Collecting Duct Cell Line Research Articles

CGAS Activation Accelerates the Progression of Autosomal Dominant Polycystic Kidney Disease.

The renal immune infiltrate observed in autosomal polycystic kidney disease contributes to the evolution of the disease. Elucidating the cellular mechanisms underlying the inflammatory response could help devise new therapeutic strategies. Here, we provide evidence for a mechanistic link between the deficiency polycystin-1 and mitochondrial homeostasis and the activation of the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)/stimulator of the interferon genes (STING) pathway. Our data identify cGAS as an important mediator of renal cystogenesis and suggest that its inhibition may be useful to slow down the disease progression. Immune cells significantly contribute to the progression of autosomal dominant polycystic kidney disease (ADPKD), the most common genetic disorder of the kidney caused by the dysregulation of the Pkd1 or Pkd2 genes. However, the mechanisms triggering the immune cells recruitment and activation are undefined. Immortalized murine collecting duct cell lines were used to dissect the molecular mechanism of cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) activation in the context of genotoxic stress induced by Pkd1 ablation. We used conditional Pkd1 and knockout cGas-/- genetic mouse models to confirm the role of cGAS/stimulator of the interferon genes (STING) pathway activation on the course of renal cystogenesis. We show that Pkd1 -deficient renal tubular cells express high levels of cGAS, the main cellular sensor of cytosolic nucleic acid and a potent stimulator of proinflammatory cytokines. Loss of Pkd1 directly affects cGAS expression and nuclear translocation, as well as activation of the cGAS/STING pathway, which is reversed by cGAS knockdown or functional pharmacological inhibition. These events are tightly linked to the loss of mitochondrial structure integrity and genotoxic stress caused by Pkd1 depletion because they can be reverted by the potent antioxidant mitoquinone or by the re-expression of the polycystin-1 carboxyl terminal tail. The genetic inactivation of cGAS in a rapidly progressing ADPKD mouse model significantly reduces cystogenesis and preserves normal organ function. Our findings indicate that the activation of the cGAS/STING pathway contributes to ADPKD cystogenesis through the control of the immune response associated with the loss of Pkd1 and suggest that targeting this pathway may slow disease progression.

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.

Vasopressin V2 receptor, tolvaptan, and ERK1/2 phosphorylation in the renal collecting duct.

Tolvaptan, a vasopressin antagonist selective for the V2-subtype vasopressin receptor (V2R), is widely used in the treatment of hyponatremia and autosomal-dominant polycystic kidney disease (ADPKD). Its effects on signaling in collecting duct cells have not been fully characterized. Here, we perform RNA-seq in a collecting duct cell line (mpkCCD). The data show that tolvaptan inhibits the expression of mRNAs that were previously shown to be increased in response to vasopressin including aquaporin-2, but also reveals mRNA changes that were not readily predictable and suggest off-target actions of tolvaptan. One such action is activation of the MAPK kinase (ERK1/ERK2) pathway. Prior studies have shown that ERK1/ERK2 activation is essential in the regulation of a variety of cellular and physiological processes and can be associated with cell proliferation. In immunoblotting experiments, we demonstrated that ERK1/ERK2 phosphorylation in mpkCCD cells was significantly reduced by vasopressin, in contrast to the increases seen in non-collecting-duct cells overexpressing V2R in prior studies. We also found that tolvaptan has a strong effect to increase ERK1/ERK2 phosphorylation in the presence of vasopressin and that tolvaptan's effect to increase ERK1/ERK2 phosphorylation is absent in mpkCCD cells in which both protein kinase A (PKA)-catalytic subunits have been deleted. Thus, it appears that the tolvaptan effect to increase ERK activation is PKA-dependent and is not due to an off-target effect of tolvaptan. We conclude that in cells expressing V2R at endogenous levels: 1) vasopressin decreases ERK1/ERK2 activation; 2) in the presence of vasopressin, tolvaptan increases ERK1/ERK2 activation; and 3) these effects are PKA-dependent.NEW & NOTEWORTHY Vasopressin is a key hormone that regulates the function of the collecting duct of the kidney. ERK1 and ERK2 are enzymes that play key roles in physiological regulation in all cells. The authors used collecting duct cell cultures to investigate the effects of vasopressin and the vasopressin receptor antagonist tolvaptan on ERK1 and ERK2 phosphorylation and activation.

#4082 TAZ-KNOCKDOWN AFFECTS THE VASOPRESSIN-INDUCED AQUAPORIN-2 (AQP2) TRAFFICKING AND PROTEIN ABUNDANCE IN KIDNEY COLLECTING DUCT CELLS

Abstract Background and Aims Transcriptional coactivator with PDZ-binding motif (TAZ), a downstream effector of the hippo signaling pathway, regulates the expression of target genes by acting as a transcription cofactor. TAZ KO mice were known to display multicystic kidneys with polyuria. We aimed to study the role of TAZ in vasopressin-induced AQP2 regulation. Method 1) siRNA-mediated knockdown of TAZ in mpkCCDc11 cells; 2) RT-qPCR, semiquantitative immunoblotting, immunocytochemistry of AQP2; 3) Next Generation Sequencing (NGS) in mpkCCDc11 cells, the mouse collecting duct cell line. Results Endogenous AQP2 expression was induced in mpkCCDc11 cells by dDAVP (10−9M) treatment. After starvation for 24 h, dDAVP (10−9M) stimulation for 15 and 30 min induced a significant AQP2 translocation to the cell membrane. In contrast, the dDAVP-induced AQP2 translocation was markedly attenuated in the cells with siRNA-mediated TAZ knockdown (TAZ-KD), despite no changes in cAMP production. Phalloidin staining demonstrated the excessive stress fiber formation in the TAZ-KD. dDAVP (10−9 M) treatment for 24 h induced AQP2 mRNA (12,608 ± 177% of the control) and AQP2 protein abundance (270 ± 18%). In contrast, dDAVP-induced increase of AQP2 mRNA (273 ± 14% of the control) and protein abundance (99 ± 17%) was significantly attenuated in TAZ-KD. Unchanged TonEBP protein abundance was observed in TAZ-KD. NGS identified several potential AQP2 transcription factors (TF), and Klf6, Irf3, Cebpb, and Nr4a1 were selected based on previous in silico database. Among them, Nr4a1 was chosen for further studies due to a significant decrease in the mRNA expression levels in TAZ-KD, as demonstrated by RT-qPCR. Conclusion TAZ is likely to affect dDAVP-induced AQP2 trafficking. This is not mediated by the changes in cAMP/PKA pathway, but other non-canonical pathways are involved. TAZ could regulate AQP2 abundance, possibly via an interaction with several TF.

Uncovering the functional role of GPR39 in the renal collecting duct

G protein-coupled receptors (GPCRs) are seven transmembrane domain receptors that comprise the largest class of proteins in the mammalian genome. GPR39 is part of the ghrelin subfamily of GPCRs, and is well-expressed in the kidney but has no reported function in renal physiology. To probe the renal role of GPR39, we first sought to localize it; in the absence of a validated immunofluorescence (IF) antibody, we used RNAscope. We found that Gpr39 localizes to nephron segments in the cortex and medulla which are positive for DBA (Dolichos Biflorus Agglutinin), a collecting duct marker. RNAScope expression of Gpr39 was highest in the inner medullary collecting duct, with lower levels in the outer medullary and cortical collecting ducts. Within the collecting duct, Gpr39 transcripts colocalized with Aqp2, indicating that Gpr39 is expressed specifically in principal cells. To address whether GPR39 is apical or basolateral, we cloned murine Gpr39 with a C-terminal EGFP tag and transiently transfected this construct into either polarized M-1 cells (murine collecting duct cell line) or polarized MDCK cells (Madin-Darby canine kidney cells); in both cases, cells were grown to confluence on permeable supports. In both cell lines, heterologous GPR39 trafficked to the basolateral membrane. Based on the localization of GPR39 to principal cells, we hypothesized that GPR39 activation may alter AQP2 expression or trafficking. To test this hypothesis in vitro, we first validated mpkCCD (mouse principal cell kidney cortical collecting duct) cells as a model. We cultured mpkCCD on transwells until polarized (TER >5kΩ·cm 2 ). In the absence of dDAVP, mpkCCD do not express AQP2 by western or IF. Basolateral treatment with 1nM dDAVP for 4 days induced AQP2 expression (western blot) and apical trafficking (confocal IF). We also confirmed that withdrawal of dDAVP (4h) resulted in a cytosolic accumulation of AQP2, while the re-introduction of dDAVP (1h) returned AQP2 to the apical membrane by IF. Moreover, we found that Gpr39 expression was unaffected by dDAVP by qPCR (n=3, ΔCt ± SEM: control = 7.9±0.3, dDAVP = 7.2±0.1) and western blot. In a preliminary study, we treated mpkCCD with 13nM of a GPR39 selective agonist, cpd1324, or with a vehicle control, and probed for changes in AQP2 by IF. Both groups were treated with 1nM dDAVP and with 10μM ZnCl2 (a GPR39 cofactor). We found that cpd1324 treatment decreased AQP2 by IF (mean integrated fluorescence density ± SEM (A.U.): cpd1324 = 233.5 ± 20.8, control = 373.8 ± 13.0). Additionally, cpd1324 treatment reduced the apical AQP2 abundance (mean integrated fluorescence density ± SEM (A.U.) of the most apical Z-slice: cpd1324 = 6.05e+05 ± 3.9e+04, control = 2.6e+06 ± 4.3e+05). These results suggest that GPR39 agonism may antagonize vasopressin-induced AQP2 trafficking and/or expression. NIH T32HL007534 This is the full abstract presented at the American Physiology Summit 2023 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.

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.

Statins attenuate cholesterol-induced ROS via inhibiting NOX2/NOX4 and mitochondrial pathway in collecting ducts of the kidney

BackgroundStatins therapy has been primarily recommended for the prevention of cardiovascular risk in patients with chronic kidney diseases. Statins has also been proved some benefits in lipid-induced kidney diseases. The current study aims to investigate the protection and underlying mechanisms of statins on renal tubular injuries induced by cholesterol overloaded.MethodsWe used tubular suspensions of inner medullary collecting duct (IMCD) cells from rat kidneys and mouse collecting duct cell line mpkCCD cells to investigate the effect of statins on reactive oxygen species (ROS) production induced by cholesterol. Protein and mRNA expression of NADPH oxidase 2 (NOX2) /NOX4 was examined by Western blot and RT-PCR in vitro studies and in rats with 5/6 nephrectomy and high-fat diet. Mitochondrial morphology and membrane potential was observed by Mito-tracker and JC-1.ResultsStatins treatment was associated with decreased NOX2 and NOX4 protein expression and mRNA levels in 5/6Nx rats with high-fat diet. Statins treatment markedly reduced the ROS production in IMCD suspensions and mpkCCD cells. Also, statins reduced NOX2 and NOX4 protein expression and mRNA levels in cholesterol overload mpkCCD cells and improved mitochondrial morphology and function.ConclusionStatins prevented ROS production induced by cholesterol in the kidney, likely through inhibiting NOXs protein expression and improving mitochondrial function. Statins may be a therapeutic option in treating obesity-associated kidney diseases.

Dynamic Compartmentalisation of Intracellular Sodium in Collecting Duct Cells

In Principal cells (PC) of the cortical collecting duct (CCD), the highly regulated and co‐ordinated reabsorption of sodium occurs through the epithelial sodium channel (ENaC) at the apical membrane and Na/K ATPase at the basolateral membrane. However, it is not known how sodium ions (Na+) are transported across the cell and if it is a regulated mechanism. We investigated trans‐cellular transport in the mouse collecting duct cell line, mCCDcl1 cells using a fluorescent sodium dye, Corona Green AM. The dye was found to accumulate in fluid‐filled domes forming between the single cell layer and the plastic dish, indicating that the cells are polarised and that Na+ ions are transported across and concentrated beneath the principal cells. Dye uptake was stimulated by aldosterone, blocked by amiloride (an ENaC inhibiter), and basolateral transport was prevented by ouabain (an Na/K ATPase blocker) thus validating the dye’s apparently faithful replication of sodium transport across the cells. Live cells exhibited a consistent pattern of sodium‐containing vesicles, of various sizes, surrounded by cytoskeleton and lipid membrane. While the smallest vesicles (~0.5µm) co‐stained with lysotracker, larger vesicles (up to 6.4µm) did not co‐stain with either lysosomal‐ or mitochondrial‐specific dyes and appeared to have internal structure, suggesting that they were multivesicular bodies. Time‐lapse confocal imaging of cells cultured on a 3D polyHYPE scaffold and subsequent movies of intracellular dynamics showed a subset of these multivesicular bodies release or take up sodium dye in a controlled manner, over the course of a few minutes. Our novel data reveal intracellular sodium compartmentalisation, which may offer new insights into intracellular sodium dynamics, possibly in response to stress, in the collecting duct.

Abstract 11704: NF-κβ Signaling Regulates Vasopressin Receptor 2 Promoter Activity

Background: Arginine vasopressin, through vasopressin receptor 2 (AVP-V2R), regulates water reabsorption via aquaporin-2 and sodium reabsorption via several mechanisms. We recently implicated V2R, and its transcriptional downregulation, in a model of B-cell deficiency with increased sodium- and water-excretion and reduced blood pressure (BP). Here, we identify mechanisms underlying the immunoregulation of V2R transcription and evaluate the importance of V2R in BP regulation. Methods &amp; Results: Using an Avpr2 promoter-luciferase reporter in an immortalized mouse inner medullary collecting duct cell line [mIMCD-3], we found that IL-6 (100ng/mL), a stimulator of NF-κβ signaling, increased promoter-reporter activity (10.4±3.2 vs 4.0±0.5 RLU; N=2-3/group); while PDTC (60μM), an inhibitor of NF-κβ signaling, reduced promoter-reporter activity (2.2 vs 4.0±0.5 RLU; N=1-3/group). Importantly, single point mutations in two putative NF-κβ binding sites in the proximal Avpr2 promoter also reduced promoter activity (2.3±0.6 vs 4.0±0.5 RLU; N=2-3/group), and blocked the stimulatory effect of IL-6 (2.4 vs 2.3±0.6 RLU; N=1-2/group). An anti-IL-6 receptor antibody (10μg/mL vs isotype, 10μg/mL) also abolished the stimulatory effect of IL-6 on promoter activity (3.2 vs 9.1 RLU; N=1/group). To determine the role of V2R in BP regulation, wild-type C57BL/6J mice (male and female; aged 10-wk) were randomized to (i) Tolvaptan (TOL), a selective V2R-antagonist or (ii) DMSO/glycerol control (50/50) via osmotic mini-pump (0.0375mg/d). Invasive hemodynamics 12-d post-implant revealed reduced mean arterial pressure (MAP) in TOL-treated mice (76±3 vs 86±3 mmHg; N=4-5/group; P&lt;0.05), while metabolic cage studies 8-11-d post-implant showed higher 24-h urine volume/body weight (UV/BW; 0.07±0.00 vs 0.05±0.00 mL/g; N=4-5/group; P&lt;0.05) and 24-h sodium excretion/BW (0.25±0.01 vs 0.17±0.01 mg/d/g; N=4-5/group; P&lt;0.01). Correlation analyses revealed inverse relationships between MAP and 24-h UV/BW (r=-0.65; N=9 pairs; P&lt;0.05) and 24-h sodium excretion/BW (r=-0.64; N=9 pairs; P&lt;0.05). Conclusion: These studies suggest that the V2R promoter is responsive to NF-κβ signaling, and that V2R serves a role in BP homeostasis through sodium- and water-handling.

MAGED2 controls vasopressin-induced aquaporin-2 expression in collecting duct cells

Mutations in the Melanoma-Associated Antigen D2 (MAGED2) cause antenatal Bartter syndrome type 5 (BARTS5). This rare disease is characterized by perinatal loss of urinary concentration capability and large urine volumes. The underlying molecular mechanisms of this disease are largely unclear. Here, we study the effect of MAGED2 knockdown on kidney cell cultures using proteomic and phosphoproteomic analyses. In HEK293T cells, MAGED2 knockdown induces prominent changes in protein phosphorylation rather than changes in protein abundance. MAGED2 is expressed in mouse embryonic kidneys and its expression declines during development. MAGED2 interacts with G-protein alpha subunit (GNAS), suggesting a role in G-protein coupled receptors (GPCR) signalling. In kidney collecting duct cell lines, Maged2 knockdown subtly modulated vasopressin type 2 receptor (V2R)-induced cAMP-generation kinetics, rewired phosphorylation-dependent signalling, and phosphorylation of CREB. Maged2 knockdown resulted in a large increase in aquaporin-2 abundance during long-term V2R activation. The increase in aquaporin-2 protein was mediated transcriptionally. Taken together, we link MAGED2 function to cellular signalling as a desensitizer of V2R-induced aquaporin-2 expression. SignificanceIn most forms of Bartter Syndrome, the underlying cause of the disease is well understood. In contrast, the role of MAGED2 mutations in a newly discovered form of Bartter Syndrome (BARTS5) is unknown. In our manuscript we could show that MAGED2 modulates vasopressin-induced protein and phosphorylation patterns in kidney cells, providing a broad basis for further studies of MAGED2 function in development and disease.

Atorvastatin does not ameliorate nephrogenic diabetes insipidus induced by lithium or potassium depletion in mice.

Acquired forms of nephrogenic diabetes insipidus (NDI) include lithium (Li)‐induced and hypokalemia‐induced NDI. Both forms are associated with AQP2 downregulation and collecting duct (CD) cellular remodeling. Statins are cholesterol‐lowering drugs appearing to increase AQP2 membrane‐translocation and improve urine concentration in other NDI models. We have investigated if statins are able to prevent or rescue the Li‐induced changes in mice and in a mouse cortical CD cell line (mCCDc1l). Biotinylation assays showed that acute (1hr) atorvastatin, simvastatin, or fluvastatin increased AQP2 membrane accumulation in mCCDc1l cells showing that the cell line responds to acute statin treatment. To see whether chronic statin treatment abolish the Li effects, mCCDc1l cells were treated with 48 h Li, combined Li/atorvastatin or combined Li/simvastatin. Li reduced AQP2, but combined Li/atorvastatin or Li/simvastatin did not prevent AQP2 downregulation. In mice, chronic (21 days) Li increased urine output and reduced urine osmolality, but combined Li/atorvastatin did not prevent these effects. In inner medulla (IM), Li reduced total AQP2 and increased pS261‐AQP2. Combined Li/atorvastatin did not abolish these changes. Atorvastatin did not prevent a Li‐induced increase in intercalated cells and proliferation in IM. In mice with already established NDI, atorvastatin had no effect on the Li‐induced changes either. Mice subjected to 14 days of potassium‐deficient diet developed polyuria and AQP2 downregulation in IM. Co‐treatment with atorvastatin did not prevent this. In conclusion, atorvastatin does not appear to be able to prevent or rescue Li‐NDI or to prevent hypokalemic‐induced NDI.

Role of hepcidin in oxidative stress and cell death of cultured mouse renal collecting duct cells: protection against iron and sensitization to cadmium

The liver hormone hepcidin regulates systemic iron homeostasis. Hepcidin is also expressed by the kidney, but exclusively in distal nephron segments. Several studies suggest hepcidin protects against kidney damage involving Fe2+ overload. The nephrotoxic non-essential metal ion Cd2+ can displace Fe2+ from cellular biomolecules, causing oxidative stress and cell death. The role of hepcidin in Fe2+ and Cd2+ toxicity was assessed in mouse renal cortical [mCCD(cl.1)] and inner medullary [mIMCD3] collecting duct cell lines. Cells were exposed to equipotent Cd2+ (0.5–5 μmol/l) and/or Fe2+ (50–100 μmol/l) for 4–24 h. Hepcidin (Hamp1) was transiently silenced by RNAi or overexpressed by plasmid transfection. Hepcidin or catalase expression were evaluated by RT-PCR, qPCR, immunoblotting or immunofluorescence microscopy, and cell fate by MTT, apoptosis and necrosis assays. Reactive oxygen species (ROS) were detected using CellROX™ Green and catalase activity by fluorometry. Hepcidin upregulation protected against Fe2+-induced mIMCD3 cell death by increasing catalase activity and reducing ROS, but exacerbated Cd2+-induced catalase dysfunction, increasing ROS and cell death. Opposite effects were observed with Hamp1 siRNA. Similar to Hamp1 silencing, increased intracellular Fe2+ prevented Cd2+ damage, ROS formation and catalase disruption whereas chelation of intracellular Fe2+ with desferrioxamine augmented Cd2+ damage, corresponding to hepcidin upregulation. Comparable effects were observed in mCCD(cl.1) cells, indicating equivalent functions of renal hepcidin in different collecting duct segments. In conclusion, hepcidin likely binds Fe2+, but not Cd2+. Because Fe2+ and Cd2+ compete for functional binding sites in proteins, hepcidin affects their free metal ion pools and differentially impacts downstream processes and cell fate.

Bifunctional protein PCBD2 operates as a co-factor for hepatocyte nuclear factor 1β and modulates gene transcription.

Hepatocyte nuclear factor 1β (HNF1β) is an essential transcription factor in development of the kidney, liver, and pancreas. HNF1β-mediated transcription of target genes is dependent on the cell type and the development stage. Nevertheless, the regulation of HNF1β function by enhancers and co-factors that allow this cell-specific transcription is largely unknown. To map the HNF1β interactome we performed mass spectrometry in a mouse kidney inner medullary collecting duct cell line. Pterin-4a-carbinolamine dehydratase 2 (PCBD2) was identified as a novel interaction partner of HNF1β. PCBD2 and its close homolog PCBD1 shuttle between the cytoplasm and nucleus to exert their enzymatic and transcriptional activities. Although both PCBD proteins share high sequence identity (48% and 88% in HNF1 recognition helix), their tissue expression patterns are unique. PCBD1 is most abundant in kidney and liver while PCBD2 is also abundant in lung, spleen, and adipose tissue. Using immunolocalization studies and biochemical analysis we show that in presence of HNF1β the nuclear localization of PCBD1 and PCBD2 increases significantly. Promoter luciferase assays demonstrate that co-factors PCBD1 and PCBD2 differentially regulate the ability of HNF1β to activate the promoters of transcriptional targets important in renal electrolyte homeostasis. Deleting the N-terminal sequence of PCBD2, not found in PCBD1, diminished the differential effects of the co-factors on HNF1β activity. All together these results indicate that PCBD1 and PCBD2 can exert different effects on HNF1β-mediated transcription. Future studies should confirm whether these unique co-factor activities also apply to HNF1β-target genes involved in additional processes besides ion transport in the kidney.

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.

Effect of information fields from written texts on cell growth and mitochondrial functions in-vitro: An exploratory study

ObjectiveMitochondria are considered a portal to receive, process and integrate external energy and information to maintain cellular homeostasis. We examined the effect of Chinese texts with positive and negative meaning on the growth and mitochondrial functions using a mouse kidney collecting duct cell line called M1 cells. MethodsTo avoid skewing the results due to differential handling of the cells or analyzing the results, we conducted experiments by keeping the texts and blanks covered in brown opaque envelopes, exposed the cells to randomly selected envelopes and examined the differences over time. All operators involved in the experiments did not know the contents of the envelopes until the end of the experiments, and all data are expressed relative to the controls. ResultsCell growth rate was not affected for the group treated with positive information but was significantly reduced by 18% when treated with negative information. At the biochemical level, positive texts significantly increased whole cell adenosine triphosphate (ATP) and glutathione (GSH) by 22% and 21% respectively. ConclusionsThis study for the first time demonstrated the effect of written words on specific biochemical measures in cultured mammalian cells.

Protective role of DJ-1 in endotoxin-induced acute kidney injury.

Acute kidney injury (AKI) is a frequent complication of sepsis and an important cause of morbidity and mortality worldwide. A cornerstone of sepsis-associated AKI is dysregulated inflammation, leading to increased tissue oxidative stress and free radical formation, which leads to multiple forms of cell death. DJ-1 is a peroxiredoxin protein with multiple functions, including its ability to control cellular oxidative stress. Although DJ-1 is expressed prominently by renal tubules, its role in AKI has not been investigated. In the present study, we examined the effect of DJ-1 deficiency in a murine model of endotoxin-induced AKI. Endotoxemia induced greater kidney injury in DJ-1-deficient mice. Furthermore, DJ-1 deficiency increased renal oxidative stress associated with increased renal tubular apoptosis and with expression of death domain-associated protein (DAXX). Similar to the in vivo model, in vitro experiments using a medullary collecting duct cell line (mIMCD3) and cytotoxic serum showed that serum obtained from wild-type mice resulted in increased expression of s100A8/s100A9, DAXX, and apoptosis in DJ-1-deficient mIMCD3 cells. Our findings demonstrate a novel renal protective role for renal tubular DJ-1 during endotoxemia through control of oxidative stress, renal inflammation, and DAXX-dependent apoptosis.

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.

RanBP3L is a NFAT5 target gene and its deficiency in principal cells promotes epithelial mesenchymal transition through MAPK signaling

Inner medullary kidney cells are exposed to an increasing hyperosmolality altering several intracellular processes. As a key regulator involved in adaption to hyperosmotic environment the transcription factor nuclear factor of activated T cells‐5 (NFAT5) was identified. Its nuclear translocation results in activation of different genes to restore homeostasis. By using primarily cultured rat renal inner medullary collecting duct cells (IMCD) and microarray analysis we identified hundreds of genes that showed differences in expression level in a time‐ and osmolality‐dependent manner. The kidney specific Ran binding protein 3 like (RanBP3L) showed the highest changes in expression level and is located in IMCDs. Within the collecting duct its expression is specific to the principal cells. Since studies describing the renal function are missing, we further characterized the cellular and physiological role of RanBP3L in a renal mouse cell line.We used the murine kidney collecting duct cell line mpKCCD creating a RanBP3L deficient cell clone by using CRISPR/Cas9. To investigate its physiological role the gene expression profiles were analyzed by Next Generation Sequencing. We further analyzed the migration and colony forming capability of the cells and used immunofluorescence to study differences in cell morphology and specific signaling pathways. These experiments were performed under isoosmolar (300 mosmol/kg) and hyperosmolar (600 mosmol/kg) conditions. Since RanBP3L expression is induced by hyperosmolality, the NFAT5 dependent expression of RanBP3L was examined through CRISPR/Cas9 mediated NFAT5 knock out.Loss of RanBP3L under 300 mosmol/kg showed no significant changes in the morphology while under 600 mosmol/kg massive changes were induced resulting in a loss of epithelial structures. This correlates with our functional analysis showing a higher migration and invasiveness capacity. Furthermore RanBP3L deficiency is associated with a massive change in gene expression, indicating a dysregulation of stress associated pathways. Immunofluorescence showed that TGF‐β stimulation in wild type induced a similar phenotype to RanBP3L knock out cells. Downstream analysis of MAPK pathway showed that the key proteins JNK, c‐JUN and Smad2 are deregulated in RanBP3L deficient cells resulting in an epithelial mesenchymal transition (EMT) like phenotype, which is a hallmark in cancer development. This matches with the data from the cancer genome atlas from patients with renal clear cell carcinoma (RCC) where low RanBP3L expression level is associated with shorter overall survival. It seems as if the expression level of RanBP3L in kidney cells is crucial for cell protection. Here we could show that RanBP3L expression is reduced in NFAT5 deficient cells, indicating that RanBP3L is a NFAT5 target gene.Taken together we have shown that RanBP3L is expressed NFAT5 dependent and prevents a MAPK associated EMT in renal cells and should be used as a prognostic marker for patients with RCC. Further experiments, like in‐vivo studies of a conditional RanBP3L knock out mouse will give us more insights to resolve the underlying pathway.Support or Funding Informationgerman research foundation

Proteolytic activation of the epithelial sodium channel: role of pro‐protein convertases and prostasin

The epithelial sodium channel (ENaC) is essential for regulating renal sodium excretion. Cleavage of the γENaC subunit increases ENaC activity both in vitro and in vivo; however, the involved endogenous proteases remain ambiguous. This study investigates the mechanistic roles of the pro‐protein convertase (PCSK) family and the serine protease prostasin in proteolytic activation of ENaC.We used the M‐1 mouse collecting duct cell line that natively expresses functional ENaC channels for pharmacological inhibition studies with pro‐protein convertase inhibitor decanoyl‐RVKR‐chloromethyl ketone (dec‐RVKR‐cmk) and serine protease inhibitor camostat mesilate (CM). Knockout cell lines of prostasin and three PCSKs were generated with the CRISPR/Cas9 system in M‐1 cells. As an index of ENaC activity, amiloride‐sensitive short‐circuit current (ISC) and transepithelial voltage (VTE) was measured on polarized cells on transwell filter supports in Ussing chambers. γENaC membrane levels and cleavage were tested by western blotting of surface biotinylated M‐1 cells with an antibody against the C‐terminal of γENaC. All values are means SE, compared with students t‐test or ANOVA as appropriate.Pro‐protein convertase inhibition decreased the amiloride‐sensitive ISC in M‐1 cells compared with controls (25.9 1.9 μA, n=5, vs. 45.7 7.0 μA, n=6, p&lt;0.05). Similarly, serine protease inhibition decreased VTE (−34.5 7.8 mV, n=3, vs. −74.2 0.9 mV, n=3, p&lt;0.05) but ISC was not significantly lower (68.0 8.5, n=3, vs. 74.0 2.3, n=3, n.s.). Preliminary evidence suggests that although both inhibitors decreased membrane expression of γENaC, only serine protease inhibitor treatment reduced γENaC cleavage (n=1). Knockout of the Psck3 (furin) gene by CRISPR completely abolished ENaC activity, while knockout of Pcsk4, Pcsk7, and prostasin all showed decreased ENaC activity compared to wild‐type M‐1 cells. Knockout of PCSKs reduced γENaC surface expression, but only knockout of prostasin reduced γENaC cleavage.We propose that PCSKs are not directly involved in proteolytic ENaC activation. Both pharmacological inhibition and knockout of prostasin decreased ENaC activity and γENaC cleavage, thus suggesting a potential role in proteolytic ENaC activation.Support or Funding Information The Danish Medical Research Council

Deletion of Von Hippel-Lindau Interferes with Hyper Osmolality Induced Gene Expression and Induces an Unfavorable Gene Expression Pattern.

Loss of von Hippel–Lindau (VHL) protein function can be found in more than 90% of patients with clear cell renal carcinoma (ccRCC). Mice lacking Vhl function in the kidneys have urine concentration defects due to postulated reduction of the hyperosmotic gradient. Hyperosmolality is a kidney-specific microenvironment and induces a unique gene expression pattern. This gene expression pattern is inversely regulated in patients with ccRCC with consequences for cancer-specific survival. Within this study, we tested the hypothesis if Vhl function influences the hyperosmolality induced changes in gene expression. We made use of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology to inhibit functional Vhl expression in murine collecting duct cell line. Loss of Vhl function induced morphological changes within the cells similar to epithelial to mesenchymal transition like phenotype. Vhl-deficient cells migrated faster and proliferated slower compared to control cells. Gene expression profiling showed significant changes in gene expression patterns in Vhl-deficient cells compared to control cells. Several genes with unfavorable outcomes showed induced and genes with favorable outcomes for patients with renal cancer reduced gene expression level. Under hyperosmotic condition, the expression of several hyperosmolality induced genes, with favorable prognostic value, was downregulated in cells that do not express functional Vhl. Taken together, this study shows that Vhl interferes with hyperosmotic signaling pathway and hyperosmolality affected pathways might represent new promising targets.