Mouse Renal Proximal Tubule Research Articles

Abstract Mo138: Renal NPFFR2-mediated increase in blood pressure is associated with downregulation of cAMP signaling and upregulation of Na + /K + -ATPase protein expression in the renal proximal tubule

Neuropeptide FF (NPFF), an octapeptide originally found in the brain, participates in the regulation of blood pressure because it is present with its receptors, NPFFR1 and NPFFR2, in the cardiovascular regulatory center in the hypothalamus. However, the role of NPFF and its receptors in the kidney on blood pressure regulation is not known. In both the human and mouse renal proximal tubule, NPFFR2, rather than NPFFR1, is the predominant NPFF receptor determined by RNA in situ hybridization and immunofluorescence microscopy. In mouse renal proximal tubule cells, AC263093 (10 -6 M, 15 min), a specific NPFFR2 agonist and NPFFR1 antagonist, inhibited the forskolin-stimulated cAMP production (Vehicle: 100±6.14%, n=6; AC263093: 72.4±6.53%, n=6, p<0.05), which was reversed by pretreatment with RF-9, an antagonist of both NPFFR1 and NPFFR2 (98.2±9.10%, n=6); RF-9, by itself, had no significant effect (100.8±11.44%, n=6), indicating linkage of NPFFR2 to the inhibitory G protein Gai in renal proximal tubule cells. In human renal proximal tubule cells, NPFF also increased Na + /K + -ATPase protein expression, determined by immunoblotting, in a time- and concentration-dependent manner. Protein expression of Na + /K + -ATPase was decreased in NPFFR2 -deficient human renal proximal tubule cells caused by specific NPFFR2 siRNA (Mock: 100±1.71%, NPFFR2 siRNA: 58.4±4.93%, n=4; P < 0.05). Furthermore, blood pressure, measured by telemetry, was increased and sodium excretion was decreased in conscious C57Bl/6 mice infused with NPFF (9.25 mmol, 0.5 mL/hr/7 days) underneath the renal capsule. The effect was probably via NPFFR2 because the blood pressure of mice was increased by the intraperitoneal injection of AC263093 (20 mg/kg/day, 7 days) and fed a high salt diet (4% NaCl), which is consistent with the decrease in blood pressure (98±4 vs 113±3 mmHg; P < 0.05; n=3-5/group) by the renal subcapsular infusion of Npffr2 siRNA in C57Bl/6 mice also fed the high salt diet. Taken together, NPFFR2 activation increases blood pressure and decreases sodium excretion that is associated with downregulation of cAMP signaling and upregulation of Na + /K + -ATPase protein expression in the renal proximal tubule.

Renal autocrine neuropeptide FF (NPFF) signaling regulates blood pressure

The kidney and brain play critical roles in the regulation of blood pressure. Neuropeptide FF (NPFF), originally isolated from the bovine brain, has been suggested to contribute to the pathogenesis of hypertension. However, the roles of NPFF and its receptors, NPFF-R1 and NPFF-R2, in the regulation of blood pressure, via the kidney, are not known. In this study, we found that the transcripts and proteins of NPFF and its receptors, NPFF-R1 and NPFF-R2, were expressed in mouse and human renal proximal tubules (RPTs). In mouse RPT cells (RPTCs), NPFF, but not RF-amide-related peptide-2 (RFRP-2), decreased the forskolin-stimulated cAMP production in a concentration- and time-dependent manner. Furthermore, dopamine D1-like receptors colocalized and co-immunoprecipitated with NPFF-R1 and NPFF-R2 in human RPTCs. The increase in cAMP production in human RPTCs caused by fenoldopam, a D1-like receptor agonist, was attenuated by NPFF, indicating an antagonistic interaction between NPFF and D1-like receptors. The renal subcapsular infusion of NPFF in C57BL/6 mice decreased renal sodium excretion and increased blood pressure. The NPFF-mediated increase in blood pressure was prevented by RF-9, an antagonist of NPFF receptors. Taken together, our findings suggest that autocrine NPFF and its receptors in the kidney regulate blood pressure, but the mechanisms remain to be determined.

Renal Function and Transporters in the Na/K-ATPase α1S/S Mouse Model of High Affnity for Cardiotonic Steroids

Genetic ablation of ATP1A1 in the mouse renal proximal tubule (RPT) revealed that Na/K-ATPase (NKA) α1 tonically inhibits sodium and water reabsorption. Mechanistically, findings in KO mouse RPT and renal epithelial cells were consistent with a prevalent role of a NKA/Src-dependent downregulation of Na+/H+ exchanger 3 (NHE3). Pharmacologically, functional activation of this pathway has been obtained in vitro and ex vivo using low concentrations of NKA archetypal ligands cardiotonic steroids (CTS). However, the physiological implication of an activation of the endogenous CTS (eCTS)/NKA/Src on renal transporters including NHE3 has not been explored. We assessed the renal impact of CTS/NKA α1 receptor activation by comparing mice expressing the naturally resistant NKA α1 and age-matched mice genetically engineered to express a mutated NKA α1 with high affnity for CTS (R111Q/D122N double substitution). Mice obtained from established colonies at the University of Cincinnati were a gift from Dr. Jerry Lingrel. CTS-sensitive (α1S/S) male mice aged 3-6 months on a C57Bl6 background and age-matched controls (α1R/R) were used. Mice were born with a normal Mendelian ratio, with no observable difference between α1S/S and α1R/R. Renal Na/K-ATPase inhibitory dose-response curve to the CTS ouabain was shifted to the left, as expected, and occurred without detectable change of total renal NKA activity, NKA α1 or β1 protein contents. Metabolic cage studies revealed that increased α1 sensitivity to CTS resulted in a significant increase in urine output in α1S/S mice compared to age-matched α1R/R controls (3.9±0.3 vs. 2.4±0.2 ml/24h; n=6, p<0.01). Consistent with an activation of the NKA/Src receptor by eCTS, western blot analyses of renal cortical preparation revealed an increased phosphorylation of NKA α1 at Y260 (Src target residue) and decreased NHE3 content (43.6 %; n=5, p<0.01). RNA-seq analysis of whole kidneys from α1S/S and α1R/R littermate mice using the Illumina NovaSeq 6000 system (Novogene) and Volcano Plot (DEG data using Python) further showed differential expression of 296 genes (147 upregulated and 149 downregulated). Gene Set Enrichment Analysis (GSEA) using the WebGestalt toolkit showed that the altered pathways were mostly involved in solute transport. Taken together, these results provide genetic evidence of the physiological role of eCTS/NKAα1 on renal function in the intact mouse. Increased affnity to eCTS is suffcient to lead to an activation of NKAα1/Src, a downregulation of NHE3, and transcriptional regulation of renal transporters associated with increased urine output. This work was supported by AHA, Predoctoral fellowship #19PRE34450095, NIH DK129937, and NIH Grant P20GM103434 to the West Virginia IDeA Network of Biomedical Research Excellence. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Molecular mechanism of NR4A1/MDM2/P53 signaling pathway regulation inducing ferroptosis in renal tubular epithelial cells involved in the progression of renal ischemia-reperfusion injury

Revealing the possible molecular mechanism of the NR4A1 (nuclear receptor subfamily 4 group A member 1)-MDM2 (MDM2 proto-oncogene)-P53 (tumor protein p53) signaling pathway that induces ferroptosis in renal tubular epithelial cells. Renal ischemia-reperfusion injury (RIRI) -related datasets were obtained from the GEO database. Differentially expressed genes in RIRI were analyzed using R language, intersected with RIRI-related genes in the GeneCard database, and retrieved from the literature to finally obtain differential ferroptosis-related genes. An in vitro cell model of RIRI was constructed using mouse renal cortical proximal tubule epithelial cells (mRTEC cells) treated with hypoxia-reoxygenation (H/R). Bioinformatic analysis showed that NR4A1 may be involved in RIRI through the induction of ferroptosis; in addition, we predicted through online databases that the downstream target gene of NR4A1, MDM2, could be targeted and regulated by ChIP and dual luciferase assays, and that NR4A1 could prevent MDM2 by inhibiting it, and NR4A1 was able to promote ferroptosis by inhibiting the ubiquitinated degradation of P53. NR4A1 expression was significantly increased in mRTEC cells in the hypoxia/reoxygenation model, and the expression of ferroptosis-related genes was increased in vitro experiments. NR4A1 reduces the ubiquitinated degradation of P53 by targeting the inhibition of MDM2 expression, thereby inducing ferroptosis and ultimately exacerbating RIRI by affecting the oxidative respiration process in mitochondria and producing oxidized lipids. This study presents a novel therapeutic approach for the clinical treatment of renal ischemia-reperfusion injury by developing drugs that inhibit NR4A1 to alleviate kidney damage caused by renal ischemia-reperfusion.

Abstract P1014: Renal Autocrine Npff, Via Its Local Receptors, Regulates Blood Pressure Through Its Interaction With The Renal Dopaminergic System

Neuropeptide FF (NPFF), a hormone originally isolated from the bovine brain, has been suggested to contribute to the pathogenesis of hypertension. However, the role of NPFF and its receptors, NPFF-R1 and NPFF-R2, in the regulation of blood pressure, via the kidney, is unknown. In this study, we found that NPFF ( NPFF , Npff ), NPFF-R1( NPFFR1 , Npffr1 ), and NPFF-R2( NPFFR2 , Npffr2 ) mRNAs and proteins were expressed in both mouse and human renal proximal tubules (RPTs), determined by RT-PCR, RNAScope, immunoblotting, and immunofluorescence microscopy. NPFF-R2, compared with NPFF-R1, was predominantly expressed in RPTs and RPT cells (RPTCs). In human and mouse RPTCs, NPFF decreased cAMP production in a concentration- and time-dependent manner, consistent with its receptors’ linkage to the inhibitory G protein, Gαi. The D 1 R, but not D 5 R, colocalized with NPFF-R2 in human RPTCs and human kidney tissue sections. The colocalization between D 1 R and NPFF-R2 was decreased by the treatment with NPFF (100 nM, 30 min) in mouse RPTCs. Consistent with these findings, D 1 R co-immunoprecipitated with NPFF-R2 in human RPTCs. Fenoldopam, a D1-like receptor agonist, increased the cellular cAMP production and intracellular Na + accumulation, which were attenuated by NPFF (100 nM, 30 min), indicating an antagonistic interaction between NPFF and D1-like receptors. Moreover, acute and chronic administration of NPFF (10 μg in 100 μL, 0.5 μL/hr) in C57Bl/6 mice increased their blood pressures (from 96.4±3 to 114±5 mmHg, n=4) that were blocked by RF9 (10 μg in 100 μL, 0.5 μL/hr), an antagonist of both NPFF-R1 and NPFF-R2. NPFF also decreased urinary sodium excretion (from 0.36±0.06 to 0.28±0.05 mEq/day, n=4). In summary, NPFF by its negative interaction with the renal dopaminergic system regulates sodium excretion and blood pressure.

KCNE1 does not shift TMEM16A from a Ca2+ dependent to a voltage dependent Cl- channel and is not expressed in renal proximal tubule

The TMEM16A (ANO1) Cl- channel is activated by Ca2+ in a voltage-dependent manner. It is broadly expressed and was shown to be also present in renal proximal tubule (RPT). KCNQ1 is an entirely different K+ selective channel that forms the cardiac IKS potassium channel together with its ß-subunit KCNE1. Surprisingly, KCNE1 has been claimed to interact with TMEM16A, and to be required for activation of TMEM16A in mouse RPT. Interaction with KCNE1 was reported to switch TMEM16A from a Ca22+-dependent to a voltage-dependent ion channel. Here we demonstrate that KCNE1 is not expressed in mouse RPT. TMEM16A expressed in RPT is activated by angiotensin II and ATP in a KCNE1-independent manner. Coexpression of KCNE1 does not change TMEM16A to a voltage gated Cl- channel and Ca2+-dependent regulation of TMEM16A is fully maintained in the presence of KCNE1. While overexpressed KCNE1 slightly affects Ca2+-dependent regulation of TMEM16A, the data provide no evidence for KCNE1 being an auxiliary functional subunit for TMEM16A.

First Author Highlights

AnnouncementFirst Author HighlightsPublished Online:17 Mar 2023https://doi.org/10.1152/ajprenal.2023.324.3.AUMoreSectionsPDF (404 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat MOLLY BINGHAMSystems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, United StatesFigure 1.Molly BinghamDownload figureDownload PowerPointMolly Bingham is a Postbaccalaureate Research Fellow at the National Cancer Institute. As an intern in the National Institute of Health’s 2021 Biomedical Engineering Summer Internship Program (led by Dr. Robert Lutz), she was mentored by Dr. Mark Knepper and studied circadian changes in gene expression in the kidney proximal tubule. Currently, Molly is investigating the tumor microenvironment in lung cancer under the mentorship of Dr. Chen Zhao. She hopes to pursue a career as a physician-scientist. When not in the laboratory or analyzing data, Molly enjoys cooking and spending time outdoors [Photo: Fig. 1].https://doi.org/10.1152/ajprenal.00231.2022JEROEN H. F. DE BAAIJDepartment of Physiology, Radboud University Medical Center, Nijmegen, The NetherlandsFigure 2.Jeroen de BaaijDownload figureDownload PowerPointJeroen de Baaij is an Associate Professor in renal physiology at the Radboud University Medical Center in Nijmegen, The Netherlands. His research focusses on the identification and characterization of new genes involved in renal magnesium reabsorption. Using a wide range of molecular, cellular, and physiological techniques, he examines new genes or new regulatory pathways in magnesium (re)absorption in the kidney. In his spare time, he writes about art and visits museums [Photo: Fig. 2].https://doi.org/10.1152/ajprenal.00298.2022KYLE DICKINSONDivision of Experimental Medicine, McGill University, Montreal, Quebec, CanadaFigure 3.Kyle DickinsonDownload figureDownload PowerPointKyle Dickinson is a researcher and laboratory manager in Dr. Julia Burnier’s laboratory at McGill University. He completed his PhD in Dr. Paul Goodyer’s laboratory in Experimental Medicine at McGill University. During his PhD, his research focused on understanding key signalling and DNA damage response pathways active in nephron progenitor cells during kidney development. His current research focuses on using liquid biopsy for cancer detection and monitoring treatment responses. Away from the laboratory bench, he enjoys sports, video games, and horror movies [Photo: Fig. 3].https://doi.org/10.1152/ajprenal.00207.2022MACKENZIE KUIDepartment of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United StatesFigure 4.Mackenzie KuiDownload figureDownload PowerPointMackenzie Kui is currently a third-year PhD student in Jennifer Pluznick’s laboratory at Johns Hopkins University. Mackenzie earned his Bachelor’s degree from the University of California-Santa Barbara, where he studied germline stem cell longevity in Drosophila diapause. After graduating, he worked as a Research Associate at City of Hope and then later at Oncovalent Therapeutics in small molecule discovery. Outside of the laboratory, Mackenzie enjoys climbing, volleyball, and swing dancing [Photo: Fig. 4].https://doi.org/10.1152/ajprenal.00272.2022ANDREW ORWICKDepartment of Pharmacology and Toxicology, University of Louisville, Louisville, KentuckyFigure 5.Andrew OrwickDownload figureDownload PowerPointAndrew Orwick is a PhD student in the Department of Pharmacology and Toxicology at the University of Louisville, mentored by Dr. Leah Siskind. He earned his PharmD from Sullivan University and practiced as a pharmacist for a number of years before deciding to go back to graduate school to obtain a PhD in the biomedical sciences. His PhD studies in chemotherapy-induced nephrotoxicity are supported by a National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases F31 predoctoral fellowship. Andrew hopes to have his own research laboratory in academia one day. In his spare time, he enjoys playing soccer and spending time with his wife and two daughters [Photo: Fig. 5].https://doi.org/10.1152/ajprenal.00317.2022This article has no references to display. Download PDF Back to Top Next FiguresReferencesRelatedInformation Related ArticlesCircadian gene expression in mouse renal proximal tubule 01 Mar 2023American Journal of Physiology-Renal PhysiologyMagnesium reabsorption in the kidney 17 Feb 2023American Journal of Physiology-Renal PhysiologyWT1 regulates expression of DNA repair gene Neil3 during nephrogenesis 17 Feb 2023American Journal of Physiology-Renal PhysiologyThe transcription factor Foxi1 promotes expression of V-ATPase and Gpr116 in M-1 cells 17 Feb 2023American Journal of Physiology-Renal PhysiologyLung cancer-kidney cross talk induces kidney injury, interstitial fibrosis, and enhances cisplatin-induced nephrotoxicity 01 Mar 2023American Journal of Physiology-Renal Physiology More from this issue > Volume 324Issue 3March 2023Pages i-ii Crossmark Copyright & PermissionsCopyright © 2023 the American Physiological Society.https://doi.org/10.1152/ajprenal.2023.324.3.AUPubMed36929919History Published online 17 March 2023 Published in print 1 March 2023 Metrics

Circadian gene expression in mouse renal proximal tubule.

Circadian variability in kidney function is well recognized but is often ignored as a potential confounding variable in physiological experiments. Here, we have created a data resource consisting of expression levels for mRNA transcripts in microdissected proximal tubule segments from mice as a function of the time of day. Small-sample RNA sequencing was applied to microdissected S1 proximal convoluted tubules and S2 proximal straight tubules. After stringent filtering, the data were analyzed using JTK-Cycle to detect periodicity. The data set is provided as a user-friendly webpage at https://esbl.nhlbi.nih.gov/Databases/Circadian-Prox2/. In proximal convoluted tubules, 234 transcripts varied in a circadian manner (4.0% of the total). In proximal straight tubules, 334 transcripts varied in a circadian manner (5.3%). Transcripts previously known to be associated with corticosteroid action and with increased flow were found to be overrepresented among circadian transcripts peaking during the "dark" portion of the day [zeitgeber time (ZT)14-22], corresponding to peak levels of corticosterone and glomerular filtration rate in mice. To ask whether there is a time-of-day dependence of protein abundances in the kidney, we carried out LC-MS/MS-based proteomics in whole mouse kidneys at ZT12 and ZT0. The full data set (n = 6,546 proteins) is available at https://esbl.nhlbi.nih.gov/Databases/Circadian-Proteome/. Overall, 293 proteins were differentially expressed between ZT12 and ZT0 (197 proteins greater at ZT12 and 96 proteins greater at ZT0). Among the regulated proteins, only nine proteins were found to be periodic in the RNA-sequencing analysis, suggesting a high level of posttranscriptional regulation of protein abundances.NEW & NOTEWORTHY Circadian variation in gene expression can be an important determinant in the regulation of kidney function. The authors used RNA-sequencing transcriptomics and LC-MS/MS-based proteomics to identify gene products expressed in a periodic manner. The data were used to construct user-friendly web resources.

Immunohistochemical localization of receptor protein tyrosine phosphatase γ in mouse renal proximal tubule, thick ascending limb and distal convoluted tubule

Most normal physiological processes require that both intracellular (i) and extracellular (o) pH be maintained within a tight range. pHo is contingent on arterial (a) pH, which is determined by the ratio of the arterial carbon dioxide to the arterial bicarbonate concentration, [CO2]a/[HCO3−]a. Pulmonary ventilation controls [CO2]a. The kidney controls [HCO3−]a by adjusting HCO3− transport rates (JHCO3) across the basal membranes of particular nephron epithelia into the blood to maintain normal pHa, or compensate for an acid‐base insult. In an effort to identify the sensor proteins for pHa, [CO2]a, or [HCO3−]a, our laboratory previously reported that, in isolated proximal tubules (PTs) from receptor protein tyrosine phosphatase γ (RPTPγ) knockout (KO) mice, JHCO3 is insensitive to changes in the basolateral [CO2] or [HCO3−]. Furthermore, RPTPγ‐KO mice have a deficiency in defending against a whole‐body metabolic acidosis. These data implicate RPTPγ as a major [CO2] or [HCO3−] sensor. RPTPγ possesses an extracellular catalytically inactive carbonic‐anhydrase‐like domain (CALD) and fibronectin type III (FNIII) domain, a single transmembrane helix, and intracellular domains D1 and D2. Dimerization of RPTPγ presumably allows the inhibitory D2 of one monomer to inhibit the otherwise catalytically active D1 of the other monomer. Thus, the dimerization/monomerization state controls downstream signaling. We hypothesize that the binding of CO2 or HCO3− to the CALD alters the CALD conformation, thereby transmitting signals that control the dimerization status of RPTPγ and thereby regulate downstream effects on JHCO3. We hypothesize that RPTPγ is localized in nephron segments important for regulating HCO3− transport/reabsorption, in the basal membranes that face the blood. To determine the location of RPTPγ in wild‐type mouse kidney cryosections, we use a chicken IgY anti‐RPTPγ antibody (Ab) raised against an epitope within the FNIII domain, and co‐stain with rabbit polyclonal Abs against the Na/HCO3 cotransporter (NBCe1‐A) as a PT marker, the Na/K/Cl cotransporter 2 (NKCC2) as a thick ascending limb (TAL) marker, and the Na/Cl cotransporter (NCC) as a distal convoluted tubule (DCT) marker. Imaging sections by confocal microscopy, we observe in 3 WT mice that RPTPγ localizes primarily to the PT basal membrane. However, 54% of PTs (NBCe1‐A positive) also exhibit apical RPTPγ staining of comparable intensity to the RPTPγ staining observed in the basal membrane. We also observe both apical and basal RPTPγ staining in Bowman’s capsules. We stained additional sections from the same animals with a rabbit polyclonal primary Ab that targets the RPTPγ CALD domain. In these sections, co‐stained with the anti‐FNIII Ab, we similarly observe both apical and basal staining in PTs. We observe RPTPγ staining in a minority of TALs, and at much lower intensity than observed in the PT. We observe minimal to no RPTPγ staining in the DCT. We validated the specificity of the RPTPγ Abs on RPTPγ‐KO kidney sections, which exhibit low background levels of fluorescence for both the anti‐FNIII and anti‐CALD Abs. In conclusion, these data confirm RPTPγ expression in PT basal membranes, but also describe the first examples of apical RPTPγ expression in renal epithelia.

Interleukin-10 attenuates renal injury after myocardial infarction in diabetes

Acute kidney injury (AKI) is a common complication after myocardial infarction (MI) and associated with significant morbidity and mortality. AKI after MI occurs more frequently in patients with diabetes, however,...

Hypouricemic Effects of Chrysanthemum indicum L. and Cornus officinalis on Hyperuricemia-Induced HepG2 Cells, Renal Cells, and Mice.

Hyperuricemia, abnormally excess accumulation of uric acid, is caused by an imbalance between the production and excretion of uric acid and is a major cause of gout. We compared the effects of extracts from Chrysanthemum indicum L. (Ci) and Cornus officinalis Siebold and Zucc. (Co) on hyperuricemia, both individually and in combination (FSU-CC), using hypoxanthine-treated human liver cancer (HepG2) cells, primary mouse renal proximal tubule cells, and potassium oxonate induced hyperuricemic mice. The Ci contained 7.62 mg/g luteolin and 0 mg/g loganin, Co contained 0 mg/g luteolin and 4.90 mg/g loganin, and FSH-CC contained 3.95 mg/g luteolin and 2.48 mg/g loganin. We found that treatment with Ci, Co, and FSU-CC suppressed the activity of xanthine oxidase and mRNA expression of xanthine dehydrogenase while inducing an increase in the expression levels of the organic anion transporter 1 (OAT1) and organic anion transporter 3 (OAT3) proteins and a decrease in the expression levels of glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1) proteins. Particularly, treatment and supplementation with FSU-CC showed stronger effects than those of supplementation with either Ci or Co alone. We observed that the excretion of creatinine and uric acid in the combination of Ci and Co was higher than that observed in their individual supplementations and was similar to that of the normal group. Therefore, our data suggest that a combination of Ci and Co may potentially be used for the development of effective natural anti-hyperuricemic functional foods.

Single-Cell Imaging for Studies of Renal Uranium Transport and Intracellular Behavior

Nephrotoxicity is the primary health effect of uranium exposure. However, the renal transport and intracellular behavior of uranium remains to be clearly elucidated. In the present study, the intracellular uranium distribution was examined with the cell lines derived from the S3 segment of mouse renal proximal tubules, which is a toxic target site of uranium, using microbeam-based elemental analysis. Uranium exposure at 100 μM for 24 h (non-toxic phase) was performed in S3 cells. Two types of measurement specimens, including those that are adhesive cell specimens and cryosection specimens, were examined for the positional relationship of the intracellular localization of uranium. Based on the combined results of single-cell imaging from the two types of cell specimens, uranium was distributed inside the cell and localized in the cytoplasm near the cell nucleus. In some cells, uranium was colocalized with phosphorus and potassium. The amount of uranium accumulated in S3 cells was estimated using thin section-standards. The mean uranium content of three adhesive cells was hundreds of femtogram per cell. Thus, we believe that single-cell imaging would be useful for studies on renal uranium transportation and cellular behavior.

Gastrin, via activation of PPARα, protects the kidney against hypertensive injury

Hypertensive nephropathy (HN) is a common cause of end-stage renal disease with renal fibrosis; chronic kidney disease is associated with elevated serum gastrin. However, the relationship between gastrin and renal fibrosis in HN is still unknown. We, now, report that mice with angiotensin II (Ang II)-induced HN had increased renal cholecystokinin receptor B (CCKBR) expression. Knockout of CCKBR in mice aggravated, while long-term subcutaneous infusion of gastrin ameliorated the renal injury and interstitial fibrosis in HN and unilateral ureteral obstruction (UUO). The protective effects of gastrin on renal fibrosis can be independent of its regulation of blood pressure, because in UUO, gastrin decreased renal fibrosis without affecting blood pressure. Gastrin treatment decreased Ang II-induced renal tubule cell apoptosis, reversed Ang II-mediated inhibition of macrophage efferocytosis, and reduced renal inflammation. A screening of the regulatory factors of efferocytosis showed involvement of peroxisome proliferator-activated receptor α (PPAR-α). Knockdown of PPAR-α by shRNA blocked the anti-fibrotic effect of gastrin in vitro in mouse renal proximal tubule cells and macrophages. Immunofluorescence microscopy, Western blotting, luciferase reporter, and Cut&tag-qPCR analyses showed that CCKBR may be a transcription factor of PPAR-α, because gastrin treatment induced CCKBR translocation from cytosol to nucleus, binding to the PPAR-α promoter region, and increasing PPAR-α gene transcription. In conclusion, gastrin protects against HN by normalizing blood pressure, decreasing renal tubule cell apoptosis, and increasing macrophage efferocytosis. Gastrin-mediated CCKBR nuclear translocation may make it act as a transcription factor of PPAR-α, which is a novel signaling pathway. Gastrin may be a new potential drug for HN therapy.

Role of Thioredoxin 1 in Impaired Renal Sodium Excretion of hD 5 R F173L Transgenic Mice.

BackgroundDopamine D5 receptor (D5R) plays an important role in the maintenance of blood pressure by regulating renal sodium transport. Our previous study found that human D5R mutant F173L transgenic (hD 5 R F173L‐TG) mice are hypertensive. In the present study, we aimed to investigate the mechanisms causing this renal D5R dysfunction in hD 5 R F173L‐TG mice.Methods and ResultsCompared with wild‐type D5R‐TG (hD 5 R WT‐TG) mice, hD 5 R F173L‐TG mice have higher blood pressure, lower basal urine flow and sodium excretion, and impaired agonist‐mediated natriuresis and diuresis. Enhanced reactive oxygen species production in hD 5 R F173L‐TG mice is caused, in part, by decreased expression of antioxidant enzymes, including thioredoxin 1 (Trx1). Na+‐K+‐ATPase activity is increased in mouse renal proximal tubule cells transfected with hD 5 R F173L, but is normalized by treatment with exogenous recombinant human Trx1 protein. Regulation of Trx1 by D5R occurs by the phospholipase C/ protein kinase C (PKC) pathway because upregulation of Trx1 expression by D5R does not occur in renal proximal tubule cells from D1R knockout mice in the presence of a phospholipase C or PKC inhibitor. Fenoldopam, a D1R and D5R agonist, stimulates PKC activity in primary renal proximal tubule cells of hD5R WT ‐TG mice, but not in those of hD 5 R F173L‐TG mice. Hyperphosphorylation of hD5RF173L and its dissociation from Gαs and Gαq are associated with impairment of D5R‐mediated inhibition of Na+‐K+‐ATPase activity in hD 5 R F173L‐TG mice.ConclusionsThese suggest that hD 5 R F173L increases blood pressure, in part, by decreasing renal Trx1 expression and increasing reactive oxygen species production. Hyperphosphorylation of hD5RF173L, with its dissociation from Gαs and Gαq, is the key factor in impaired D5R function of hD 5 R F173L‐TG mice.

Abstract 093: Salt Resistance and Renal Gpr83 Function

The G protein coupled receptor 83 (Gpr83) is an orphan receptor that has been associated with essential hypertension. Gpr83 is expressed in the kidney but its function is unknown. We found that Gpr83 is expressed in mouse renal proximal and distal convoluted tubules, as well as in human renal proximal tubule cells (hRPTCs). In C57Bl/6J mice on normal salt diet, the lack of one (Gpr83+/-) or both Gpr83 (Gpr83-/-) alleles resulted in an increase in systolic blood pressure (SBP, ~20 mm Hg (P<0.05; n=4/group, measured under anesthesia) compared with Gpr83+/+ littermates, suggesting that Gpr83 is needed to keep a normal BP. Renal specific Gpr83 silencing by the renal subcapsular infusion of Gpr83 siRNA (3 μg/day; 7 days) increased SBP in C57Bl/6J mice on a normal salt diet, relative to mice treated with nonsilencing siRNA (120±5 vs 98±6 mmHg; P<0.05; n=4/group). A high salt (4% NaCl) diet increased renal Gpr83 transcription by 2 fold (P<0.05; n=4/group) in SJL/J and BALB/c salt resistant mice, relative to C57Bl/6J salt sensitive mice. Gpr83 gene down-regulation in mouse and human renal proximal tubule cells decreased about 50% the expression of nuclear factor of activated T cells 5 (NFAT5). The renal expressions of NFAT5 and the NFAT5 targets UT-A3, Tau T and BGT1 were increased by a high salt diet in salt resistant mice. The neuropeptide PEN (PEN), a ligand of Gpr83, is also expressed in hRPTCs (0.43±0.03 mRNA PEN/mRNA GAPDH). We found that Gpr83 stimulation with PEN (1 μM, 3 hr) decreased the transcription of NFAT5 (0.59±0.05 vs 1.0±0.2fold, P<0.05; n=3-4/group), which was abolished by Gpr83 silencing. Our results suggest that Gpr83 may protect against the development of salt sensitivity. Gpr83 mediated regulation of NFAT5 may play a role in salt-sensitive hypertension.

ATP induces PAD4 in renal proximal tubule cells via P2X7 receptor activation to exacerbate ischemic AKI.

We previously demonstrated that renal tubular peptidylarginine deiminase-4 (PAD4) is induced after ischemia-reperfusion (IR) injury and this induction of PAD4 exacerbates ischemic acute kidney injury (AKI) by promoting renal tubular inflammation and neutrophil infiltration. However, the mechanisms of renal tubular PAD4 induction after IR remain unknown. Here, we tested the hypothesis that ATP, a proinflammatory danger-associated molecular pattern (DAMP) ligand released from necrotic cells after IR injury, induces renal tubular PAD4 and exacerbates ischemic AKI via P2 purinergic receptor activation. ATP as well as ATPγS (a nonmetabolizable ATP analog) induced PAD4 mRNA, protein, and activity in human and mouse renal proximal tubule cells. Supporting the hypothesis that ATP induces renal tubular PAD4 via P2X7 receptor activation, A804598 (a selective P2X7 receptor antagonist) blocked the ATP-mediated induction of renal tubular PAD4 whereas BzATP (a selective P2X7 receptor agonist) mimicked the effects of ATP by inducing renal tubular PAD4 expression and activity. Moreover, ATP-mediated calcium influx in renal proximal tubule cells was blocked by A804598 and was mimicked by BzATP. P2X7 activation by BzATP also induced PAD4 expression and activity in mouse kidney in vivo. Finally, supporting a critical role for PAD4 in P2X7-mediated exacerbation of renal injury, BzATP exacerbated ischemic AKI in PAD4 wild-type mice but not in PAD4-deficient mice. Taken together, our studies show that ATP induces renal tubular PAD4 via P2X7 receptor activation to exacerbate renal tubular inflammation and injury after IR.

Gastrin stimulates renal dopamine production by increasing the renal tubular uptake of l-DOPA.

Gastrin is a peptide hormone that is involved in the regulation of sodium balance and blood pressure. Dopamine, which is also involved in the regulation of sodium balance and blood pressure, directly or indirectly interacts with other blood pressure-regulating hormones, including gastrin. This study aimed to determine the mechanisms of the interaction between gastrin and dopamine and tested the hypothesis that gastrin produced in the kidney increases renal dopamine production to keep blood pressure within the normal range. We show that in human and mouse renal proximal tubule cells (hRPTCs and mRPTCs, respectively), gastrin stimulates renal dopamine production by increasing the cellular uptake of l-DOPA via the l-type amino acid transporter (LAT) at the plasma membrane. The uptake of l-DOPA in RPTCs from C57Bl/6J mice is lower than in RPTCs from normotensive humans. l-DOPA uptake in renal cortical slices is also lower in salt-sensitive C57Bl/6J than in salt-resistant BALB/c mice. The deficient renal cortical uptake of l-DOPA in C57Bl/6J mice may be due to decreased LAT-1 activity that is related to its decreased expression at the plasma membrane, relative to BALB/c mice. We also show that renal-selective silencing of Gast by the renal subcapsular injection of Gast siRNA in BALB/c mice decreases renal dopamine production and increases blood pressure. These results highlight the importance of renal gastrin in stimulating renal dopamine production, which may give a new perspective in the prevention and treatment of hypertension.

TNF-α Deficiency Prevents Renal Inflammation and Oxidative Stress in Obese Mice

Background/Aims: Obese patients and experimental animals exhibit high levels of inflammatory cytokines, such as tumor necrosis factor (TNF)-α. However, the role of TNF-α in the pathophysiologic process in obesity induced kidney damage is still unknown. Methods: We used TNF-α deficient mice and wild-type (WT) C57/BJ6 mice controls to study the effect of TNF-α on inflammation and oxidative stress in kidney by the model of high-fat diet (HFD) and primary isolated mouse renal proximal tubule cells treated with a mixture of free fatty acids (FFA). Results: Compared with the chow diet group, HFD-fed WT mice had higher urinary albumin and increased levels of renal fibrosis, glomerulosclerosis, inflammation, oxidative stress and apoptosis in the kidney. These changes were co-related with increased expression of TNF-α in the kidney and were attenuated by TNF-α deficiency. In vitro, accumulation of intracellular lipids induced TNF-α expression and oxidative stress in FFA treated primary proximal tubule cells. However, TNF-α inhibition with siRNA or TNF-α deficiency decreased the lipid induced oxidative stress in these cells. Conclusion: These findings suggest that TNF-α plays an important role in the HFD induced kidney damage, and targeting TNF-α and/or its receptors could be a promising therapeutic regimen for progressive nephropathy.

Abstract P185: Role of Ucp2 in the Oxidative/nitrosative Stress-mediated Hypertension Associated with Dj-1 Depletion

DJ-1 -/- mice, relative to wild-type (WT) littermates, have increased blood pressure (BP) ( DJ-1 -/- :130±4 vs WT:100±3 mmHg, n=6/8). and renal expressions of nitro-tyrosine (+76±31% of WT mice, n=5) and malondialdenyde (+63±23% of WT mice, n=4). Tempol, a superoxide dismutase mimetic, decreased the BP of DJ-1 -/- mice ( DJ-1 -/- : before tempol:119±3; after tempol:100±1 mmHg vs WT, n=4) and renal malondialdehyde production ( DJ-1 -/- : before tempol:+40±5%; after tempol:-24±5% vs WT, n=4) but increased serum nitrate/nitrite levels (+72±30%, n=4), indicating the presence of both oxidative and nitrosative stress. Lack of DJ-1 makes some cells vulnerable to endoplasmic reticulum (ER) stress. However, renal mRNA expression of ER stress markers, GRP94, ATF-4, ATF-6, sXBP-1, CHOP, caspase-12, and caspase-3 was not different between DJ-1 -/- and WT (n=7) mice. Markers of inflammation, IL-6, TNF α, MCP-1, NFκB, and T-cell and macrophage infiltration, were also not increased in the kidney of DJ-1 -/- mice. By contrast, renal mitochondrial (mt) HSP60, but not mtHSP40, was increased in DJ-1 -/- mice (2.9±0.1 fold, n=4) but there were no changes in the renal mRNA expressions of Nix/BNIP3L, BNIP3, PINK, FIS1, MFN1, MFN2, PPRC1, NRF-1, and PGC1, indicating that mt oxidative stress did not affect mt function. The renal expression of UCP2, which is involved in the control of mt-reactive oxygen species production, was elevated in DJ-1 -/- mice (4.1±1.1 fold of WT, n=4). Silencing UCP2 in mouse renal proximal tubule cells (-0.46.5±0.01 fold) increased the expression of ER stress and apoptosis markers CHOP (2±0.4 fold), ATF4 (2.6±0.6 fold), caspase-3 (2.3±0.4 fold), and caspase-12 (1.7±0.2 fold)(n=3). There were no differences in renal renin expression, sodium excretion, and serum creatinine between DJ-1 -/- and WT mice (n=5). There were no abnormalities in renal morphology, including fibrosis, in the kidneys of DJ-1 -/- mice. However, urinary KIM-1 was increased in DJ-1 -/- mice (148±22% of WT mice, n=4) and decreased by tempol (-58±3%, n=4); renal UCP2 expression was also partially normalized by tempol (1.8±0.2 fold of WT, n=4). UCP2 may protect from the development of renal ER stress and damage in the mt oxidative/nitrosative stress associated with DJ-1 depletion.

The Synergistic Roles of Cholecystokinin B and Dopamine D5 Receptors on the Regulation of Renal Sodium Excretion.

Renal dopamine D1-like receptors (D1R and D5R) and the gastrin receptor (CCKBR) are involved in the maintenance of sodium homeostasis. The D1R has been found to interact synergistically with CCKBR in renal proximal tubule (RPT) cells to promote natriuresis and diuresis. D5R, which has a higher affinity for dopamine than D1R, has some constitutive activity. Hence, we sought to investigate the interaction between D5R and CCKBR in the regulation of renal sodium excretion. In present study, we found D5R and CCKBR increase each other’s expression in a concentration- and time-dependent manner in the HK-2 cell, the specificity of which was verified in HEK293 cells heterologously expressing both human D5R and CCKBR and in RPT cells from a male normotensive human. The specificity of D5R in the D5R and CCKBR interaction was verified further using a selective D5R antagonist, LE-PM436. Also, D5R and CCKBR colocalize and co-immunoprecipitate in BALB/c mouse RPTs and human RPT cells. CCKBR protein expression in plasma membrane-enriched fractions of renal cortex (PMFs) is greater in D5R-/- mice than D5R+/+ littermates and D5R protein expression in PMFs is also greater in CCKBR-/- mice than CCKBR+/+ littermates. High salt diet, relative to normal salt diet, increased the expression of CCKBR and D5R proteins in PMFs. Disruption of CCKBR in mice caused hypertension and decreased sodium excretion. The natriuresis in salt-loaded BALB/c mice was decreased by YF476, a CCKBR antagonist and Sch23390, a D1R/D5R antagonist. Furthermore, the natriuresis caused by gastrin was blocked by Sch23390 while the natriuresis caused by fenoldopam, a D1R/D5R agonist, was blocked by YF476. Taken together, our findings indicate that CCKBR and D5R synergistically interact in the kidney, which may contribute to the maintenance of normal sodium balance following an increase in sodium intake.