Rat Renal Proximal Tubule Research Articles

Mesenchymal Stem Cell-conditioned Medium Protecting Renal Tubular Epithelial Cells by Inhibiting Hypoxia-inducible Factor-1α and Nuclear Receptor Coactivator-1.

Acute kidney injury (AKI) is characterized by inflammatory infiltration and damage and death of renal tubular epithelial cells (RTECs), in which hypoxia plays an important role. Deferoxamine (DFO) is a well-accepted chemical hypoxia-mimetic agent. Mesenchymal stem cell-conditioned medium (MSC-CM) can reduce local inflammation and repair tissue. In this study, we explored the effect and molecular mechanism of MSC-CM-mediated protection of RTECs under DFO-induced hypoxia. Rat renal proximal tubule NRK-52E cells were treated with different concentrations of DFO for 24 hours, followed by evaluation of RTEC injury, using a Cell Counting Kit-8 (CCK-8) to detect cell viability and western blotting to evaluate the expression of transforming growth factor- beta 1 (TGF-β1), α-smooth muscle actin (α-SMA), and hypoxia-inducible factor-1 alpha (HIF-1α) in NRK-52E cells. Then, three groups of NRK-52E cells were used in experiments, including normal control (NC), 25 μM DFO, and 25 μM DFO + MSC-CM. MSC-CM was obtained from the human umbilical cord. MSC-CM was used to culture cells for 12 hours before DFO treatment, then fresh MSC-CM and 25 μM DFO were added, and cells were cultured for another 24 hours before analysis. Western blotting and cellular immunofluorescence staining showed culture of NRK-52E cells in 25 μM DFO for 24 hours induced HIF-1α and nuclear receptor coactivator-1 (NCoA-1), simulating hypoxia. MSC-CM could inhibit the DFO-induced up-regulation of α-SMA, TGF-β1, HIF-1α and NCoA-1. Our results suggest that MSC-CM has a protective effect on RTECs by down-regulating HIF-1α and NCoA-1, which may be the harmful factors in renal injury.

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.

Evidence That Binding of Cyclic GMP to the Extracellular Domain of NKA (Sodium-Potassium ATPase) Mediates Natriuresis.

Extracellular renal interstitial guanosine cyclic 3',5'-monophosphate (cGMP) inhibits renal proximal tubule (RPT) sodium (Na+) reabsorption via Src (Src family kinase) activation. Through which target extracellular cGMP acts to induce natriuresis is unknown. We hypothesized that cGMP binds to the extracellular α1-subunit of NKA (sodium-potassium ATPase) on RPT basolateral membranes to inhibit Na+ transport similar to ouabain-a cardiotonic steroid. Urine Na+ excretion was measured in uninephrectomized 12-week-old female Sprague-Dawley rats that received renal interstitial infusions of vehicle (5% dextrose in water), cGMP (18, 36, and 72 μg/kg per minute; 30 minutes each), or cGMP+rostafuroxin (12 ng/kg per minute) or were subjected to pressure-natriuresis±rostafuroxin infusion. Rostafuroxin is a digitoxigenin derivative that displaces ouabain from NKA. Renal interstitial cGMP and raised renal perfusion pressure induced natriuresis and increased phosphorylated SrcTyr416 and Erk 1/2 (extracellular signal-regulated protein kinase 1/2)Thr202/Tyr204; these responses were abolished with rostafuroxin coinfusion. To assess cGMP binding to NKA, we performed competitive binding studies with isolated rat RPTs using bodipy-ouabain (2 μM)+cGMP (10 µM) or rostafuroxin (10 µM) and 8-biotin-11-cGMP (2 μM)+ouabain (10 μM) or rostafuroxin (10 µM). cGMP or rostafuroxin reduced bodipy-ouabain fluorescence intensity, and ouabain or rostafuroxin reduced 8-biotin-11-cGMP staining. We cross-linked isolated rat RPTs with 4-N3-PET-8-biotin-11-cGMP (2 μM); 8-N3-6-biotin-10-cAMP served as negative control. Precipitation with streptavidin beads followed by immunoblot analysis showed that RPTs after cross-linking with 4-N3-PET-8-biotin-11-cGMP exhibited a significantly stronger signal for NKA than non-cross-linked samples and cross-linked or non-cross-linked 8-N3-6-biotin-10-cAMP RPTs. Ouabain (10 μM) reduced NKA in cross-linked 4-N3-PET-8-biotin-11-cGMP RPTs confirming fluorescence staining. 4-N3-PET-8-biotin-11-cGMP cross-linked samples were separated by SDS gel electrophoresis and slices corresponding to NKA molecular weight excised and processed for mass spectrometry. NKA was the second most abundant protein with 50 unique NKA peptides covering 47% of amino acids in NKA. Molecular modeling demonstrated a potential cGMP docking site in the ouabain-binding pocket of NKA. cGMP can bind to NKA and thereby mediate natriuresis.

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.

Predicting changes in renal metabolism after compound exposure with a genome-scale metabolic model

The kidneys are metabolically active organs with importance in several physiological tasks such as the secretion of soluble wastes into the urine and synthesizing glucose and oxidizing fatty acids for energy in fasting (non-fed) conditions. Once damaged, the metabolic capability of the kidneys becomes altered. Here, we define metabolic tasks in a computational modeling framework to capture kidney function in an update to the iRno network reconstruction of rat metabolism using literature-based evidence. To demonstrate the utility of iRno for predicting kidney function, we exposed primary rat renal proximal tubule epithelial cells to four compounds with varying levels of nephrotoxicity (acetaminophen, gentamicin, 2,3,7,8-tetrachlorodibenzodioxin, and trichloroethylene) for six and twenty-four hours, and collected transcriptomics and metabolomics data to measure the metabolic effects of compound exposure. For the transcriptomics data, we observed changes in fatty acid metabolism and amino acid metabolism, as well as changes in existing markers of kidney function such as Clu (clusterin). The iRno metabolic network reconstruction was used to predict alterations in these same pathways after integrating transcriptomics data and was able to distinguish between select compound-specific effects on the proximal tubule epithelial cells. Genome-scale metabolic network reconstructions with coupled omics data can be used to predict changes in metabolism as a step towards identifying novel metabolic biomarkers of kidney function and dysfunction.

Phosphorus Localization and Its Involvement in the Formation of Concentrated Uranium in the Renal Proximal Tubules of Rats Exposed to Uranyl Acetate.

Although the kidneys comprise a critical target of uranium exposure, the dynamics of renal uranium distribution have remained obscure. Uranium is considered to function physiologically in the form of uranyl ions that have high affinity for phosphate groups. The present study applied microbeam-based elemental analysis to precisely determine the distribution of phosphorus and uranium in the kidneys of male Wistar rats exposed to uranium. One day after a single subcutaneous injection of uranyl acetate (2 mg/kg), areas of concentrated phosphorus were scattered in the S3 segments of the proximal tubule of the kidneys, whereas the S3 segments in control rats and in rats given a lower dose of uranium (0.5 mg/kg) contained phosphorus without concentrated phosphorus. Areas with concentrated phosphorus contained uranium 4- to 14-fold more than the mean uranium concentration (126–472 vs. 33.1 ± 4.6 μg/g). The chemical form of uranium in the concentrated phosphorus examined by XAFS was uranium (VI), suggesting that the interaction of uranyl ions with the phosphate groups of biomolecules could be involved in the formation of uranium concentration in the proximal tubules of kidneys in rats exposed to uranium.

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.

Modulation of NHE3 Activity in Rat Renal Proximal Tubules during the Estrous Cycle Phases and Direct Impact of Estradiol and Progesterone

Estradiol (E2) and progesterone (P4) are two of the major steroid hormones involved in the control of reproductive functions in females. Among other effects, E2 and P4 can modulate sodium reabsorption along the nephron, altering the hydroelectrolytic balance, leading to hydrosaline retention. Na/H exchanger, isoform 3, is the main route for sodium bicarbonate reabsorption in renal proximal tubules (PT). The aims of the present study are to evaluate the activity of NHE3 in the different estrous cycle phases and to investigate the direct effects of E2 and P4 on NHE3 activity in kidneys of control (sham) or ovariectomized rats (OVX). All the protocols were approved by the Committee on Animal Research and Ethics from the Ceara State University (protocol #2722214/2016). The estrous cycle of female Wistar rats were evaluated during 15 days and animals randomly assigned to OVX or sham group. Thereafter, two weeks after surgery, the activity of NHE3 was evaluated in vivo by using the stationary microperfusion (SM) technique and after this procedure, the kidneys were stored for evaluation of NHE3 protein abundance by Western Blot (WB). In a first set of experiments, PT were punctured to perfuse control solution (PCS) in order to compare the different phases of the estrous cycle against OVX‐group. In a second set, PTs were perfused with E2 or P4 to compare with OVX animals. SM allows the measurement of H+ secretion in proximal tubules, representing an indirect measure of NHE3 function. It was observed that OVX presented lower H+ secretory activity than all cyclic groups and the estrous group presented higher secretion rates: [JHCO3‐ nmol.cm‐2.s‐1 OVX 1.74 ± 0.07 (10); Proestrus 1.90 ± 0.12 (12)a; Estrus 2.35 ± 0.08 (13)ab; Metaestrus 1.97 ± 0.12 (15)ac and Diestrus 1.89 ± 0.08 (14)ac, where a p <0.05 vs OVX, b p <0.05 vs Proestrus and c p <0.05 vs Estrus]. In addition, an increase in proximal H+ secretion/bicarbonate reabsorption was observed in all E2 and P4 concentrations when administered intratubularly and compared to the OVX group: [JHCO3‐, nmol.cm‐2s‐ OVX+Vehicle 1.84 ± 0.06 (11); E2 (0.1 nM) 2.18 ± 0.07 (15)a; E2 (1 nM) 2.24 ± 0.08 (11)a; E2 (10 nM) 2.20 ± 0.09 (12)a; P4 (0.1 nM), 2.18 ± 0.11 (13)a, P4 (10 nM), 2.10 ± 0.07 (10)a, where a p <0.05 vs OVX + Vehicle]. NHE3 total/villin expression in brush border membranes of PT is lower in the Diestrus group when compared to all other groups: OVX 0.79 ± 0.26 (n=5); Proestrus 0.64 ± 0.08 (n=6); Estrus 0.76 ± 0.30 (n=6); Metaestrus 0.69 ± 0.12 (n=6); Diestrus 0.22 ± 0.12 (n=6)*, p <0.05 vs OVX, Proestrus, Estrus and Metaestrus. Significative changes in NHE3 activity occurs in the different phases of estrous cycles with higher activity in the estrus and lower in diestrus. In addition, it was observed a positive regulation of NHE3 by E2 and P4 in PT which may contribute to sodium retention if this stimulus is maintained for a long‐term.Support or Funding InformationCNPq/FUNCAPThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

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.

Characterization of injury in isolated rat proximal tubules during cold incubation and rewarming.

Organ shortage leads to an increased utilization of marginal organs which are particularly sensitive to storage-associated damage. Cold incubation and rewarming-induced injury is iron-dependent in many cell types. In addition, a chloride-dependent component of injury has been described. This work examines the injury induced by cold incubation and rewarming in isolated rat renal proximal tubules. The tissue storage solution TiProtec® and a chloride-poor modification, each with and without iron chelators, were used for cold incubation. Incubation was performed 4°C for up to 168 h, followed by rewarming in an extracellular buffer (3 h at 37°C). After 48, 120 and 168 h of cold incubation LDH release was lower in solutions containing iron chelators. After rewarming, injury increased especially after cold incubation in chelator-free solutions. Without addition of iron chelators LDH release showed a tendency to be higher in chloride-poor solutions. Following rewarming after 48 h of cold incubation lipid peroxidation was significantly decreased and metabolic activity was tendentially better in tubules incubated with iron chelators. Morphological alterations included mitochondrial swelling and fragmentation being partially reversible during rewarming. ATP content was better preserved in chloride-rich solutions. During rewarming, there was a further decline of ATP content in the so far best conditions and minor alterations under the other conditions, while oxygen consumption was not significantly different compared to non-stored control tubules. Results show an iron-dependent component of preservation injury during cold incubation and rewarming in rat proximal renal tubules and reveal a benefit of chloride for the maintenance of tubular energy state during cold incubation.

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.

Expression of Cyt-c-Mediated Mitochondrial Apoptosis-Related Proteins in Rat Renal Proximal Tubules during Development

Background: Apoptosis regulates embryogenesis, organ metamorphosis and tissue homeostasis. Mitochondrial signaling is an apoptotic pathway, in which Cyt-c and Apaf-1 are transformed into an apoptosome, which activates procaspase-9 and triggers apoptosis. This study evaluated Cyt-c, Apaf-1 and caspase-9 expression during renal development. Methods: Kidneys from embryonic (E) 16-, 18-, and 20-day-old fetuses and postnatal (P) 1-, 3-, 5-, 7-, 14-, and 21-day-old pups were obtained. Immunohistochemical analysis, dual-labeled immunofluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique assay and Western blot were performed in addition to histological analysis. Results: Immunohistochemistry showed that Cyt-c was strongly expressed in proximal and distal tubules (DTs) at all time points. Caspase-9 and Apaf-1 were strongly expressed in proximal tubules (PTs) but only weakly expressed in DTs. Dual-labeled immunofluorescence showed that most tubules expressed both Cyt-c and Apaf-1, except for some tubules that only expressed Cyt-c. The TUNEL assay showed a greater percentage of apoptotic cells in PTs compared to DTs. Apaf-1 and cleaved caspase-9 protein expression gradually increased during the embryonic period and peaked during the early postnatal period but apparently declined from P7. Cyt-c protein expression was weak during the embryonic period but obviously increased after P1. Conclusion: This study showed that PTs are more sensitive to apoptosis than DTs during rat renal development, even though both tubule segments contain a large number of mitochondria. Furthermore, Cyt-c-mediated mitochondrial apoptosis-related proteins play an important role in PTs during the early postnatal kidney development.

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

Rapid regulation of NHE3 activity may occur through subcellular redistribution of the transporter between the microdomains of the proximal tubule (PT) microvilli (MV). Myosin motor proteins that move along actin filaments on the MV may mediate NHE3 translocation in response to a variety of stimuli. The present study was undertaken to test the following hypothesis: (i) that myosin IIA may prevent NHE3 stimulation by angiotensin II (Ang II), which is known to increase NHE3 activity at least in part by translocating NHE3 from the base to the body of the PT MV; (ii) that myosin VI may prevent NHE3 inhibition by parathyroid hormone (PTH), which is known to reduce NHE3 activity at least in part by translocating NHE3 from the body to the base of the PT MV. To this end, NHE3 activity of Wistar rats was evaluated by net bicarbonate reabsorption (JHCO3−) of in vivo renal proximal tubules using H+ ion‐sensitive microelectrodes in the presence or absence of 10−10 M Ang II, 10−6 M PTH, 10−5 M blebbistatin (a specific myosin IIA inhibitor) or 4×10−6 M 2,4,6‐triiodophenol (TIP) (a specific myosin VI inhibitor). As expected, Ang II increased [4.36 ± 0.18 nmol/cm2.s (N=25)] whereas PTH reduced [1.18 ± 0.10 nmol/cm2.s (N=17)] NHE3‐mediated bicarbonate reabsorption compared to CTRL [2.74 ± 0.15 nmol/cm2.s (N=9)]. Simultaneous tubular perfusion of Ang II with blebbistatin completely prevent Ang II‐induced NHE3 stimulation [2.92 ± 0.14 (N=21) vs. 4.36 ± 0.18 nmol/cm2.s (N=25), P < 0.001]. Likewise, simultaneous tubular perfusion of PTH with TIP was capable of blocking the inhibitory effect of PTH [2.91 ± 0.15 (N=22) vs. 1.18 ± 0.10 nmol/cm2.s (N=17), P < 0.001] on NHE3 activity. Neither blebbistatin [2.51 ± 0.11 (N=15) vs. 2.74 ± 0.15 nmol/cm2.s, (N=9)] nor TIP [2.49 ± 0.18 (N=14) vs. 2.74 ± 0.15 nmol/cm2.s (N=9)] alone affected baseline NHE3 activity. These results suggest that myosin IIA and VI may play a role in mediating hormonal regulation of NHE3 in the rat kidney proximal tubule.Support or Funding InformationFAPESP

The Sodium-Bicarbonate Cotransporter 1 (NBC1) and Monocarboxylate Transporter 1 (MCT1) in the Rat Kidney are Regulated in Response to Metabolic Acidosis

The present work was designed to study the effect of metabolic acidosis induced by ammonium chloride on the expression of sodium-bicarbonate cotransporter 1 (NBC1) and Monocarboxylate transporter 1 (MCT1) in rat kidney. Following the treatment of NH4Cl, blood pH and bicarbonate concentration were significantly decreased, while significant increase in chloride concentration was developed. Moreover, urine analysis revealed decrease in urine pH and significant increase in ammonia in acidotic rats. The protein expression of NBC1 and MCT1 was determined in the rat kidney by immunofluorescence confocal laser-scanning microscopy and Western blotting analyses. In the experimental group, the expression level of NBC1 was significantly increased in the renal proximal tubules, whereas, the expression of MCT1 was significantly decreased. Western blotting data confirmed the immunofluorescence confocal laser-scanning microscopy. In conclusion, metabolic acidosis causes adaptive increases in the basolateral NBC1 in the rat renal proximal tubule that are likely responsible for the increased ability of the proximal tubule to reabsorb filtered bicarbonates. Additionally, acidosis leads to decrease the MCT1 protein expression level in renal proximal tubule that may explain the increase in lactate excretion in the urine of acidotic rats due to the reduction in lactate co-transport.

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.