Cortical Collecting Duct Cell Line Research Articles

Tmprss2 maintains epithelial barrier integrity and transepithelial sodium transport.

The mouse cortical collecting duct cell line presents a tight epithelium with regulated ion and water transport. The epithelial sodium channel (ENaC) is localized in the apical membrane and constitutes the rate-limiting step for sodium entry, thereby enabling transepithelial transport of sodium ions. The membrane-bound serine protease Tmprss2 is co-expressed with the alpha subunit of ENaC. αENaC gene expression followed the Tmprss2 expression, and the absence of Tmprss2 resulted not only in down-regulation of αENaC gene and protein expression but also in abolished transepithelial sodium transport. In addition, RNA-sequencing analyses unveiled drastic down-regulation of the membrane-bound protease CAP3/St14, the epithelial adhesion molecule EpCAM, and the tight junction proteins claudin-7 and claudin-3 as also confirmed by immunohistochemistry. In summary, our data clearly demonstrate a dual role of Tmprss2 in maintaining not only ENaC-mediated transepithelial but also EpCAM/claudin-7-mediated paracellular barrier; the tight epithelium of the mouse renal mCCD cells becomes leaky. Our working model proposes that Tmprss2 acts via CAP3/St14 on EpCAM/claudin-7 tight junction complexes and through regulating transcription of αENaC on ENaC-mediated sodium transport.

Analysis of the Hypoxic Response in a Mouse Cortical Collecting Duct-Derived Cell Line Suggests That Esrra Is Partially Involved in Hif1α-Mediated Hypoxia-Inducible Gene Expression in mCCDcl1 Cells.

The kidney is strongly dependent on a continuous oxygen supply, and is conversely highly sensitive to hypoxia. Controlled oxygen gradients are essential for renal control of solutes and urine-concentrating mechanisms, which also depend on various hormones including aldosterone. The cortical collecting duct (CCD) is part of the aldosterone-sensitive distal nephron and possesses a key function in fine-tuned distal salt handling. It is well known that aldosterone is consistently decreased upon hypoxia. Furthermore, a recent study reported a hypoxia-dependent down-regulation of sodium currents within CCD cells. We thus investigated the possibility that cells from the cortical collecting duct are responsive to hypoxia, using the mouse cortical collecting duct cell line mCCDcl1 as a model. By analyzing the hypoxia-dependent transcriptome of mCCDcl1 cells, we found a large number of differentially-expressed genes (3086 in total logFC< −1 or >1) following 24 h of hypoxic conditions (0.2% O2). A gene ontology analysis of the differentially-regulated pathways revealed a strong decrease in oxygen-linked processes such as ATP metabolic functions, oxidative phosphorylation, and cellular and aerobic respiration, while pathways associated with hypoxic responses were robustly increased. The most pronounced regulated genes were confirmed by RT-qPCR. The low expression levels of Epas1 under both normoxic and hypoxic conditions suggest that Hif-1α, rather than Hif-2α, mediates the hypoxic response in mCCDcl1 cells. Accordingly, we generated shRNA-mediated Hif-1α knockdown cells and found Hif-1α to be responsible for the hypoxic induction of established hypoxically-induced genes. Interestingly, we could show that following shRNA-mediated knockdown of Esrra, Hif-1α protein levels were unaffected, but the gene expression levels of Egln3 and Serpine1 were significantly reduced, indicating that Esrra might contribute to the hypoxia-mediated expression of these and possibly other genes. Collectively, mCCDcl1 cells display a broad response to hypoxia and represent an adequate cellular model to study additional factors regulating the response to hypoxia.

Loss of Adam10 Disrupts Ion Transport in Immortalized Kidney Collecting Duct Cells.

The kidney cortical collecting duct (CCD) comprises principal cells (PCs), intercalated cells (IC), and the recently discovered intermediate cell type. Kidney pathology in a mouse model of the syndrome of apparent aldosterone excess revealed plasticity of the CCD, with altered PC:intermediate cell:IC ratio. The self-immortalized mouse CCD cell line, mCCDcl1, shows functional characteristics of PCs, but displays a range of cell types, including intermediate cells, making it ideal to study plasticity. We knocked out Adam10, a key component of the Notch pathway, in mCCDcl1 cells, using CRISPR-Cas9 technology, and isolated independent clones, which exhibited severely affected sodium transport capacity and loss of aldosterone response. Single-cell RNA sequencing revealed significantly reduced expression of major PC-specific markers, such as Scnn1g (γ-ENaC) and Hsd11b2 (11βHSD2), but no significant changes in transcription of components of the Notch pathway were observed. Immunostaining in the knockout clone confirmed the decrease in expression of γ-ENaC and importantly, showed an altered, diffuse distribution of PC and IC markers, suggesting altered trafficking in the Adam10 knockout clone as an explanation for the loss of polarization.

Investigating the Transitional Cell Type in the Kidney Collecting Duct

The kidney cortical collecting duct (CCD) has long been thought to contain two distinct cell types, principal cells (PC) and intercalated cells (IC), which facilitate 4-5% of sodium and 10% of water reabsorption in the kidney. Recent single cell RNA-Seq data has identified a distinct transitional cell expressing both PC and IC markers and a number of distinct markers. In this study, the presence of CCD cell types was investigated in mice and rats under the disease model of the syndrome of apparent mineralocorticoid excess (SAME, conferred by a mutation of the Hsd11b2gene, expressed by PCs) and also in two CCD derived cell lines. Inactivation of HSD11B2in humans results in a severe, and often fatal, phenotype in early neonatal life, whilst milder mutations, with residual enzyme activity cause hypertension, sodium retention and hypokalemia in adulthood. The Hsd11b2 gene knockout (KO) model of SAME in mice and rats was studied in the context of CCD cell plasticity and it was hypothesised to reduce the proportion of PCs within the CCD. Cells in the CCD were counted in WT and Hsd11b2 KO animals following immunofluorescent staining for PC- and IC-specific markers (Aqp2 and V-ATPase B1, respectively) in paraffin-embedded kidney sections. ImageJ and GraphPadPrism were used to analyse data, which are expressed as mean ± SEM. Significance was assessed using Tukey's multiple comparisons test, p<0.0001. PC proportion was significantly reduced in KO mice CCDs compared to controls at 4 weeks (52.1 ± 1.9 % vs. 73.0 ± 1.9 %) and 17 weeks (54.1 ± 1.8 % vs. 72.2 ± 1.3 %). A significant increase in the number of cells expressing both PC and IC markers was observed between WT and KO at 4 weeks (8.4 ± 1.2 % vs.25.9 ± 2.0 %) and at 17 weeks (6.5 ± 0.8 % vs. 22.1 ± 2.0 %). Similarly, in the rat KO a significant reduction in the proportion of PCs was noted compared to controls at 5 weeks (60.69 ± 2.51 % vs. 73.30 ± 1.84 %) and 24 weeks (38.40 ± 2.07 % vs. 66.80 ± 1.94 %). This study shows CCD cell plasticity in models of SAME, but also provides further evidence for a PC+/IC+cell in the kidney. The mCCDcl1­ and mpkCCDcl4­ cell lines are typically used as models of PC function. Immunofluorescent staining using antibodies against PC, IC and transitional cell markers (Aqp2, V-ATPase B1 and Parm1, respectively) was carried out on fixed, polarised cells and images analysed using ImageJ software. It was found that both cell line lines contained cells that co-expressed PC and IC markers, similar to the cell type seen in mouse and rat kidneys. Furthermore, a large proportion of cells in both cell lines expressed the transitional cell marker, Parm1. These results provide further support for the presence of a transitional cell type in the CCD, especially in pathophysiological states. The observation of similar cells in CCD cell lines also indicates their use as in vitro models for investigating the role and plasticity of CCD cell types.

Knock‐out of Adam10 in mCCD cl1 cells gives insight into the effects of Notch pathway disruption for kidney collecting duct cell plasticity

The kidney cortical collecting duct (CCD) is responsible for the fine tuning of sodium, volume, and acid-base homeostasis, and comprises of principal and intercalated cells (PCs and ICs) as well as a newly discovered ‘intermediate’ cell type (1). Plasticity between PCs and ICs has been reported previously in mice in which genes for factors implicated in the Notch pathway, such as Adam10, have been knocked out (KO) (2). In vivo, disruption of the Notch pathway leads to a decreasing PC:IC ratio. The self-immortalized mouse CCD cell line, mCCDcl1, shows functional characteristics of PCs and a response to physiological concentrations of hormones such as aldosterone; it also has been shown to express both PC and IC markers, a phenotype transmitted through single cell cloning (3). These characteristics make mCCDcl1 cells a useful platform for the study of plasticity in the CCD. Adam10 was knocked out in mCCDcl1 cells using CRISPR-Cas9 technology and independent clones were isolated and expanded for study. Despite mCCDcl1 cells being polyploid, our strategy enabled all alleles for each gene to be successfully targeted. Clones were cultured on Transwell filters over 10 days and their electrophysiological properties measured (trans-epithelial resistance Rte, voltage Vte, calculated short-circuit current Isc). Both Adam10 KO clones showed a slightly reduced baseline Rte compared to control (0.90±0.19 kΩ.cm2 vs 1.12±0.18 kΩ.cm2) but a complete reduction in baseline Isc (0.3±0.1 μA/cm2 vs -8.1±0.4 μA/cm2 for control) as well as aldosterone response, decreased by 95% (fold change 0.19±0.03 down 3.70±0.11 for control). Single-cell RNA sequencing principal component analysis shows different clustering of KO clones compared to WT. Potentially explaining the loss of sodium transport function, a significant drop in expression for the gamma subunit of the epithelial sodium channel (γ-ENaC) (between ~40% and ~60% depending on the cluster) was detected in the KO clones, as well as a decrease in the expression of 11ßHSD2 (between ~20% and ~60%). Immunostaining in KO clones confirmed the decrease in expression of γ-ENaC and showed an altered distribution and localisation of γ-ENaC, Aqp2, and V-ATPase B1 compared to WT mCCDcl1, overall suggesting a loss of polarisation. These data suggest that genomic modification of mCCDcl1 cells provides an effective means to investigate the mechanisms through which CCD cells mediate their plasticity and can identify new pathways involved in regulating cell phenotype as well as specific effects of pathway disruption on collecting duct cells functions. (1) Park et al.; Single-cell transcriptomics of the mouse kidney reveals potential cellular targets of kidney disease; Science; 360: 758-763, 2018. (2) Guo et al.; Adam10 Mediates the Choice between Principal Cells and Intercalated Cells in the Kidney; JASN; 26: 149-159, 2015. (3) Assmus et al.; mCCDcl1 cells show plasticity consistent with the ability to transition between principal and intercalated cells; AJP-renal; 314 (5): 820-831, 2018.

Site-1 Protease-Derived Soluble (Pro)Renin Receptor Contributes to Angiotensin II-Induced Hypertension in Mice.

Activation of PRR ([pro]renin receptor) contributes to enhancement of intrarenal RAS and renal medullary α-ENaC and thus elevated blood pressure during Ang II (angiotensin II) infusion. The goal of the present study was to test whether such action of PRR was mediated by sPRR (soluble PRR), generated by S1P (site-1 protease), a newly identified PRR cleavage protease. F1 B6129SF1/J mice were infused for 6 days with control or Ang II at 300 ng/kg per day alone or in combination with S1P inhibitor PF-429242 (PF), and blood pressure was monitored by radiotelemetry. S1P inhibition significantly attenuated Ang II-induced hypertension accompanied with suppressed urinary and renal medullary renin levels and expression of renal medullary but not renal cortical α-ENaC expression. The effects of S1P inhibition were all reversed by supplement with histidine-tagged sPRR termed as sPRR-His. Ussing chamber technique was performed to determine amiloride-sensitive short-circuit current, an index of ENaC activity in confluent mouse cortical collecting duct cell line cells exposed for 24 hours to Ang II, Ang II + PF, or Ang II + PF + sPRR-His. Ang II-induced ENaC activity was blocked by PF, which was reversed by sPRR-His. Together, these results support that S1P-derived sPRR mediates Ang II-induced hypertension through enhancement of intrarenal renin level and activation of ENaC.

Deletion of Elavl1 in renal collecting duct cells is associated with a specific gene expression pattern resulting in major functional changes

IntroductionThe action of vasopressin and aldosterone on water and sodium reabsorption in the renal collecting duct includes the action of the RNA binding protein human antigen R also known as Embryonic Lethal Abnormal Vision Like 1 (Elavl1). Elavl1 modifies the protein expression of the epithelial sodium channel EnaC by stabilizing the mRNA. Under hypertonic conditions it down regulates the expression of the mineralocorticoid receptor (MR), the endogenous target of aldosterone. So far, the role of Elavl1 on the expression of other factors has not been studied, nor whether hypertonicity has an effect on Elavl1 mediated gene expression. Within this study we identified hundreds of genes that show differences in expression upon Elavl1 knock out.MethodsWe used the CRISPR‐Cas9 technology to delete Elavl1. First we cloned specific guideRNAs targeting Elavl1 and scrambled gRNA into the lentiV2‐vector and used them to produce virus particles in HEK293T‐cells. With those particles we transduced a mouse cortical collecting duct cell line (mpkccd). The successful induction of mutation in the Elavl1 locus was verified by Sanger‐Sequencing. Single cell clones were selected and the deletion on protein level was confirmed by immunofluorescence analysis. The Elavl1‐deficient cells were used for functional analysis like cell proliferation, migration and colony forming capacity. Using next generation sequencing we analyzed the gene expression profile comparing Elavl1 deficient to control cells and identified signaling pathways, which were deregulated by deletion of Elavl1.ResultsThe successful deletion of Elavl1 was functionally associated with significantly slower proliferation, migration and lower colony forming capacity. As expected, Elavl1 deficiencies lead to down regulation of the MR gene. Beside MR the expression of more than 440 genes is significantly (p&lt;0,001) up and more than 2700 genes are down regulated. Classification by gene ontology terms showed that gene members of the ontology categories like “enzyme binding”, „rna binding”,„cell stress” and „cell death” are significantly enriched. KEGG pathway analysis showed that the differentially expressed genes are involved in metabolic and cancer associated pathways, such as „PI3K‐AKT‐Signaling pathway” or „HIF1A Signaling pathway”.DiscussionWithin this study we showed a comprehensive analysis of Elval1 function in renal collecting duct cells. Beside mayor physiological differences, Elavl1 deficiency is associated with massive changes in gene expression. Further analyses are needed to investigate its role for in vivo renal function.

Knockout of Adam10 and Tfcp2l1 in mCCDcl1 Collecting Duct Cells Demonstrates Notch‐Dependent Cell Plasticity and Phenotype

The kidney cortical collecting duct (CCD) is responsible for the fine tuning of sodium, volume, and acid‐base homeostasis, and comprises of principal and intercalated cells (PCs and ICs) as well as a newly discovered ‘intermediate’ cell type (1). Plasticity between PCs and ICs has been reported previously in mice in which genes for factors implicated in the Notch pathway, such as Adam10 and Tfcp2l1, have been knocked out (KO) (2, 3). In vivo, disruption of the Notch pathway leads to a decreasing PC:IC ratio. The self‐immortalized mouse CCD cell line, mCCDcl1, shows functional characteristics of PCs and a response to physiological concentrations of hormones such as aldosterone; it also has been shown to express both PC and IC markers, a phenotype transmitted through single cell cloning (4). These characteristics make mCCDcl1 cells a useful platform for the study of plasticity in the CCD. Tfcp2l1 and Adam10 were knocked out in mCCDcl1 cells using CRISPR‐Cas9 technology and independent clones were isolated and expanded for study. Despite mCCDcl1 cells being polyploid, our strategy enabled all alleles for each gene to be successfully targeted and the knockout clones were verified using T7 endonuclease analysis, DNA sequencing and immunocytochemistry. Clones were cultured on Transwell filters over 10 days and their electrophysiological properties measured (trans‐epithelial resistance Rte, voltage Vte, calculated short‐circuit current Isc). Both Adam10 KO clones showed a slightly reduced baseline Rte compared to control (0.90±0.19 kΩ.cm2 vs 1.12±0.18 kΩ.cm2) but a complete reduction in baseline Isc (0.3±0.1 mA/cm2 vs −8.1±0.4 mA/cm2 for control) as well as aldosterone response, decreased by 95% ( fold change 0.19±0.03 down 3.70±0.11 for control). Both Tfcp2l1 KO clones failed to form a resistive cell monolayer, with a dramatic decrease in Rte compared to control (down to 0.10±0.02 kΩ.cm2) as well as a complete disappearance of baseline and epithelial sodium channel (ENaC) mediated amiloride‐sensitive current. These data suggest that genomic modification of mCCDcl1 cells provides an effective means to investigate the mechanisms through which CCD cells mediate their plasticity and, in combination with single cell RNA‐Seq data, will identify new pathways involved in regulating cell phenotype.Support or Funding InformationBritish Heart Foundation Centre for Research Excellence (BHF, RE/13/3/30183) Kidney Research UK (KRUK, RP/026/20180305)

Inverse Regulation of Lipocalin-2/24p3 Receptor/SLC22A17 and Lipocalin-2 Expression by Tonicity, NFAT5/TonEBP and Arginine Vasopressin in Mouse Cortical Collecting Duct Cells mCCD(cl.1): Implications for Osmotolerance.

The rodent collecting duct (CD) expresses a 24p3/NGAL/lipocalin-2 (LCN2) receptor (SLC22A17) apically, possibly to mediate high-affinity reabsorption of filtered proteins by endocytosis, although its functions remain uncertain. Recently, we showed that hyperosmolarity/-tonicity upregulates SLC22A17 in cultured mouse inner-medullary CD cells, whereas activation of toll-like receptor 4 (TLR4), via bacterial lipopolysaccharides (LPS), downregulates SLC22A17. This is similar to the upregulation of Aqp2 by hyperosmolarity/-tonicity and arginine vasopressin (AVP), and downregulation by TLR4 signaling, which occur via the transcription factors NFAT5 (TonEBP or OREBP), cAMP-responsive element binding protein (CREB), and nuclear factor-kappa B, respectively. The aim of the study was to determine the effects of osmolarity/tonicity and AVP, and their associated signaling pathways, on the expression of SLC22A17 and its ligand, LCN2, in the mouse (m) cortical collecting duct cell line mCCD(cl.1). Normosmolarity/-tonicity corresponded to 300 mosmol/L, whereas the addition of 50–100 mmol/L NaCl for up to 72 h induced hyperosmolarity/-tonicity (400–500 mosmol/L). RT-PCR, qPCR, immunoblotting and immunofluorescence microscopy detected Slc22a17/SLC22A17 and Lcn2/LCN2 expression. RNAi silenced Nfat5, and the pharmacological agent 666-15 blocked CREB. Activation of TLR4 was induced with LPS. Similar to Aqp2, hyperosmotic/-tonic media and AVP upregulated Slc22a17/SLC22A17, via activation of NFAT5 and CREB, respectively, and LPS/TLR4 signaling downregulated Slc22a17/SLC22A17. Conversely, though NFAT5 mediated the hyperosmolarity/-tonicity induced downregulation of Lcn2/LCN2 expression, AVP reduced Lcn2/LCN2 expression and predominantly apical LCN2 secretion, evoked by LPS, through a posttranslational mode of action that was independent of CREB signaling. In conclusion, the hyperosmotic/-tonic upregulation of SLC22A17 in mCCD(cl.1) cells, via NFAT5, and by AVP, via CREB, suggests that SLC22A17 contributes to adaptive osmotolerance, whereas LCN2 downregulation could counteract increased proliferation and permanent damage of osmotically stressed cells.

Murine epithelial sodium (Na+) channel regulation by biliary factors

The epithelial sodium channel (ENaC) mediates Na+ transport in several epithelia, including the aldosterone-sensitive distal nephron, distal colon, and biliary epithelium. Numerous factors regulate ENaC activity, including extracellular ligands, post-translational modifications, and membrane-resident lipids. However, ENaC regulation by bile acids and conjugated bilirubin, metabolites that are abundant in the biliary tree and intestinal tract and are sometimes elevated in the urine of individuals with advanced liver disease, remains poorly understood. Here, using a Xenopus oocyte-based system to express and functionally study ENaC, we found that, depending on the bile acid used, bile acids both activate and inhibit mouse ENaC. Whether bile acids were activating or inhibiting was contingent on the position and orientation of specific bile acid moieties. For example, a hydroxyl group at the 12-position and facing the hydrophilic side (12α-OH) was activating. Taurine-conjugated bile acids, which have reduced membrane permeability, affected ENaC activity more strongly than did their more membrane-permeant unconjugated counterparts, suggesting that bile acids regulate ENaC extracellularly. Bile acid-dependent activation was enhanced by amino acid substitutions in ENaC that depress open probability and was precluded by proteolytic cleavage that increases open probability, consistent with an effect of bile acids on ENaC open probability. Bile acids also regulated ENaC in a cortical collecting duct cell line, mirroring the results in Xenopus oocytes. We also show that bilirubin conjugates activate ENaC. These results indicate that ENaC responds to compounds abundant in bile and that their ability to regulate this channel depends on the presence of specific functional groups.

ENaC activation by bile acids depends on specific moieties, but not on membrane permeability

The epithelial sodium channel (ENaC) mediates Na+ transport in several epithelia, including the aldosterone‐sensitive distal nephron, distal colon, and biliary epithelium. Numerous factors regulate the activity of the channel, including extracellular ligands, post‐translational modifications, and membrane‐resident lipids. Bile acids are abundant in the biliary tree and intestinal tract, and can be elevated in the urine of patients with advanced liver disease. Using Xenopus oocytes, we found that bile acids both activated and inhibited mouse ENaC, dependent on the bile acid. Whether bile acids were activating or inhibiting depended on the position and stereochemistry of specific moieties. Taurine conjugated bile acids had stronger effects than their more membrane permeant unconjugated counterparts, suggesting that bile acids regulate ENaC extracellularly. Bile acids that increased ENaC currents had a hydroxyl group at position 12, facing the hydrophilic side. Bile acids that decreased ENaC currents had a hydroxyl group at position 6. Bile acid dependent activation of ENaC currents was mildly voltage‐dependent, suggesting that regulation occurs in the outer leaflet of the membrane. Bile acids also regulated ENaC in a cortical collecting duct cell line, mirroring results in Xenopus oocytes. These results suggest that bile acids interact directly with ENaC near the interface between the outer leaflet and the extracellular solution.Support or Funding InformationThis work was supported by NIDDK, National Institutes of Health, Grant R01 DK098204 (to O.B.K), and a grant from the Pittsburgh Liver Research Center. The Pittsburgh Center for Kidney Research was supported by Grant DK P30 DK079307 from NIDDK, National Institutes of Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Atorvastatin does not ameliorate lithium‐induced nephrogenic diabetes insipidus in mice

Lithium (Li) is an important mood‐stabilizing drug used in the treatment of affective disorders. However, as a significant adverse effect patients can develop Nephrogenic Diabetes Insipidus (NDI). NDI is characterized by the inability of the kidney to concentrate urine in response to vasopressin, resulting in severe polyuria and an increased long‐term risk of chronic kidney disease. Li‐NDI is associated with downregulation of the water channel AQP2 and a cellular remodeling of the kidney collecting duct. Effective and safe treatment of Li‐NDI is still lacking. Statins are cholesterol‐lowering drugs, which appear to increase AQP2 membrane‐translocation and improve urine concentration ability in other NDI models.This project will investigate if atorvastatin is able to prevent the Li‐induced changes in mice using a mouse cortical collecting duct cell line (mCCD) and C57BL/6 mice.Biotinylation assays showed that acute (1hr) treatment with simvastatin (p&lt;0.05) or fluvastatin (p&lt;0.0001) increased AQP2 membrane accumulation compared to controls in the mCCD cells. This confirms that the mCCD cell line respond to acute statin treatment as shown before for these two statins in other cellular and animal models. In order to see whether chronic statin treatment abolish the effects of Li, cells were treated with Li and Li/atorvastatin for 48 hrs. Li reduced total AQP2 levels (p&lt;0.01) compared to controls as expected, but combined Li/atorvastatin treatment did not prevent the Li‐induced downregulation of AQP2. Chronic (21 days) Li treatment of mice increased urine output (p&lt;0.0001) and reduced urine osmolality (p&lt;0.0001) compared to controls (receiving normal food). Atorvastatin (given in combination with Li for 21 days) decreased levels of triglycerides in the blood (p&lt;0.05) compared to controls. Combined Li/atorvastatin treatment did not prevent the effects of Li on urine output and urine osmolality. In kidney inner medulla, western blotting showed that Li reduced total AQP2 levels (p&lt;0.001) and increased pS261‐AQP2 levels (p&lt;0.05), and further showed a tendency to pERK1/2 upregulation (p=0.06) compared to controls as shown previously. However, combined Li/atorvastatin treatment did not abolish any of these changes. Immunohistochemistry further showed that atorvastatin did not prevent the Li‐induced increase in intercalated cells and increased proliferation in the inner medulla.In conclusion, both chronic in vitro and in vivo experiments showed that atorvastatin does not appear to have positive effects on prevention of Li‐NDI in mice.Support or Funding InformationDanish Medical Research Council, Beckett Fonden, Aase and Ejnar Danielsens Fonden, Knud and Edith Eriksens MindefondThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Stabilization of Hypoxia Inducible Factor by Cobalt Chloride Can Alter Renal Epithelial Transport

Around 600,000 people in the United States alone have polycystic kidney disease (PKD) which is characterized by the formation of fluid‐filled pouch‐like structures, called cysts. These cysts tend to expand over the years, finally leading to kidney failure. Previously, kidney cysts were reported to be hypoxic [1]. Cyst expansion, stagnant fluid accumulation, and insufficient vascular supply can result in localized chronic ischemia‐hypoxia in cysts and as well as in normal epithelia adjacent to a cyst. We hypothesize that in normal epithelia near a cyst, the stabilization of Hypoxia Inducible Factor 1α (HIF1α), a major regulator of cellular response to hypoxia, can cause altered paracellular and transcellular transport in normal renal epithelia, transforming the normal absorptive epithelia to a secretory and leaky phenotype, leading to cyst expansion.Using cobalt chloride (CoCl2), HIF1α was stabilized in cellular nucleus of a mouse cortical collecting duct cell line (mCCD 1296 (d)), which resulted in an increased level of erythropoietin, an effector molecule of HIF1α. The mCCD monolayers have high transepithelial resistance (TER) value around 3000 Ω‐cm2 and around 95% amiloride sensitive voltage value. Equivalent current (Ieq) was calculated to compare active ion transport and FITC‐conjugated dextran molecules (3 and 70 kDa) were used to quantify transepithelial transport in terms of permeability values. MTT assay was used in order to make sure CoCl2 treated and untreated groups maintained same levels of metabolically active cells. Our results showed that TER values decreased significantly after 48 and 72 hours of HIF stabilization by CoCl2. The decrease of TER value was consistent with the increase in the permeability of large‐size 70 kDa FITC‐dextran molecules, supporting HIF stabilization actually altered paracellular transport. Western blot analysis showed HIF stabilization caused a significant decrease in the protein level of one of the major tight junction proteins, zonula occludin 1 (ZO1). Decrease in the ZO1 protein level was consistent with the decreased TER value and the increased paracellular permeability. HIF stabilization by CoCl2 for 48 hours caused almost complete loss of active sodium ion transport, and very interestingly, 72 hours of HIF stabilization caused a switch in the direction of active ion transport. Western blot analysis showed that HIF stabilization caused a decrease in the protein level of sodium‐potassium‐ATPase (Na+‐K+‐ATPase) α1 subunit, the catalytic subunit of the enzyme responsible for active sodium ion transport. Decreased level of Na+‐K+‐ATPase α1 subunit due to HIF stabilization was consistent with the loss of active sodium ion transport. Thus, HIF stabilization by CoCl2 can affect Na+‐K+‐ATPase α1 subunit and ZO1 protein levels, leading to alterations of transcellular and paracellular transport through normal kidney epithelial monolayers. Thus, our results indicate that localized ischemia‐hypoxia can transform a normal absorptive epithelia to a secretory and leaky epithelia and may contribute to cyst expansion.Support or Funding InformationThis research was supported by the Dr. John Vitullos Pilot and Bridge Funding Program at the Center for Gene Regulation in Health and Disease (GRHD), NIH DK092716 award (both to AR) and Cleveland State University Dissertation Research Award to SN.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

AQP2 can modulate the pattern of Ca2+ transients induced by store-operated Ca2+ entry under TRPV4 activation.

There is increasing evidence indicating that aquaporins (AQPs) exert an influence in cell signaling by the interplay with the TRPV4 Ca2+ channel. Ca2+ release from intracellular stores and plasma membrane hyperpolarization due to opening of Ca2+ -activated potassium channels (KCa) are events that have been proposed to take place downstream of TRPV4 activation. A major mechanism for Ca2+ entry, activated after depletion of intracellular Ca2+ stores and driven by electrochemical forces, is the store-operated Ca2+ entry (SOCE). The consequences of the interplay between TRPV4 and AQPs on SOCE have not been yet investigated. The aim of our study was to test the hypothesis that AQP2 can modulate SOCE by facilitating the interaction of TRPV4 with KCa channels in renal cells. Using fluorescent probe techniques, we studied intracellular Ca2+ concentration and membrane potential in response to activation of TRPV4 in two rat cortical collecting duct cell lines (RCCD1 ), one not expressing AQPs (WT-RCCD1 ) and the other transfected with AQP2 (AQP2-RCCD1 ). We found that AQP2 co-immunoprecipitates with TRPV4 and with the small-conductance potassium channel (SK3). We also showed that AQP2 is crucial for the activation of SK3 by TRPV4, leading to hyperpolarization of the plasma membrane. This seems to be relevant to modulate the magnitude of SOCE and is accompanied by TRPV4 translocation to the plasma membrane only in AQP2 expressing cells. These findings open the perspective to further investigate whether the interplay between different AQPs with TRPV4 and KCa channels can be an important mechanism to modulate SOCE with physiological relevance.

Renal Epithelial Function Can Be Altered by Hypoxia Inducible Mechanisms

Polycystic Kidney Disease (PKD) is characterized by fluid filled cysts which interfere with renal function and can lead to kidney failure. In cystic epithelia, hypoxia inducible factor 1α (HIF1α) and HIF2α were reported to be elevated [1]. Cyst expansion, stagnant fluid accumulation, and insufficient vascular supply may result in localized chronic ischemia‐hypoxia in cysts and as well as in normal epithelia adjacent to a cyst. We hypothesize that in normal epithelia near a cyst, the stabilization of HIF1α, a major regulator of cellular response to hypoxia, can cause altered paracellular and transcellular epithelial transport. Thus, increased level of HIF1α can be a triggering factor in the transformation of normal renal epithelia from an absorptive to a secretory phenotype, leading to cyst expansion.Cobalt chloride (CoCl2) was used at a concentration of 100 μM to stabilize HIF1α in the cellular nucleus. Using western blots, we confirmed that increased HIF1α levels in the nuclear extracts by CoCl2 also increased cellular erythropoietin (EPO) levels. Application of a HIF1α inhibitor, methyl 3‐[[2‐[4‐(2‐adamantyl)phenoxy]acetyl]amino]‐4‐hydroxybenzoate was found to suppress the increased level of EPO expression by CoCl2. Transcellular and paracellular transport studies were performed in a mouse cortical collecting duct cell line (mCCD 1296 (d)) which has a high monolayer transepithelial resistance (TER) value around 3000 Ω‐cm2 and a detectable 95% amiloride sensitive voltage value. A few paracellular transport studies were also carried out in Mardin‐Darby Canine Kidney (MDCK) epithelial cell line which has relatively low monolayer TER value around 400 Ω‐cm2 with no detectable voltage value. Short circuit current (Isc) was calculated using Ohm's law (I = V/R) to compare active ion transport. TER and voltage values were recorded using an Endohm chamber. Higher TER values in mCCD cells can reflect reduced paracellullar permeability which was verified by permeability assay using FITC‐conjugated dextran molecules (3 and 70 kDa). MTT assay was used in order to make sure CoCl2 treated and untreated groups maintained same level of cellular viability. Cells exposed to CoCl2 showed altered active ion transport in mCCD cells. Treatment with CoCl2 caused increased 70 kDa FITC‐dextran paracellular permeability both in mCCD and MDCK monolayers. In MDCK cell line, we performed a paracellular permeability experiment in which we used the HIF1α inhibitor along with CoCl2. The HIF1α inhibitor was found to block the effect of CoCl2 treatment on FITC‐dextran permeability to a certain extent. Thus, CoCl2 can alter epithelial transport most likely due to hypoxia inducible mechanisms. Currently, we are trying to strengthen the connection between HIF1α levels and renal epithelial function using HIF1α inhibitors in active and passive transport experiments in the presence of CoCl2 or another HIFα stabilizer, the iron chelator desferrioxamine.Support or Funding InformationThe project is supported by National Institute of Diabetes and Digestive and Kidney Diseases 1R15DK092716 to AR and Cleveland State University Dissertation Research Award to SN.

Characterisation of a Mouse Kidney Cortical Collecting Duct Cell Line Suggests Plasticity Between Cell Types

The kidney collecting duct is composed of principal (PC) and intercalated (IC) cells responsible for the reabsorption of water and sodium, and the acid‐base balance, respectively. The immortalized mCCDcl1 cell line is widely used as a model for PCs since cells respond to glucocorticoid (MR) and mineralocorticoid (MR) hormones, such as aldosterone, and give reproducible electrophysiological responses.The effect of culture media on sodium transport in the mCCDcl1 cell line was tested by electrophysiological methods and immunostaining. The cells were cultured on filters in two media: the original medium published by Gaeggeler et al.(2005), and an alternative medium using a different source of sodium selenite supplement (Gibco). The cells transepithelial voltage and resistance across monolayers of cells was measured using an epithelial volt‐ohm‐meter, allowing calculation of the equivalent short circuit current by Ohm's law. Basal currents recorded from cells cultured in the original medium measured −9.97μA/cm2, were amiloride‐sensitive and stimulated by ~2.6 fold following aldosterone treatment (−26.78μA/cm2). Cells cultured in the alternative medium exhibited negligible current and no response to aldosterone was observed.Immunostaining was performed in the alternative media on the mCCDcl1 cells and on isolated subclones using antibodies against PC and IC markers: Aquaporin 2 (Aqp2) and the alpha subunit of the epithelial sodium channel (α‐ENaC) for PC, Connexin 30 (Cx30) and vacuolar H+ ATPase (V‐ATPase) for IC. Both the original cell line and the clonal sub‐lines exhibited varied expression of Aqp2 and Cx30, with evidence of co‐expression in sub‐populations of cells. 41.8% of cells from the parental sub‐line express both PC and IC markers whilst 23.8 to 58.7% cells in the clonal sub‐lines express both PC and IC markers. Expression of both PC and IC markers was confirmed by PCR analyses, and RNA sequencing. Immunostaining for α‐ENaC revealed staining across the apical membrane of the cells when cultured in the original medium and perinuclear staining when cells were cultured in the alternative medium.These results show that the provenance of the culture media supplements has a direct consequence on the phenotype of the cells and correlates with their capacity for sodium transport. Secondly, mCCDcl1 cells can present characteristics of both principal and intercalated cells and may have the capacity to switch between the two phenotypes.Support or Funding InformationBritish Heart Foundation Center of Research Excellence PhD Studentship

AQP2-Induced Acceleration of Renal Cell Proliferation Involves the Activation of a Regulatory Volume Increase Mechanism Dependent on NHE2.

We have previously shown in renal cells that expression of the water channel Aquaporin 2 (AQP2) increases the rate of cell proliferation by shortening the transit time through the S and G2 /M phases of the cell cycle. This acceleration is due, at least in part, to a down-regulation of regulatory volume decrease (RVD) mechanisms when volume needs to be increased in order to proceed into the S phase. We hypothesize that in order to increase cell volume, RVD mechanisms may be overtaken by regulatory volume increase mechanisms (RVI). In this study, we investigated if the isoform 2 of the Na+ /H+ exchanger (NHE2), the main ion transporter involved in RVI responses, contributed to the AQP2-increased renal cell proliferation. Three cortical collecting duct cell lines were used: WT-RCCD1 (not expressing AQPs), AQP2-RCCD1 (transfected with AQP2), and mpkCCDc14 (with inducible AQP2 expression). We here demonstrate, for the first time, that both NHE2 protein activity and expression were increased in AQP2-expressing cells. NHE2 inhibition decreased cell proliferation and delayed cell cycle progression by slowing S and G2 /M phases only if AQP2 was expressed. Finally, we observed that only in AQP2-expressing cells a NHE2-dependent RVI response was activated in the S phase. These observations suggest that the AQP2-increased proliferation involves the activation of a regulatory volume increase mechanism dependent on NHE2. Therefore, we propose that the accelerated proliferation of AQP2-expressing cells requires a coordinated modulation of the RVD/RVI activity that contributes to cell volume changes during cell cycle progression. J. Cell. Biochem. 118: 967-978, 2017. © 2016 Wiley Periodicals, Inc.

TRPM4 Is Expressed in and Carries a Nonselective Cation Current Through the Apical Membrane of Mouse Cortical Collecting Duct Principal Cells

Maintaining daily water, K+‐ and Na+‐balance is an important function of principal cells of the mouse cortical collecting duct (CCD). Under physiological conditions, these cells express a Ca2+ ‐activated nonselective cation channel (NSCCa), the molecular identity of which has been unknown. This study sought to identify whether the nonselective cation channel, TRPM4, functions as a NSCCa in principal cells of mouse cortical collecting duct.Using immunohistochemistry and western blotting, we show the presence of TRPM4 channel protein in the mouse cortical collecting duct cell line mpkCCDc14. Single channel recordings showed a current that is carried by monovalent cations and is Ca2+ sensitive and exhibits equal permeability for Na+ and K+. The corresponding current–voltage relationship of this channel was linear with a slope conductance 23.4 pS, which is the same as that observed from heterologously expressed TRPM4 channels. TRPM4 is inhibited by ATP and stimulated by PIP2. Application of 1 mM ATP to the “cytoplasmic” bath solution of an inside‐out patch almost completely inhibited channel activity but washing out ATP fully restored the channel activity. The channel activity significantly increased after adding 1 μM PI(4,5)P2 to the “cytoplasmic” bath solution. The TRPM4 inhibitor 9‐phenanthrol (100 μM) strongly inhibited the endogenous current. These biophysical properties of NSCCa are identical to those described for TRPM4. Acute application of H2O2 (100 μM and 500 μM) was unable to stimulate TRPM4 activity inside‐out patches; however, using a cell surface biotinylation assay, we showed that treating the mpkCCDc14 with 100 μM H2O2 for 24hrs significantly decreased cell surface levels of TRPM4 protein.Overall, we conclude that TRPM4 is the molecular identity of NSCCa in mpkCCDc14. Further study will be needed to determine the physiological relevance of this channel.Support or Funding InformationNIH R01‐DK100582 to HM

Aldosterone-induced expression of ENaC-α is associated with activity of p65/p50 in renal epithelial cells.

The amiloride-sensitive epithelial sodium channel (ENaC), located in the apical membrane in the cortical collecting duct of the kidney, mediates the fine-tuned regulation of external Na+ balance. Expression of the alpha-subunit of ENaC (ENaC-α) is regulated by a number of factors in the lung, including transcription factor nuclear factor kappa B (NF-κB). In the present study, we examined the effect of IKKβ/p65/p50 on ENaC-α in a murine cortical collecting duct cell line that endogenously expresses ENaC, mpkCCDc14 (CCD) cells. Aldosterone exposure led to up-regulation of ENaC-α and IKKβ, and nuclear p65 and p50. Knockdown of IKKβ or p65 exhibited >60% reduction of aldosterone-induced ENaC-α mRNA levels. Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated a specific interaction between p65/p50 and ENaC-α gene promoter, which was further confirmed using luciferase reporter-gene vectors transiently transfected into CCD cells. Taken together these data support an important role for p65/p50 in the direct regulation of ENaC-α transcription and have important implications for understanding the role of NF-κB in the regulation of renal function.

Effect of Insulin on Transepithelial Na + Transport in Renal Principal Cells Grown Under Normo‐glycemic, Hyperinsulinemic Conditions

Metabolic Syndrome is associated with hyperinsulinemia, hyperglycemia and hypertension. It is our hypothesis that hyperinsulinemia contributes to hypertension by stimulating renal Na+ reabsorption via the epithelial Na+ channel (ENaC). A mouse cortical collecting duct cell line (mCCD) with the characteristics of the renal principal cells was used to study potential interactions between glucose and insulin in regulating ENaC activity. Electrophysiological studies were used to explore the natriferic (Na+ retaining) response of the mCCD cells to insulin stimulation in cells grown with different insulin doses. In cells grown chronically (14 days) in high insulin (45 mU/ml) but normal (3.15 gr/L) glucose, the basal level of transepithelial Na+ transport was significantly reduced compared to cells grown in normal glucose with no insulin. The basal level of transepithelial Na+ flux was, however, not reduced by short-term (24 hours) incubation with high insulin, normal glucose media after the cells were confluent. Despite the changes in basal transport, acute stimulation with insulin in cells mounted in an Ussing chamber exhibited statistically identical increases in Na+ flux measured as an incremental change in transepithelial ion flux that was independent of the basal level of transport. These initial studies indicate the natriferic response to acute changes in insulin is unaltered by a previous hyperinsulinemic condition if the cells are grown in normal glucose. Extrapolating these studies to metabolic syndrome, we would predict that hyperinsulinemia per se is not sufficient to cause an increase in insulin-stimulated Na+ retention.