.NIH grant DK077133 and SNSF grant PBZHP3‐135919.