Rat Kidney Proximal Tubule Research Articles

Distinct concentration-dependent oxidative stress profiles by cadmium in a rat kidney proximal tubule cell line

Levels and chemical species of reactive oxygen/nitrogen species (ROS/RNS) determine oxidative eustress and distress. Abundance of uptake pathways and high oxygen consumption for ATP-dependent transport makes the renal proximal tubule particularly susceptible to cadmium (Cd2+)-induced oxidative stress by targeting ROS/RNS generation or antioxidant defence mechanisms, such as superoxide dismutase (SOD) or H2O2-metabolizing catalase (CAT). Though ROS/RNS are well-evidenced, the role of distinct ROS profiles in Cd2+ concentration-dependent toxicity is not clear. In renal cells, Cd2+ (10–50 µM) oxidized dihydrorhodamine 123, reaching a maximum at 2–3 h. Increases (up to fourfold) in lipid peroxidation by TBARS assay and H2O2 by Amplex Red were evident within 30 min. ROS and loss in cell viability by MTT assay with 50 µM Cd2+ could not be fully reversed by SOD mimetics Tempol and MnTBAP nor by SOD1 overexpression, whereas CAT expression and α-tocopherol were effective. SOD and CAT activities were attenuated below controls only with >6 h 50 µM Cd2+, yet augmented by up to 1.5- and 1.2-fold, respectively, by 10 µM Cd2+. Moreover, 10 µM, but not 25–50 µM Cd2+, caused 1.7-fold increase in superoxide anion (O2•−), detected by dihydroethidium, paralled by loss in cell viability, that was abolished by Tempol, MnTBAP, α-tocopherol and SOD1 or CAT overexpression. H2O2-generating NADPH oxidase 4 (NOX4) was attenuated by ~50% with 10 µM Cd2+ at 3 h compared to upregulation by 50 µM Cd2+ (~1.4-fold, 30 min), which was sustained for 24 h. In summary, O2•− predominates with low–moderate Cd2+, driving an adaptive response, whereas oxidative stress by elevated H2O2 at high Cd2+ triggers cell death signaling pathways.HighlightsDifferent levels of reactive oxygen species are generated, depending on cadmium concentration.Superoxide anion predominates and H2O2 is suppressed with low cadmium representing oxidative eustress.High cadmium fosters H2O2 by inhibiting catalase and increasing NOX4 leading to oxidative distress.Superoxide dismutase mimetics and overexpression were less effective with high versus low cadmium.Oxidative stress profile could dictate downstream signalling pathways.

Protein Kinase C-δ Mediates Kidney Tubular Injury in Cold Storage-Associated Kidney Transplantation.

Kidney injury associated with cold storage is a determinant of delayed graft function and the long-term outcome of transplanted kidneys, but the underlying mechanism remains elusive. We previously reported a role of protein kinase C-δ (PKCδ) in renal tubular injury during cisplatin nephrotoxicity and albumin-associated kidney injury, but whether PKCδ is involved in ischemic or transplantation-associated kidney injury is unknown. To investigate PKCδ's potential role in injury during cold storage-associated transplantation, we incubated rat kidney proximal tubule cells in University of Wisconsin (UW) solution at 4°C for cold storage, returning them to normal culture medium at 37°C for rewarming. We also stored kidneys from donor mice in cold UW solution for various durations, followed by transplantation into syngeneic recipient mice. We observed PKCδ activation in both in vitro and in vivo models of cold-storage rewarming or transplantation. In the mouse model, PKCδ was activated and accumulated in mitochondria, where it mediated phosphorylation of a mitochondrial fission protein, dynamin-related protein 1 (Drp1), at serine 616. Drp1 activation resulted in mitochondrial fission or fragmentation, accompanied by mitochondrial damage and tubular cell death. Deficiency of PKCδ in donor kidney ameliorated Drp1 phosphorylation, mitochondrial damage, tubular cell death, and kidney injury during cold storage-associated transplantation. PKCδ deficiency also improved the repair and function of the renal graft as a life-supporting kidney. An inhibitor of PKCδ, δV1-1, protected kidneys against cold storage-associated transplantation injury. These results indicate that PKCδ is a key mediator of mitochondrial damage and renal tubular injury in cold storage-associated transplantation and may be an effective therapeutic target for improving renal transplant outcomes.

Role of Myosins II and VI in Mediating Hormonal Regulation of NHE3 Activity in the Rat Renal Proximal Tubule

Rapid regulation of NHE3 activity may occur through redistribution of the transporter between the body and the base of the proximal tubule microvilli (PT MV). Myosins are motor proteins that move along actin filaments. The present study tested the hypothesis that myosins II (myo II), which moves cargo away from cell center, and VI (myo VI), which moves cargo toward cell center, may mediate NHE3 redistribution within the MV in response to stimuli. Renal PT NHE3 activity was evaluated in Wistar rats by net bicarbonate reabsorption (JHCO3−) in the presence or absence of 10−10 M angiotensin II (Ang II), 10−4 M forskolin (FSK), a cAMP‐elevating agent, 10−5 M blebbistatin (a specific myo II inhibitor) or 4×10−6 M 2,4,6‐triiodophenol (TIP) (a myo VI inhibitor). As expected, Ang II increased [4.36 ± 0.18 nmol/cm2.s (N = 5)] whereas FSK reduced [1.26 ± 0.10 nmol/cm2.s (N = 5)] JHCO3− compared to CTRL [2.74 ± 0.13 nmol/cm2.s (N = 4)]. Simultaneous tubular perfusion of Ang II with blebbistatin prevent Ang II‐induced NHE3 stimulation [2.92 ± 0.14 nmol/cm2.s (N = 5), P < 0.001]. Simultaneous tubular perfusion of FSK with TIP reduced the inhibition with FSK alone [1.78 ± 0.15 (N = 4) vs. 1.26 ± 0.10 nmol/cm2.s (N = 5), P < 0.05] on JHCO3−. The localization of NHE3 and myosins IIB and VI was assessed by confocal immunofluorescence in Wistar rats kidney after a 30 min pressor dose of Ang II (50 ng/kg/min). NHE3 and myo VI shift from base to body of MV after Ang II; myo IIB remains mostly enriched at the MV base. To assess NHE3 activity in OKP cells, experimental pH recovery points were fitted to simple exponential decay curves (y = A·e(−kt) + y0) in order to predict the rate constant (k) of each recovery curve. FSK reduced NHE3 activity [0.025 ± 0.003 s−1 (N = 4)] compared to CTRL [0.048 ± 0.007 s−1 (N = 5)]. siRNA for myo VI efficiently reduced its expression by 64 %; the inhibitory effect of FSK on NHE3 activity was completely abolished by siRNA myo VI [0.043 ± 0.005 s−1 (N = 4)]. These results provide further evidence for myosin II and VI regulation of NHE3 redistribution in the rat kidney proximal tubule.Support or Funding InformationFAPESPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Initial autophagic protection switches to disruption of autophagic flux by lysosomal instability during cadmium stress accrual in renal NRK-52E cells

The renal proximal tubule (PT) is the major target of cadmium (Cd2+) toxicity where Cd2+ causes stress and apoptosis. Autophagy is induced by cell stress, e.g., endoplasmic reticulum (ER) stress, and may contribute to cell survival or death. The role of autophagy in Cd2+-induced nephrotoxicity remains unsettled due to contradictory results and lack of evidence for autophagic machinery damage by Cd2+. Cd2+-induced autophagy in rat kidney PT cell line NRK-52E and its role in cell death was investigated. Increased LC3-II and decreased p62 as autophagy markers indicate rapid induction of autophagic flux by Cd2+ (5-10µM) after 1h, accompanied by ER stress (increased p-PERK, p-eIF2α, CHOP). Cd2+ exposure exceeding 3h results in p62/LC3-II accumulation, but diminished effect of lysosomal inhibitors (bafilomycin A1, pepstatin A +E-64d) on p62/LC3-II levels, indicating decreased autophagic flux and cargo degradation. At 24h exposure, Cd2+ (5-25µM) activates intrinsic apoptotic pathways (Bax/Bcl-2, PARP-1), which is not evident earlier (≤6h) although cell viability by MTT assay is decreased. Autophagy inducer rapamycin (100nM) does not overcome autophagy inhibition or Cd2+-induced cell viability loss. The autophagosome-lysosome fusion inhibitor liensinine (5μM) increases CHOP and Bax/Bcl-2-dependent apoptosis by low Cd2+ stress, but not by high Cd2+. Lysosomal instability by Cd2+ (5μM; 6h) is indicated by increases in cellular sphingomyelin and membrane fluidity and decreases in cathepsins and LAMP1. The data suggest dual and temporal impact of Cd2+ on autophagy: Low Cd2+ stress rapidly activates autophagy counteracting damage but Cd2+ stress accrual disrupts autophagic flux and lysosomal stability, possibly resulting in lysosomal cell death.

Estrogen directly and specifically downregulates NaPi-IIa through the activation of both estrogen receptor isoforms (ERα and ERβ) in rat kidney proximal tubule.

We have previously demonstrated that estrogen (E2) downregulates phosphate transporter NaPi-IIa and causes phosphaturia and hypophosphatemia in ovariectomized rats. In the present study, we examined whether E2 directly targets NaPi-IIa in the proximal tubule (PT) and studied the respective roles of estrogen receptor isoforms (ERα and ERβ) in the downregulation of NaPi-IIa using both in vivo and an in vitro expression systems. We found that estrogen specifically downregulates NaPi-IIa but not NaPi-IIc or Pit2 in the kidney cortex. Proximal tubules incubated in a "shake" suspension with E2 for 24 h exhibited a dose-dependent decrease in NaPi-IIa protein abundance. Results from OVX rats treated with specific agonists for either ERα [4,4',4″;-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol, PPT] or ERβ [4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol, DPN] or both (PPT + DPN), indicated that only the latter caused a sharp downregulation of NaPi-IIa, along with significant phosphaturia and hypophosphatemia. Lastly, heterologous expression studies demonstrated that estrogen downregulated NaPi-IIa only in U20S cells expressing both ERα and ERβ, but not in cells expressing either receptor alone. In conclusion, these studies demonstrate that rat PT cells express both ERα and ERβ and that E2 induces phosphaturia by directly and specifically targeting NaPi-IIa in the PT cells. This effect is mediated via a mechanism involving coactivation of both ERα and ERβ, which likely form a functional heterodimer complex in the rat kidney proximal tubule.

Glutathione and mitochondria determine acute defense responses and adaptive processes in cadmium-induced oxidative stress and toxicity of the kidney.

Cadmium (Cd(2+)) induces oxidative stress that ultimately defines cell fate and pathology. Mitochondria are the main energy-producing organelles in mammalian cells, but they also have a central role in formation of reactive oxygen species, cell injury, and death signaling. As the kidney is the major target in Cd(2+) toxicity, the roles of oxidative signature and mitochondrial function and biogenesis in Cd(2+)-related stress outcomes were investigated in vitro in cultured rat kidney proximal tubule cells (PTCs) (WKPT-0293 Cl.2) for acute Cd(2+) toxicity (1-30 µM, 24 h) and in vivo in Fischer 344 rats for sub-chronic Cd(2+) toxicity (1 mg/kg CdCl2 subcutaneously, 13 days). Whereas 30 µM Cd(2+) caused ~50 % decrease in cell viability, apoptosis peaked at 10 µM Cd(2+) in PTCs. A steep, dose-dependent decline in reduced glutathione (GSH) content occurred after acute exposure and an increase of the oxidized glutathione (GSSG)/GSH ratio. Quantitative PCR analyses evidenced increased antioxidative enzymes (Sod1, Gclc, Gclm), proapoptotic Bax, metallothioneins 1A/2A, and decreased antiapoptotic proteins (Bcl-xL, Bcl-w). The positive regulator of mitochondrial biogenesis Pparγ and mitochondrial DNA was increased, and cellular ATP was unaffected with Cd(2+) (1-10 µM). In vivo, active caspase-3, and hence apoptosis, was detected by FLIVO injection in the kidney cortex of Cd(2+)-treated rats together with an increase in Bax mRNA. However, antiapoptotic genes (Bcl-2, Bcl-xL, Bcl-w) were also upregulated. Both GSSG and GSH increased with chronic Cd(2+) exposure with no change in GSSG/GSH ratio and augmented expression of antioxidative enzymes (Gpx4, Prdx2). Mitochondrial DNA, mitofusin 2, and Pparα were increased indicating enhanced mitochondrial biogenesis and fusion. Hence, these results demonstrate a clear involvement of higher mitochondria copy numbers or mass and mitochondrial function in acute defense against oxidative stress induced by Cd(2+) in renal PTCs as well as in adaptive processes associated with chronic renal Cd(2+) toxicity.

G-protein βγ subunit dimers modulate kidney repair after ischemia-reperfusion injury in rats.

Heterotrimeric G-proteins play a crucial role in the control of renal epithelial cell function during homeostasis and in response to injury. In this report, G-protein βγ subunit (Gβγ) dimer activity was evaluated during the process of tubular repair after renal ischemia-reperfusion injury (IRI) in male Sprague Dawley rats. Rats were treated with a small molecule inhibitor of Gβγ activity, gallein (30 or 100 mg/kg), 1 hour after reperfusion and every 24 hours for 3 additional days. After IRI, renal dysfunction was prolonged after the high-dose gallein treatment in comparison with vehicle treatment during the 7-day recovery period. Renal tubular repair in the outer medulla 7 days after IRI was significantly (P < 0.001) attenuated after treatment with high-dose gallein (100 mg/kg) in comparison with low-dose gallein (30 mg/kg), or the vehicle and fluorescein control groups. Gallein treatment significantly reduced (P < 0.05) the number of proliferating cell nuclear antigen-positive tubular epithelial cells at 24 hours after the ischemia-reperfusion phase in vivo. In vitro application of gallein on normal rat kidney (NRK-52E) proximal tubule cells significantly reduced (P < 0.05) S-phase cell cycle entry compared with vehicle-treated cells as determined by 5'-bromo-2'-deoxyuridine incorporation. Taken together, these data suggest that Gβγ signaling contributes to the maintenance and repair of renal tubular epithelium and may be a novel therapeutic target for the development of drugs to treat acute kidney injury.

Abstract 102: Aldosterone Evokes Senescence-associated Secreting Phenotype And Subsequent Apoptosis In Proximal Tubular Cells

We previously reported that aldosterone/mineralocorticoid receptor activation caused cell senescence via p21, a cell cycle regulator, -dependent pathway in proximal tubules of rat kidney and human proximal tubular cells. However, functional influences of cell senescent on the kidney are still unclear. In the present study, we hypothesized that aldosterone-induced cell senescence caused cell apoptosis by increasing the secretion of apoptotic factors (n=6 in all groups). Aldosterone increased senescent cells at day 3 and day 5 in human proximal tubular cells, and the changes were accompanied by the increase in p21 protein expression (day 3: 2.9±0.7 fold, day 5: 6.5±0.9 fold, p&lt;0.05) and TNF-α mRNA (day 3: 2.3±0.3 fold, day 5: 2.4±0.6 fold, p&lt;0.05). In addition, aldosterone also increased secretion of TNF-α into the culture medium compared with vehicle-treated group (day 3: 15.7±1.2 vs 7.2±1.6 pg/ml; day 5: 33.3±5.7 vs 11.8±2.3 pg/ml; p&lt;0.05, respectively): gene silencing of p21 abolished these changes. Meanwhile, 3-day treatment with aldosterone showed no sign of cell apoptosis, which was evaluated by terminal nick end labeling (TUNEL) and annexin V/propidium iodide (PI) staining, and 5-day treatment with aldosterone increased cell apoptosis (TUNEL: aldosterone, 6.4±1.5%; vehicle, 0.6±0.3%; annexin V/PI: aldosterone, 5.8±0.8%; vehicle, 0.8±0.3%; p&lt;0.05). Importantly, p21 siRNA and TNF-α neutralizing antibody abolished these aldosterone-induced apoptotic changes (TUNEL: 1.4±0.1 and 0.9±0.5%; annexin V/PI: 0.9±0.3 and 1.5±0.9%; respectively). These results indicate that aldosterone increases TNF-α synthesis and secretion in proximal tubular cells via p21/senescence-dependent pathway, and that the secreted TNF-α plays an important role as an autocrine/paracrine factor to mediate cell apoptosis, which may be involved in aldosterone-induced renal damage.

Aldosterone induces p21-regulated apoptosis via increased synthesis and secretion of tumour necrosis factor-α in human proximal tubular cells

1. Aldosterone has been shown to mediate p21-dependent cellular senescence in rat kidney proximal tubules in vivo and in cultured human proximal tubular cells. The p21-induced senescent cells express higher levels of apoptotic cytokines, such as tumour necrosis factor (TNF)-α compared with non-senescent cells. The aim of the present study was to investigate the hypothesis that aldosterone increases proximal tubular apoptosis by increasing the secretion of apoptosis-inducing factors through a p21-dependent mechanism. 2. Human proximal tubular cells were incubated with aldosterone (10 nmol/L) and cell senescence was detected by senescence-associated β-galactosidase staining and expression of p21. Apoptosis was analysed by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling and annexin/propidium iodide staining, whereas p21 localization was determined by immunofluorescence. 3. Exposure of cells to aldosterone for 3 or 5 days increased senescence-associated β-galactosidase staining, p21 and TNF-α mRNA expression and secretion of TNF-α into the culture medium. These changes were abolished by gene silencing of p21. Aldosterone failed to increase the number of apoptotic cells on day 3, but did increase them on day 5. A neutralizing antibody against TNF-α prevented the aldosterone-induced apoptotic changes. Aldosterone did not affect localization of p21. 4. These findings indicate that aldosterone increases TNF-α synthesis and secretion in proximal tubular cells via p21/senescence-dependent cell phenotypic changes and that the TNF-α secreted plays an important role as a paracrine factor in mediating cell apoptosis, indicating a possible involvement in aldosterone-induced renal damage.

Localization and Functional Characterization of the Rat Oatp4c1 Transporter in an In Vitro Cell System and Rat Tissues

The organic anion transporting polypeptide 4c1 (Oatp4c1) was previously identified as a novel uptake transporter predominantly expressed at the basolateral membrane in the rat kidney proximal tubules. Its functional role was suggested to be a vectorial transport partner of an apically-expressed efflux transporter for the efficient translocation of physiological substrates into urine, some of which were suggested to be uremic toxins. However, our in vitro studies with MDCKII cells showed that upon transfection rat Oatp4c1 polarizes to the apical membrane. In this report, we validated the trafficking and function of Oatp4c1 in polarized cell systems as well as its subcellular localization in rat kidney. Using several complementary biochemical, molecular and proteomic methods as well as antibodies amenable to immunohistochemistry, immunofluorescence, and immunobloting we investigated the expression pattern of Oatp4c1 in polarized cell systems and in the rat kidney. Collectively, these data demonstrate that rat Oatp4c1 traffics to the apical cell surface of polarized epithelium and localizes primarily in the proximal straight tubules, the S3 fraction of the nephron. Drug uptake studies in Oatp4c1-overexpressing cells demonstrated that Oatp4c1-mediated estrone-3-sulfate (E3S) uptake was pH-dependent and ATP-independent. These data definitively demonstrate the subcellular localization and histological location of Oatp4c1 and provide additional functional evidence that reconciles expression-function reports found in the literature.

ERK1/2-dependent bestrophin-3 expression prevents ER-stress-induced cell death in renal epithelial cells by reducing CHOP

Upon endoplasmic reticulum (ER) stress induction, cells endeavor to survive by engaging the adaptive stress response known as the unfolded protein response or by removing aggregated proteins via autophagy. Chronic ER stress culminates in apoptotic cell death, which involves induction of pro-apoptotic CHOP. Here, we show that bestrophin-3 (Best-3), a protein previously associated with Ca2+-activated Cl− channel activity, is upregulated by the ER stressors, thapsigargin (TG), tunicamycin (TUN) and the toxic metal Cd2+. In cultured rat kidney proximal tubule cells, ER stress, CHOP and cell death were induced after 6h by Cd2+ (25μM), TG (3μM) and TUN (6μM), were associated with increased cytosolic Ca2+ and downstream formation of reactive oxygen species and attenuated by the Ca2+ chelator BAPTA-AM (10μM), the antioxidant α-tocopherol (100μM), or overexpression of catalase (CAT). Immunofluorescence staining showed Best-3 expression in the plasma membrane, nuclei and intracellular compartments, though not in the ER, in cultured cells and rat kidney cortex sections. Best-3 mRNA was augmented by ER stress and signaled through increased Ca2+, oxidative stress and ERK1/2 phosphorylation, because it was attenuated by α-tocopherol, CAT expression, BAPTA-AM, calmodulin kinase inhibitor calmidazolium (40μM), ERK1/2 inhibitor U0126 (10μM), and ERK1/2 RNAi. Knockdown of Best-3 resulted in decreased cell number consequentially of cell death, as determined by nuclear staining and PARP-1 cleavage. Furthermore, reduced ER stress-cell death by Best-3 overexpression is attributed to diminished CHOP. Since Best-3 overexpression did not affect upstream signaling pathways, we hypothesize that Best-3 possibly interferes with CHOP transcription. From our novel observations, we conclude that ERK1/2-dependent Best-3 activation regulates cell fate decisions during ER stress by suppressing CHOP induction and death.

Renal Glycosuria without Hyperglycemia in Cyclosporine-Treated Rats

Background/Aims The pathogenesis of post-transplant diabetes mellitus is thought to be partly related to the direct toxic effect of cyclosporine on pancreatic β-cells and the resultant decrease in insulin synthesis and secretion. As a result of hyperglycemia , glycosuria may be found in cyclosporine administration. However, mechanism of glycosuria in cyclosporine nephrotoxicity was not clear Methods Cyclosporine was subcutaneously injected to male Sprague-Dawley rats at a daily dose of 7.5 mg/kg (n=6) for 6 weeks. Biochemical data were obtained from urine and plasma samples. Results Cyclosporine treatment caused a remarkable increase in urine volume and a decrease in urine osmolality. Urinary excretion of glucose was remarkably elevated by cyclosporine administration (7±3.0 mg/dL versus 12896±3218 mg/dL; P Conclusion Glycosuria may occur without hyperglycemia in cyclosporine administration. We suggest that cyclosporine may decrease tubular reabsorption of glucose in renal tubular epithelial cells, and then glycosuria could be induced by the altered glucose transporter expressions. We will analyze the glucose transporters in proximal tubule of rat kidney.

ERK1/2‐dependent bestrophin‐3 (Best‐3) expression prevents ER‐stress‐induced cell death of renal epithelial cells by reducing CHOP

Upon ER stress induction, cells endeavor to survive by engaging the adaptive stress response, known as the unfolded protein response, or by removing aggregated proteins. Chronic ER stress leads to apoptosis through induction of pro‐apoptotic CHOP. Here, we show that Best‐3, a Ca2+‐activated Cl−channel, is upregulated by the ER stressors, thapsigargin (TG, 3 μM), tunicamycin (TUN, 6 μM) and the toxic metal Cd2+ (25 μM). In cultured rat kidney proximal tubule cells, ER stress after 6 h by Cd2+, TG and TUN was downstream of reactive oxygen species formation and attenuated by Ca2+ chelator BAPTA‐AM (10 μM) or general antioxidant α‐tocopherol (100 μM). Immunofluorescence staining localized Best‐3 to the plasma membrane, nuclei and intracellularly, though not in the ER, in cultured cells and rat kidney cortex sections. ER stress augmented Best‐3 mRNA and was decreased by α‐tocopherol, BAPTA‐AM, calmodulin kinase inhibitor calmidazolium (40 μM), ERK1/2 inhibitor U0126 (10 μM), and ERK1/2 RNAi. Knockdown of Best‐3 resulted in decreased cell number consequentially of cell death, as determined by nuclear staining and PARP‐1 cleavage. Furthermore, Best‐3 overexpression reduced cell death by Cd2+, TG and TUN, attributed to diminished CHOP. From our novel data, we conclude that ERK1/2‐dependent Best‐3 activation regulates cell fate decisions during ER stress by suppressing CHOP induction and death.Funded by DFG TH345/10‐1

Antioxidant Protection against Pathological Mycotoxins Alterations on Proximal Tubules in Rat Kidney

Background: Ochratoxin A (OTA) was one of the mycotoxins and received attention worldwide because of the hazard it posed to human and animal health, where the kidney was the primary target organ for OTA toxicity. In the other hand, dates served as a good source of natural antioxidants and could potentially be considered as a functional food.Methods: The study was performed in the department of biology in King Abdulaziz University. Animals were gavage administrated and divided into four groups: first group received (sodium bicarbonate), second group received (289 µg OTA /kg B.W. /day), third group received (1mg Ajwa/kg B.W. / day) and fourth group received (289 µg OTA /kg B.W./day+ 1mg Ajwa /kg B.W. / day). Serum (creatinine - urea) levels were measured in each group at the time of tissue collection, some biopsies were fixed in 10% buffered formalin solution for light microscopy processing stained with Haematoxylin and Eosin (H&amp; E.), Periodic Acid-Schiff (PAS) and Masson´s Trichrome (M.T.).Other biopsies were immediately collected into electron microscopy processing. Results: After 28 days, a significant decrease in body weight, kidney weight and relative weight was detected in OTA treated group. Also, Serum (creatinine-urea) level were elevated. The normal cyto-architecture of proximal tubules were lost exhibiting damaged bruch border, degenerated, binucleated and karyomegalic cells. The most destructed ultra-structure was the mitochondria which severely swollen with disintegrated membranes. In Ajwa Date extract-group the proximal tubules were normal, whereas in Ajwa date extract + OTA -group the severity of the lesions was significantly reduced. Conclusion: The present results indicated that, Ajwa date have protective effects and ameliorated the lesions of Ochratoxin nepherotoxicity which might lead to kidney failure. Key words: Ochratoxin A., Ajwa date, proximal tubules, light –structure, ultra –structure, biochemical analysis, morphometry.

Validation of the histological localization and subcellular polarity of the Oatp4c1 transporter in the rat kidney

Recently, the organic anion transporting polypeptide 4c1 (4c1) was identified as a novel uptake transporter predominantly expressed on the basolateral membrane of rat kidney proximal tubules. However, in‐vitro studies with MDCKII cells showed that upon transfection 4c1 polarizes to the apical membrane. We used multiple antibodies (N=4) to validate the subcellular localization of 4c1 in rat tissues and in MDCKII and LLCPK1 cells expressing rat 4c1. Antibodies amenable to immunohistochemistry were generated using N‐ and C‐terminal peptide sequences. Apical and basolateral protein markers were used for colocalization studies. Immunofluorescence studies showed that 4c1 polarizes to the apical surface in the in‐vitro cell models. In rat kidney, colocalization of 4c1 with aquaporin 1 but not calbindin in the subcortical region suggest that apical 4c1 expression is localized in proximal straight tubules. 4c1 also colocalizes with P‐glycoprotein and breast cancer resistance protein, but not with multidrug resistance protein 4 in rat kidney. These results were verified by western blot analysis of brush‐border membrane vesicles isolated from the kidney cortex and conclusively demonstrate the apical expression of 4c1 in the rat kidney. To investigate the physiological role of 4c1, future studies will focus on identifying substrates and the driving force for 4c1 mediated transport.

Ferroportin 1 is expressed basolaterally in rat kidney proximal tubule cells and iron excess increases its membrane trafficking.

Ferroportin 1 (FPN1) is an iron export protein expressed in liver and duodenum, as well as in reticuloendothelial macrophages. Previously, we have shown that divalent metal transporter 1 (DMT1) is expressed in late endosomes and lysosomes of the kidney proximal tubule (PT), the nephron segment responsible for the majority of solute reabsorption. We suggested that following receptor mediated endocytosis of transferrin filtered by the glomerulus, DMT1 exports iron liberated from transferrin into the cytosol. FPN1 is also expressed in the kidney yet its role remains obscure. As a first step towards determining the role of renal FPN1, we localized FPN1 in the PT. FPN1 was found to be located in association with the basolateral PT membrane and within the cytosolic compartment. FPN1 was not expressed on the apical brush-border membrane of PT cells. These data support a role for FPN1 in vectorial export of iron out of PT cells. Furthermore, under conditions of iron loading of cultured PT cells, FPN1 was trafficked to the plasma membrane suggesting a coordinated cellular response to export excess iron and limit cellular iron concentrations.

Regulation of the creatine transporter by AMP-activated protein kinase in kidney epithelial cells

The metabolic sensor AMP-activated protein kinase (AMPK) regulates several transport proteins, potentially coupling transport activity to cellular stress and energy levels. The creatine transporter (CRT; SLC6A8) mediates creatine uptake into several cell types, including kidney epithelial cells, where it has been proposed that CRT is important for reclamation of filtered creatine, a process critical for total body creatine homeostasis. Creatine and phosphocreatine provide an intracellular, high-energy phosphate-buffering system essential for maintaining ATP supply in tissues with high energy demands. To test our hypothesis that CRT is regulated by AMPK in the kidney, we examined CRT and AMPK distribution in the kidney and the regulation of CRT by AMPK in cells. By immunofluorescence staining, we detected CRT at the apical pole in a polarized mouse S3 proximal tubule cell line and in native rat kidney proximal tubules, a distribution overlapping with AMPK. Two-electrode voltage-clamp (TEV) measurements of Na(+)-dependent creatine uptake into CRT-expressing Xenopus laevis oocytes demonstrated that AMPK inhibited CRT via a reduction in its Michaelis-Menten V(max) parameter. [(14)C]creatine uptake and apical surface biotinylation measurements in polarized S3 cells demonstrated parallel reductions in creatine influx and CRT apical membrane expression after AMPK activation with the AMP-mimetic compound 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside. In oocyte TEV experiments, rapamycin and the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (ZMP) inhibited CRT currents, but there was no additive inhibition of CRT by ZMP, suggesting that AMPK may inhibit CRT indirectly via the mammalian target of rapamycin pathway. We conclude that AMPK inhibits apical membrane CRT expression in kidney proximal tubule cells, which could be important in reducing cellular energy expenditure and unnecessary creatine reabsorption under conditions of local and whole body metabolic stress.

Protein localization of SLC26A2 (DTDST) in rat kidney

The SLC26 family represents a group of integral membrane anion transport proteins. Mutations in one member of this protein family, SLC26A2 (DTDST or diastrophic dysplasia sulfate transporter), result in various chondrodysplasias due to undersulfation of proteoglycans in chondrocytes, a major site of DTDST protein expression. DTDST mRNA has been detected in the kidney, but protein expression has not been characterized. Our objective for this study was to determine the protein localization of this sulfate transporter in the kidney. We used immunofluorescence (IMF) techniques with an anti-DTDST monoclonal antibody to examine kidneys harvested from adult rats. Double labeling was performed with antibodies directed against megalin, which is found in the microvillus membrane and coated pits of the proximal tubule. IMF analysis indicated that DTDST protein expression was limited to the microvillus membrane of proximal tubule cells in the renal cortex but absent in glomeruli and other nephron segments. DTDST was also detected in isolated rat kidney proximal tubule microvillus membranes by Western blot analysis, confirming the immunofluorescent localization of the DTDST transporter to this nephron segment. The functional role of the DTDST protein in the kidney is unknown, but it may play a role in proximal tubule sulfate transport.

CFTR and ClC‐5 Chloride Channels Modulate Plasma Membrane H+‐ATPase in Proximal Tubule Cells

H+ transport across membranes by H‐ATPase creates a lumen‐positive voltage, generating an electrochemical potential gradient that limits pump activity. This DP is normally dissipated by a Cl‐ movement, which provides an electric shunt compensating for the transferred positive charge. This work was performed to identify the associated Cl−‐conducting proteins involved in the regulation of plasma membrane (PM) H‐ATPase from rat kidney proximal tubule (PT). Through in vivo stationary microperfusion, it was observed that ATPase‐mediated HCO3− reabsorption was significantly reduced in the presence of the Cl− channels inhibitor NPPB. This effect was confirmed in vitro by measuring the cell pH recovery rates after NH4Cl pulse in a cultured cell line derived from PT, IRPTC. In these cells, even after abolishing the membrane potential with valinomycin, ATPase activity was seen to be still dependent on Cl−. siRNA‐mediated CFTR and ClC‐5 channels knockdown significantly reduced H‐ATPase activity. These results indicate that PM H‐ATPase activity from PT depends on both CFTR and ClC‐5 chloride channels.Supported by FAPESP

Evidence for Aldosterone‐mediated regulation of Na‐K ATPase in kidney proximal tubules

Recent investigations from several laboratories have shown MR expression and increased NHE3, NHE1, and NBCe1 activities in kidney proximal tubules (PT) after aldosterone (Aldo) stimulation. Non‐genomic effects of Aldo have been demonstrated in human kidney PT cells (HK‐2). We hypothesize that Aldo increases Na+/K+ ATPase (Na/K) activity in PT. In the present study we aim to 1) determine if MR is present in human and rat kidney PT 2) determine if Aldo regulates Na/K expression and activity in rat basolateral membranes 3) determine if Aldo increases Na/K subunit in human kidney PT cells. IHC analysis of human and rat kidneys showed presence of MR in PT. Adrenalectomy (ADX) resulted in decreased MR, Na/K, and NHE1 in PT and β and γ ENaC in DCT and CCD. Treatment with Aldo restored MR, Na/K, and NHE1 expression in PT and ENaC expression in DCT and CCD. In human kidney PT cells (HK2, HKC‐5,‐8, and ‐11) Aldo caused translocation of MR into nucleus. Aldo increased Na/K expression and activity in human kidney PT cells. The effects of Aldo on Na/K activity in human kidney PT cells was blocked by specific inhibitors of MR (spironolactone) and by 11β‐HSD inhibitor carbonexolone but not by a GR inhibitor (mifepristone). Treatment with Aldo resulted in decrease in intracellular pH in human kidney PT cells. These results suggest that Aldo may regulate Na/K and NHE1 expression and activity in human and rat kidney PT.Funded by VAMR and AHA