Rat Renal Proximal Tubule Cells Research Articles

Membranous translocation of murine double minute 2 promotes the increased renal tubular immunogenicity in ischemia-reperfusion-induced acute kidney injury.

Kidneys from donors with prolonged warm and cold ischemia are prone to posttransplant T cell-mediated rejection (TCMR) due to ischemia-reperfusion injury (IRI). However, the precise mechanisms still remain obscure. Renal tubular epithelial cells (TECs) are the main target during IRI. Meanwhile, we have previously reported that murine double minute 2 (MDM2) actively participates in TEC homeostasis during IRI. In this study, we established a murine model of renal IRI and a cell model of hypoxia-reoxygenation by culturing immortalized rat renal proximal tubule cells (NRK-52E) in a hypoxic environment for different time points followed by 24 h of reoxygenation and incubating NRK-52E cells in a chemical anoxia-recovery environment. We found that during renal IRI MDM2 expression increased on the membrane of TECs and aggregated mainly on the basolateral side. This process was accompanied by a reduction of a transmembrane protein, programmed death ligand 1 (PD-L1), a coinhibitory second signal for T cells in TECs. Using mutant plasmids of MDM2 to anchor MDM2 on the cell membrane or nuclei, we found that the upregulation of membrane MDM2 could promote the ubiquitination of PD-L1 and lead to its ubiquitination-proteasome degradation. Finally, we set up a coculture system of TECs and CD4+ T cells in vitro; our results revealed that the immunogenicity of TECs was enhanced during IRI. In conclusion, our findings suggest that the increased immunogenicity of TECs during IRI may be related to ubiquitinated degradation of PD-L1 by increased MDM2 on the cell membrane, which consequently results in T-cell activation and TCMR.NEW & NOTEWORTHY Ischemic acute kidney injury (AKI) donors can effectively shorten the waiting time for kidney transplantation but increase immune rejection, especially T cell-mediated rejection (TCMR), the mechanism of which remains to be elucidated. Our study demonstrates that during ischemia-reperfusion injury (IRI), the translocation of tubular murine double minute 2 leads to basolateral programmed death ligand 1 degradation, which ultimately results in the occurrence of TCMR, which may provide a new therapeutic strategy for preventing AKI donor-associated TCMR.

Maslinic Acid Attenuates Ischemia/Reperfusion-Induced Acute Kidney Injury by Suppressing Inflammation and Apoptosis Through Inhibiting NF-κB and MAPK Signaling Pathway.

Inflammation and apoptosis are the major contributors to the mechanisms of acute kidney injury (AKI) due to renal ischemia-reperfusion injury (IRI). Maslinic acid (MA), a pentacyclic triterpene acid mostly found in dietary plants, the current study was to demonstrate the renoprotective effect of MA on IRI-induced AKI, and to investigate the role of inflammation and apoptosis-related signaling pathways as a molecular mechanism. C57BL/6J mice were subjected to IRI for 72 h, and MA was daily administered by intraperitoneal injection during this period. In parallel, rat renal proximal tubule cells (NRK52E) were prophylactically treated with MA and then exposed to hydrogen peroxide (H2O2). MA treatment significantly inhibited the mRNA expression of interleukin (IL-1β), tumor necrosis factor-α (TGF-α), monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecule-1(ICAM-1). Also, MA reduced the expression of Bax/Bcl2 ratio and cleaved caspase-3. In NRK52 cells, MA inhibited the IκBα degradation, blocked NF-κB/p65 phosphorylation, and nuclear translocation. The phosphorylation of ERK, JNK, and p38 was attenuated by MA in IRI-induced kidney injury and H2O2-stimulated NRK52 cells. The expression levels of IL-1β, MCP-1, and ICAM-1 were upregulated in H2O2-stimulated NRK52E cells, which was attenuated by NF-κB inhibitor. H2O2 treatment increased the Bax/Bcl2 ratio and cleaved caspase-3 in NRK52E cells, which was counteracted by MAPK inhibitors. Together, our data demonstrate that MA suppresses IR-induced AKI injury through NF-κB and MAPK signaling pathways and that MA is a promising agent in the treatment of kidney diseases.

Abstract 129: The Microbiome Product Urolithin a Abrogates the Tgf-β Egfr Pai-1 Pathway by Inhibiting Egfr Activation and Expression

Introduction: Ellagitanins are highly abundant in various foods including fruits, nuts and wines, and particularly high in commercial muscadine grape-derived supplements. The microbiome metabolizes ellagitanins to urolithins. Urolithin A (UA), the final product of the microbiome processing pathway may contribute to the cardioprotective effects of ellagitanins; however, the cellular signaling mechanisms of UA are unclear. Since the TGFβ-PAI-1 pathway contributes to fibrosis, cellular senescence and progressive kidney injury, we evaluated the effects of UA on TGFβ-induced stimulation of PAI-1 in rat renal proximal tubule cells (rPTCs - NRK52E). We hypothesize that UA may be an endogenous anti-fibrotic compound that attenuates the TGFβ-PAI-1 response. Results: TGFβ (5 ng/ml) treatment markedly increased PAI-1 levels 10-fold [43.9 ± 0.7 vs 3.5 ± 0.3 ng/ml; P<0.001, n=3] in the rPTCs at 24 hrs. The TGFβ receptor (TGFβR) kinase (ALK5) inhibitor SB525334 or UA abolished the increase in PAI-1 to TGFβ [SB: 1.3 ± 0.3; UA: 2.0 ± 0.4 ng/ml; P<0.001, n=3]; UA exhibits an IC50 of 3.1 μM to abrogate the TGFβ-PAI-1 response. Since the TGFβ pathway may transactivate the EGFR, treatment with the EGFR kinase inhibitors AG1478 (AG, 1 μM) and Lapatinib (LAP, 1 μM) also abrogated the PAI-1 response to TGFβ [AG: 3.3 ± 0.4; LAP: 6.2 ± 1.4 ng/ml; P<0.001, n=3]. Addition of EGF alone increased PAI-1 to a similar extent as TGFβ [46.6 ± 2.7 ng/ml; P<0.001, n=3], and treatment with UA or AG abolished the EGF-PAI-1 response [2.4 ± 0.1 and 1.3 ± 0.1 ng/ml, respectively; P<0.001, n=3]. Given the dependence on EGFR signaling for PAI-1, we determined whether UA directly attenuates EGFR stimulation. Indeed, UA treatment inhibited peak EGFR phosphorylation to EGF at 5 mins [10 μM: 67 ± 4%; 50 μM: 71 ± 5%; P<0.05, n=3], and reduced total EGFR expression at 24 hrs [10 μM: 27 ± 11%; 50 μM: 66 ± 7%; P<0.05, n=6]. UA was devoid of cytotoxic effects (LDH release). Conclusion: The present study is the first to establish that UA abrogates the TGFβ-EGFR-PAI-1 pathway via the inhibition of EGFR stimulation and expression. Moreover, the potency of UA to attenuate PAI-1 is comparable to its physiological levels. The microbiome product UA may convey anti-fibrotic actions within the kidney by targeting the EGFR system.

Rat Kidney Slices for Evaluation of Apical Membrane Transporters in Proximal Tubular Cells

Kidney slice has been often used as a tool reflecting basolateral transport in renal tubular epithelial cells. Recently, we reported that several important apical reabsorptive transporters such as Octn1/2, Sglt1/2, and Pept1/2 were functional in mouse kidney slices as well as transporter activities in basolateral side, which have been well accepted. Because rats are often used for preclinical pharmacodynamic and pharmacokinetic studies as well as mice, it is important to confirm applicability of rat kidney slices for evaluation of apically expressed transporters. The present study investigates usefulness of kidney slices from rats for evaluation of apical membrane transporters for efflux (multidrug resistance 1a, mdr1a) as well as influx (Octn1/2, Sglt1/2, Pept1/2). Na+-dependent uptake of ergothioneine (Octn1), carnitine (Octn2), and methyl-α-D-glucopyranoside (Sglt1/2) by rat kidney slices was observed, and the uptake was decreased by selective inhibitors. In addition, uptake of glycyl-sarcosine (Pept1/2) showed H+-dependence and was decreased by selective inhibitor. Furthermore, accumulation of mdr1a substrate azasetron was increased in the presence of zosuquidar, an mdr1a inhibitor, while strain differences existed. In conclusion, rat kidney slices should be useful for evaluation of renal drug disposition regulated by transporters in apical as well as basolateral membranes of rat renal proximal tubule cells.

Angiotensin II type 2 receptor inhibits expression and function of insulin receptor in rat renal proximal tubule cells

Both renin–angiotensin systems and insulin participate in kidney-involved blood pressure regulation. Activation of angiotensin II type 2 receptor (AT2R) decreases sodium reabsorption in renal proximal tubule (RPT) cells, whereas insulin produces the opposite effect. We presume that AT2R has an inhibitory effect on insulin receptor expression in RPT cells, which may affect renal sodium transport and therefore be of physiological or pathological significance. Our present study found that activation of AT2R inhibited insulin receptor expression in a concentration and time-dependent manner in RPT cells from Wistar-Kyoto (WKY) rats. In the presence of a protein kinase C (PKC) inhibitor (PKC inhibitor peptide 19–31, 10−6 mol/L) or a phosphatidylinositol 3 kinase inhibitor (wortmannin, 10−6 mol/L), the inhibitory effect of AT2R on insulin receptor was blocked, indicating that both PKC and phosphatidylinositol 3 kinase were involved in the signaling pathway. There was a linkage between AT2R and insulin receptor which was determined by both laser confocal microscopy and coimmunoprecipitation. However, the effect of AT2R activation on insulin receptor expression was different in RPT cells from spontaneously hypertensive rats (SHRs). Being contrary to the effect in WKY RPT cells, AT2R stimulation increased insulin receptor in SHR RPT cells. Insulin (10−7 mol/L, 15 minutes) enhanced Na+-K+-ATPase activity in both WKY and SHR RPT cells. Pretreatment with CGP42112 decreased the stimulatory effect of insulin on Na+-K+-ATPase activity in WKY RPT cells, whereas pretreatment with CGP42112 increased it in SHR RPT cells. It is suggested that activation of AT2R inhibits insulin receptor expression and function in RPT cells. The lost inhibitory effect of AT2R on insulin receptor expression may contribute to the pathophysiology of hypertension.

Abstract P321: Clozapine-induced Hypertension: Role of the Dopamine Type 4 Receptor (D4R) in Human Renal Proximal Tubule Cells (RPTC)

Clozapine (CLZ), a potent D 4 R antagonist that is an effective antipsychotic drug for the treatment of schizophrenia, is associated with hypertension. The hypertensive side effect of CLZ may be through inhibition of the D 4 R because germline deletion of D 4 R in mice causes hypertension. The hypertension caused by germline deletion of D 4 R may, in part, be due to increased renal expression of the angiotensin type 1 receptor (AT 1 R) and renal expression of sodium exchangers, transporters, and pumps, e.g., NHE3, NKCC2, NCC, and Na + K + /ATPase, α subunit. CLZ administered subcutaneously (20mg/kg/day/x3 days, n=5) in mice induced hypertension, shifted the pressure-natriuresis plot to the right, and increased the renal expressions of NCC and Na + K + /ATPase, α subunit, relative to vehicle-treated mice (n=5). Activation of D 4 R decreased AT 1 R expression and Na + K + /ATPase activity in rat renal proximal tubule cells (RPTCs). Therefore, we studied two groups: subjects 18 to 75 years old with a DSM-IV diagnosis of schizophrenia or schizoaffective disorder treated with CLZ for >2 months (CLZ, N=32) and untreated healthy controls ( HC , N=20) without psychiatric illness. There were no significant differences in blood pressure (BP) between the two groups since more than half of the CLZ participants received antihypertensive medication. We studied RPTCs cultured from the participant’s urine (CLZ, N=18; HC, N=9) and treated those cells with 100 nM CLZ. CLZ decreased D 4 R expression in both HC and CLZ groups ( HC , VEH 1.04±0.17 vs CLZ 0.73±0.09, n=9, paired t-test, P<0.05; CLZ, VEH 1.02±0.09 vs CLZ 0.75±0.06, n=16, paired t-test, P<0.01). CLZ also increased reactive oxygen species (Rosstar550 assay) in the HC but not CLZ-treated group ( HC , VEH 0.92±0.07 vs CLZ 1.02±0.09, n=8, paired t-test , P<0.05; CLZ VEH 1.09±0.18 vs CLZ 1.07±0.18, n=14). The density of plasma membrane AT 1 R tended to be higher in CLZ-treated hypertensive (CLX HP) than the CLZ-treated normotensive participants (CLZ NP): ( HC , 0.28±0.08 n=8; CLZ NP, 0.10±0.02 n=4; CLZ HP, 0.81±0.31 n=7). Thus, CLZ may induce hypertension through a D 4 R/AT 1 R-mediated mechanism similar to that reported in D 4 R knockout mice. Additional studies will determine if CLZ increases sodium transport in human RPTCs.

Sarpogrelate hydrochloride ameliorates diabetic nephropathy associated with inhibition of macrophage activity and inflammatory reaction in db/db mice.

The aim of this study was to evaluate the effects of sarpogrelate hydrochloride (SH), a selective serotonin 2A receptor antagonist, on diabetic nephropathy in a type 2 diabetes mouse model. We treated db/m and db/db mice with SH (30 mg/kg/day) for 12 weeks. Rat renal proximal tubule cells (NRK-52E) and mouse macrophages (Raw 264.7) were stimulated by high glucose (30 mM glucose) or LPS (100 ng/ml) with or without SH (20 μM). We found that SH treatment increased serum adiponectin level and decreased urinary albumin, macrophage infiltration to glomeruli, and renal inflammatory and fibrosis signals, which were highly expressed in diabetic mice. Proximal tubule cells treated with high glucose (30 mM) also showed increased inflammatory and fibrosis signals. However, SH (20 μM) treatment reduced these changes. Moreover, SH treatment inhibited LPS-stimulated macrophage migration and activation. These findings suggest that SH ameliorates diabetic nephropathy not only by suppressing macrophage infiltration, but also by anti-inflammatory and anti-fibrotic effects.

Differential roles of 3-Hydroxyflavone and 7-Hydroxyflavone against nicotine-induced oxidative stress in rat renal proximal tubule cells.

Plant flavonoids are well known as antioxidants against oxidative stress induced by exposure to external pollutants. Nicotine (NIC) is one of those agents which increases renal oxidative stress, an important factor in the pathogenesis of renal epithelial injury in smokers. Although several studies had been conducted on flavonoids and oxidative stress, the mechanism of the protective pathways are not fully understood. Here, we present studies on antioxidant properties of two mono-hydroxyflavone isomers, 3-hydroxyflanove (3HF)- and 7-hydroxyflavone (7HF), against nicotine-associated oxidative stress and injury in cultured renal proximal tubule cells and correlate their antioxidant properties with their chemical structure. Our data clearly demonstrates, for the first time, that while both 3HF and 7HF protect renal cells from NIC-associated cytotoxicity, the mechanism of their action is different: 3HF elicits protective activity via the PKA/CREB/MnSOD pathway while 7HF does so via the ERK/Nrf2/HO-1 pathway. Molecular docking and dynamics simulations with two major signaling pathway proteins showed significant differences in the binding energies of 3HF (-5.67 and -7.39 kcal.mol-1) compared to 7HF (-5.41 and -8.55 kcal.mol-1) in the matrices of CREB and Keap1-Nrf2 proteins respectively, which corroborate with the observed differences in their protective properties in the renal cells. The implications of this novel explorative study is likely to promote the understanding of the mechanisms of the antioxidative functions of different flavones.

Coenzyme Q10 protects renal proximal tubule cells against nicotine-induced apoptosis through induction of p66shc-dependent antioxidant responses.

Chronic nicotine exposure (via smoking, E-cigarettes) increases oxidative stress in the kidney that sensitizes it to additional injury in experimental models and in the renal patient. The pro-apoptotic p66shc protein-via serine36 phosphorylation that facilitates its mitochondrial translocation and therein cytochrome c binding-generates oxidative stress that leads to injury of renal proximal tubule cells during chronic nicotine exposure. Coenzyme Q10-a clinically safe antioxidant-has been used against nicotine/smoke extract-associated oxidative stress in various non-renal cells. This study explored the anti-oxidant/anti-apoptotic effect of Coenzyme Q10 on nicotine-induced oxidative stress and its impact on p66shc in cultured rat renal proximal tubule cells (NRK52E). We studied the anti-oxidant effect of 10µM Coenzyme Q10 using various mutants of the p66shc gene and also determined the induction of selected anti-oxidant entities (antioxidant response element, promoter of the manganese superoxide dismutase gene) in reporter luciferase assay during oxidative stress induced by 200µM nicotine. Our studies revealed that Coenzyme Q10 strongly inhibits nicotine-mediated production of reactive oxygen species and consequent apoptosis that requires serine36 phosphorylation but not mitochondrial translocation/cytochrome c binding of p66shc. While both nicotine and Coenzyme Q10 stimulates the p66shc promoter, only nicotine exposure results in mitochondrial translocation of p66shc. In contrast, the Coenzyme Q10-stimulated and non-mitochondrial p66shc activates the anti-oxidant manganese superoxide dismutase promoter via the antioxidant response elements and hence, rescues cells from nicotine-induced oxidative stress and consequent apoptosis.

Aristolochic acids - Induced transcriptomic responses in rat renal proximal tubule cells in vitro.

Aristolochic acids (AAs) are the active components of herbal drugs derived from Aristolochia species that have been used for medicinal purposes since antiquity. However, AAs have recently been discovered to be highly nephrotoxic and induced urothelial cancer in humans and malignant tumors in the kidney and urinary tract of rodents. In this study, we exposed rat renal proximal tubule cells in vitro to a sub-cytotoxic level of AAs at three different time points (6 h, 24 h and 72 h). We then analyzed the gene expression profile after the compound exposure. Functional analysis with Ingenuity Pathways Analysis and DAVID tools revealed that at the late time point (72 h) there are many significantly altered genes involved in cancer-related pathways such as p53 signaling.MIAMI-compliant microarray data are deposited in the NCBI GEO database under accession number GSE68687 and can be found at: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE68687.

Transcriptomic alterations induced by Monuron in rat and human renal proximal tubule cells in vitro and comparison to rat renal-cortex in vivo

Monuron (1,1-dimethyl-3-(4-chlorophenyl)urea) is a widely used herbicide in developing countries although concerns have been raised about its toxicity and carcinogenicity.

Catalase overexpression prevents nuclear factor erythroid 2-related factor 2 stimulation of renal angiotensinogen gene expression, hypertension, and kidney injury in diabetic mice.

This study investigated the impact of catalase (Cat) overexpression in renal proximal tubule cells (RPTCs) on nuclear factor erythroid 2–related factor 2 (Nrf2) stimulation of angiotensinogen (Agt) gene expression and the development of hypertension and renal injury in diabetic Akita transgenic mice. Additionally, adult male mice were treated with the Nrf2 activator oltipraz with or without the inhibitor trigonelline. Rat RPTCs, stably transfected with plasmid containing either rat Agt or Nrf2 gene promoter, were also studied. Cat overexpression normalized systolic BP, attenuated renal injury, and inhibited RPTC Nrf2, Agt, and heme oxygenase-1 (HO-1) gene expression in Akita Cat transgenic mice compared with Akita mice. In vitro, high glucose level, hydrogen peroxide, and oltipraz stimulated Nrf2 and Agt gene expression; these changes were blocked by trigonelline, small interfering RNAs of Nrf2, antioxidants, or pharmacological inhibitors of nuclear factor-κB and p38 mitogen-activated protein kinase. The deletion of Nrf2-responsive elements in the rat Agt gene promoter abolished the stimulatory effect of oltipraz. Oltipraz administration also augmented Agt, HO-1, and Nrf2 gene expression in mouse RPTCs and was reversed by trigonelline. These data identify a novel mechanism, Nrf2-mediated stimulation of intrarenal Agt gene expression and activation of the renin-angiotensin system, by which hyperglycemia induces hypertension and renal injury in diabetic mice.

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<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<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<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.

Regulation of renalase expression by D5 dopamine receptors in rat renal proximal tubule cells

The dopaminergic and sympathetic systems interact to regulate blood pressure. Our previous studies showed regulation of α1-adrenergic receptor function by D1-like dopamine receptors in vascular smooth muscle cells. Because renalase could regulate circulating epinephrine levels and dopamine production in renal proximal tubules (RPTs), we tested the hypothesis that D1-like receptors regulate renalase expression in kidney. The effect of D1-like receptor stimulation on renalase expression and function was measured in immortalized RPT cells from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs). We found that the D1-like receptor agonist fenoldopam (10(-7)-10(-5) mol/l) increased renalase protein expression and function in WKY RPT cells but decreased them in SHR cells. Fenoldopam also increased renalase mRNA levels in WKY but not in SHR cells. In contrast, fenoldopam increased the degradation of renalase protein in SHR cells but not in WKY cells. The regulation of renalase by the D1-like receptor was mainly via the D5 receptor because silencing of the D5 but not D1 receptor by antisense oligonucleotides blocked the stimulatory effect of the D1-like receptor on renalase expression in WKY cells. Moreover, inhibition of PKC, by the PKC inhibitor 19-31, blocked the stimulatory effect of fenoldopam on renalase expression while stimulation of PKC, by a PKC agonist (PMA), increased renalase expression, indicating that PKC is involved in the process. Our studies suggest that the D5 receptor positively regulates renalase expression in WKY but not SHR RPT cells; aberrant regulation of renalase by the D5 receptor may be involved in the pathogenesis of hypertension.

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<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<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<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<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<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<0.05, n=4 vs SHR) and in SHR CAV1beta 5.0±0.3 (p<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.

Substrate- and Cell Contact-Dependent Inhibitor Affinity of Human Organic Cation Transporter 2: Studies with Two Classical Organic Cation Substrates and the Novel Substrate Cd2+

Polyspecific organic cation transporter Oct2 from rat (gene Slc22A2) has been previously shown to transport Cs(+). Here we report that human OCT2 (hOCT2) is able to transport Cd(2+) showing substrate saturation with a Michaelis-Menten constant (Km) of 54 ± 5.8 μM. Uptake of Cd(2+) by hOCT2 was inhibited by typical hOCT2 ligands (unlabeled substrates and inhibitors), and the rate of uptake was decreased by a point mutation in a substrate binding domain of hOCT2. Incubation of hOCT2 overexpressing human embryonic kidney 293 cells (HEK-hOCT2-C) or rat renal proximal tubule cells expressing rOct2 (NRK-52E-C) with Cd(2+) resulted in an increased level of apoptosis that was reduced by OCT2 inhibitory ligand cimetidine(+). HEK-hOCT2-C exhibited different functional properties when they were confluent or had been dissociated by removal of Ca(2+) and Mg(2+). Only confluent HEK-hOCT2-C transported Cd(2+), and confluent and dissociated cells exhibited different potencies for inhibition of uptake of 1-methyl-4-phenylpyridinium(+) (MPP(+)) by Cd(2+), MPP(+), tetraethylammonium(+), cimetidine(+), and corticosterone. In confluent HEK-hOCT2-C, largely different inhibitor potencies were obtained upon comparison of inhibition of Cd(2+) uptake, 4-[4-(dimethylamino)styryl]-N-methylpyridinium(+) (ASP(+)) uptake, and MPP(+) uptake using substrate concentrations far below the respective Km values. Employing a point mutation in the previously identified substrate binding site of rat Oct1 produced evidence that short distance allosteric effects between binding sites for substrates and inhibitors are involved in substrate-dependent inhibitor potency. Substrate-dependent inhibitor affinity is probably a common property of OCTs. To predict interactions between drugs that are transported by OCTs and inhibitory drugs, it is necessary to employ the specific transported drug rather than a model substrate for in vitro measurements.

764 SYNERGISTIC EFFECTS OF RENAL DOPAMINE AND GASTRIN RECEPTORS IN HYPERTENSION

764 SYNERGISTIC EFFECTS OF RENAL DOPAMINE AND GASTRIN RECEPTORS IN HYPERTENSION

Shank2 redistributes with NaPilla during regulated endocytosis.

Serum phosphate levels are acutely impacted by the abundance of sodium-phosphate cotransporter IIa (NaPiIIa) in the apical membrane of renal proximal tubule cells. PSD-95/Disks Large/Zonula Occludens (PDZ) domain-containing proteins bind NaPiIIa and likely contribute to the delivery, retention, recovery, and trafficking of NaPiIIa. Shank2 is a distinctive PDZ domain protein that binds NaPiIIa. Its role in regulating NaPiIIa activity, distribution, and abundance is unknown. In the present in vivo study, rats were maintained on a low-phosphate diet, and then plasma phosphate levels were acutely elevated by high-phosphate feeding to induce the recovery, endocytosis, and degradation of NaPiIIa. Western blot analysis of renal cortical tissue from rats given high-phosphate feed showed NaPiIIa and Shank2 underwent degradation. Quantitative immunofluorescence analyses, including microvillar versus intracellular intensity ratios and intensity correlation quotients, showed that Shank2 redistributed with NaPiIIa during the time course of NaPiIIa endocytosis. Furthermore, NaPiIIa and Shank2 trafficked through distinct endosomal compartments (clathrin, early endosomes, lysosomes) with the same temporal pattern. These in vivo findings indicate that Shank2 is positioned to coordinate the regulated endocytic retrieval and downregulation of NaPiIIa in rat renal proximal tubule cells.

Reduced 22Na+ Uptake and Dysregulation of NHERF2/Akt/NOS Pathway in Rat Renal Proximal Tubule Cells Stably Overexpressing p22phox

We showed that superoxide (O2−) reduced renal proximal tubule (PT) reabsorption (Hypertension. 2009 Oct.), but, the molecular mechanisms are unknown. We tested the hypothesis that O2− impairs Na+‐H+ exchanger 3 (NHE3) activity. A stably p22phox transfected rat PT cell line (s‐p22phox), compared to empty vector transfected wild type cells (Wt), had increased p22phox mRNA and protein by 4.1‐ and 1.59‐fold, and O2− by 2.2‐ fold but reduced 22Na+uptake by 36% (all P<0.05). Immunoblot assay of s‐p22phox cells showed no change in NHE3 but increased expression of inhibitory Na+–H+ exchanger regulatory factor 2 (NHERF2), neuronal and inducible nitric oxide synthase (nNOS & iNOS) by 183%, 293% and 372%, respectively (all P<0.05). Since activated Akt regulates NOS and NHERF2, we compared the Akt in both cell types. Total Akt (t‐Akt) protein abundance was similar but the phosphorylated Akt at ser473 (p‐Aktser473) was markedly increased leading to a 207% higher p‐Akt/t‐Akt ratio in s‐p22phox cells. Knockdown of NHEFR2 by specific siRNA in s‐p22phox cells increased significantly (25±4.9%; P<0.05) 22Na+uptake. In conclusion, overexpression of p22phox in PT cells inhibits NHE3 transport activity and upregulates NHERF2, nNOS and iNOS, and phosphorylated Akt. This dysregulation of NHERF2/Akt/NOS signaling pathways by O2− impairs PT function. This provides a novel insight into the molecular mechanisms underlying the effects of oxidative stress on ion and fluid homeostasis in the kidney.