Inner Medullary Collecting Duct Cells Research Articles

#1236 Translation reprogramming in autosomal dominant polycystic kidney disease

Abstract Background and Aims Global regulation of mRNA translation has emerged as a central mechanism driving cellular adaptation to innate or acquired metabolic challenges. tRNA modifications are emerging as key drivers in the reprogramming of mRNA translation. Metabolic dysregulation is a central feature in the pathophysiology of PKD1-associated autosomal dominant polycystic kidney disease (ADPKD). Here, we aim at investigating the putative role of mRNA translation regulation in the development of cysts in ADPKD. Method The classical murine Inner Medullary Collecting Duct (IMCD) cells knocked out for Pkd1 by CRISPR-Cas9 were used. We combined proteomics and transcriptomics analyses of wild-type versus Pkd1-KO IMCD cells. A series of bioinformatic tools was used to identify pathways and processes dysregulated at the level of mRNA translation. Moreover, global mRNA translation rates were assessed in Pkd1-KO cells by measuring translating ribosomes and nascent protein synthesis by flow cytometry labelling methods. Finally, systematic analyses of tRNA regulation (sequencing and modification) were performed in Pkd1-KO cells. Results The proposed combination of analyses highlighted pathways differentially regulated in Pkd1-KO cells. We focused on pathways and proteins significantly upregulated in Pkd1-KO as compared to controls cells. On top of expected up-regulated pathways, we also uncovered a series of specific cellular and molecular pathways that are specifically and significantly upregulated in Pkd1-KO cells, including intracellular vesicular metabolism and ciliary transport. Interestingly, the pathway “tRNA modification” was significantly enriched in Pkd1-KO cells. Given the role of tRNA modifications in translation reprogramming during cell adaptation, we further validated that several enzymes known to regulate tRNA modification were differentially expressed in Pkd1-KO cells. In order to investigate the changes in tRNA modifications taking place in Pkd1-KO cells, we performed an unbiased analysis of tRNA modifications (LC-MS). This uncovered reproducible differences in the abundance of specific tRNA modifications between control and Pkd1-KO IMCD cells. Conclusion Our data uncovered different patterns of tRNA modifications between Pkd1-KO versus wild-type IMCD cells. This suggests that a specific tRNA-modification-dependent reprogramming of mRNA translation may occur in ADPKD. These data will be further validated using patient derived ADPKD cyst models.

TNF inhibits AQP2 expression via a miR137-dependent pathway.

As miR-137 is a regulator of aquaporin (AQP)2 expression and tumor necrosis factor (TNF) inhibits the expression of several extrarenal AQPs, we tested the hypothesis that TNF inhibits AQP2 in the kidney via a miR-137-dependent mechanism. AQP2 mRNA and protein expression decreased ∼70% and 53%, respectively, in primary renal inner medullary collecting duct (IMCD) cells transfected with a miRNA mimic of mmu-miR-137, suggesting that miR-137 directly targets AQP2 mRNA in these cells. Exposure of IMCD cells for 2 h to 400 mosmol/kgH2O medium increased mmu-miR-137 mRNA expression about twofold, conditions that also increased TNF production approximately fourfold. To determine if the increase in mmu-miR-137 mRNA expression was related to the concomitant increase in TNF, IMCD cells were transfected with a lentivirus construct to silence TNF. This construct decreased mmu-miR-137 mRNA expression by ∼63%, suggesting that TNF upregulates the expression of miR-137. Levels of miR-137 also increased approximately twofold in IMCD tubules isolated from male mice given 1% NaCl in the drinking water for 3 days. Intrarenal lentivirus silencing of TNF increased AQP2 mRNA levels and protein expression concomitant with a decrease in miR-137 levels in tubules isolated from mice given NaCl. The changes in AQP2 expression levels affected the diluting ability of the kidney, which was assessed by measuring urine osmolality and urine volume, as the decrease in these parameters after renal silencing of TNF was prevented on intrarenal administration of miR-137. The study reveals a novel TNF function via a miR-137-dependent mechanism that regulates AQP2 expression and function.NEW & NOTEWORTHY An emerging intratubular tumor necrosis factor system, functioning during normotensive noninflammatory conditions, acts as a breaking mechanism that attenuates both the increases in Na+-K+-2Cl- cotransporter and aquaporin-2 induced by arginine vasopressin, thereby contributing to the regulation of electrolyte balance and blood pressure. A greater appreciation for the role of cytokines as mediators of immunophysiological responses may help reveal the relationship between the immune system and other physiological systems.

TNF inhibits AQP2 expression via a miR137-dependent pathway

We previously showed that production of tumor necrosis factor-alpha (TNF) by the kidney, under normotensive, non-inflammatory conditions, inhibits expression of angiotensinogen in the proximal tubule and NKCC2 isoform A in the thick ascending limb of Henle’s loop via micro-RNA (miRNA)-dependent mechanisms. As miR-137 is a regulator of aquaporin-2 (AQP2) expression and TNF inhibits the expression of several extrarenal aquaporins, we tested the hypothesis that TNF inhibits AQP2 in the kidney via a miR-137-dependent mechanism. Target seed regions of mmu-miR-137 are conserved in the 3'-untranslated region (3’-UTR) of mouse AQP2 mRNA and luciferase assays showed that mmu-miR-137 directly targeted the 3’-UTR of mouse AQP2. Moreover, AQP2 expression was decreased by approximately 70% (P<0.05) in primary renal inner medullary collecting duct (IMCD) cells transfected with a miRNA mimic of mmu-miR-137, indicating that miR-137 directly targets AQP2 mRNA in these cells. The decrease in AQP2 mRNA accumulation was accompanied by a 53% decrease (P<0.05) in AQP2 protein expression when mmu-miR-137 was overexpressed in IMCD cells. Exposure of IMCD cells for 2 hr to 400 mosmol/kg H2O medium, made by increasing the osmolality by adding NaCl, increased mmu-miR-137 mRNA expression about 2-fold (P<0.05) as determined by quantitative RT-PCR analysis. These conditions also increased TNF production approximately 2-fold (P<0.05). To determine if the increase in mmu-miR-137 mRNA expression was related to the concomitant increase in TNF, cells were transfected with either a lentivirus construct to silence TNF or a control construct. The silencing construct, EGFP-shTNF-ex4, but not U6 control, decreased mmu-miR-137 mRNA expression in the IMCD cells exposed to 400 mosmol/kg H2O medium by approximately 63% (P<0.05) indicating that TNF upregulates mmu-miR-137 mRNA expression in IMCD cells. The levels of miR-137 also increased approximately 2-fold (P<0.05) in inner medulla isolated from age- and sex-matched male or female mice given 1% NaCl in the drinking water (HS) for 3 days. Six days after intrarenal injection of lentivirus EGFP-shTNF-ex4 to silence TNF, renal AQP2 mRNA levels increased approximately 3-fold (P<0.05) in inner medullary tissue isolated from male or female mice given HS compared with their respective control groups. In addition, renal silencing of TNF in male or female mice given HS exhibited an increase in AQP2 protein expression the inner medulla compared with control group as determined by Western blot analysis. Similarly, EGFP-shTNF-ex4, but not U6, decreased miR-137 levels in the inner medulla in male or female mice given HS. Taken together, these data reveal a novel mechanism by which TNF mediates the inhibition of AQP2 via miR-137, indicating that miR-137 may be important for studying TNF-regulated effects involving the AQP2 pathway on renal water excretion and blood pressure regulation. NIH grants R01 HL133077 and HL153525 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.

The nuclear factor of activated T cells 5 (NFAT5) contributes to the renal corticomedullary differences in gene expression

The corticomedullary osmotic gradient between renal cortex and medulla induces a specific spatial gene expression pattern. The factors that controls these differences are not fully addressed. Adaptation to hypertonic environment is mediated by the actions of the nuclear factor of activated T-cells 5 (NFAT5). NFAT5 induces the expression of genes that lead to intracellular accumulation of organic osmolytes. However, a systematical analysis of the NFAT5-dependent gene expression in the kidneys was missing. We used primary cultivated inner medullary collecting duct (IMCD) cells from control and NFAT5 deficient mice as well as renal cortex and inner medulla from principal cell specific NFAT5 deficient mice for gene expression profiling. In primary NFAT5 deficient IMCD cells, hyperosmolality induced changes in gene expression were abolished. The majority of the hyperosmolality induced transcripts in primary IMCD culture were determined to have the greatest expression in the inner medulla. Loss of NFAT5 altered the expression of more than 3000 genes in the renal cortex and more than 5000 genes in the inner medulla. Gene enrichment analysis indicated that loss of NFAT5 is associated with renal inflammation and increased expression of kidney injury marker genes, like lipocalin-2 or kidney injury molecule-1. In conclusion we show that NFAT5 is a master regulator of gene expression in the kidney collecting duct and in vivo loss of NFAT function induces a kidney injury like phenotype.

Comprehensive analysis of NFAT5 associated gene expression in the renal collecting duct

BackgroundThe renal cortico‐medullary osmotic gradient is a crucial factor for the urine concentration mechanism and is mainly generated by sodium chloride and urea. Due to this, the cells in the renal inner medulla are challenged with a hypertonic environment. The adaptation of these cells is mediated by the action of the tonicity‐responsive enhancer‐binding factor (TonEBP, also known as nuclear factor of activated T cells 5, NFAT5). NFAT5 induces the expression of genes that lead to accumulation of organic osmolytes within the cells. The cortico‐medullary osmotic gradient also induces a specific spatial gene expression pattern within the kidneys. So far, a systematic analysis of the contribution of NFAT5 on gene expression in the kidneys was missing, which was accomplished here.MethodsWe performed next generation RNA sequencing (NGS) using NFAT5 deficient and wild type control primary cultivated inner medullary collecting duct (IMCD) cells. As a second approach, we used renal cortex and renal inner medulla from principal cell specific NFAT5 deficient mice and control mice for NGS analysis. Differentially expressed genes were analyzed by pathway, functional and gene enrichment analysis.ResultsIn primary IMCD cells hyperosmolality induced massive changes in gene expression. Aquaproin‐2 (Aqp2) was one of the transcripts with the highest level of induction. Loss of NFAT5 abolished hyperosmolality‐induced expression of Aqp2. Most of the hyperosmolality induced transcripts had the highest expression in the inner medulla. Loss of NFAT5 affected the expression of more than 3,000 genes in renal cortex and more than 5,000 genes in the inner medulla, compared to control kidneys. Again, loss of NFAT5 was associated with decreased expression of Aqp2. We observed increased expression of kidney injury marker genes, like lipocalin‐2 or kidney injury molecule 1. Gene enrichment analysis also indicated that loss of NFAT5 is associated with a renal inflammation‐like phenotype.ConclusionThis is the first study addressing the role of NFAT5 on hypertonicty‐induced changes in gene expression in the kidney. NFAT5 is the master regulator of osmolality induced Aqp2 gene expression and in vivoloss of function induces a kidney injury‐like phenotype through currently unknown mechanisms.

HDAC3 inhibition improves urinary-concentrating defect in hypokalaemia by promoting AQP2 transcription.

This study investigated whether enhanced histone acetylation, achieved by inhibiting histone deacetylases (HDACs), could prevent decreased aquaporin-2 (AQP2) expression during hypokalaemia. Male Wistar rats were fed a potassium-free diet with or without 4-phenylbutyric acid (4-PBA) or the selective HDAC3 inhibitor RGFP966 for 4days. Primary renal inner medullary collecting duct (IMCD) cells and immortalized mouse cortical collecting duct (mpkCCD) cells were cultured in potassium-deprivation medium with or without HDAC inhibitors. 4-PBA increased the levels of AQP2 mRNA and protein in the kidney inner medullae in hypokalaemic (HK) rats, which was associated with decreased urine output and increased urinary osmolality. The level of acetylated H3K27 (H3K27ac) protein was decreased in the inner medullae of HK rat kidneys; this decrease was mitigated by 4-PBA. The H3K27ac levels were decreased in IMCD and mpkCCD cells cultured in potassium-deprivation medium. Decreased H3K27ac in the Aqp2 promoter region was associated with reduced Aqp2 mRNA levels. HDAC3 protein expression was upregulated in mpkCCD and IMCD cells in response to potassium deprivation, and the binding of HDAC3 to the Aqp2 promoter was also increased. RGFP966 increased the levels of H3K27ac and AQP2 proteins and enhanced binding between H3K27ac and AQP2 in mpkCCD cells. Furthermore, RGFP966 reversed the hypokalaemia-induced downregulation of AQP2 and H3K27ac and alleviated polyuria in rats. RGFP966 increased interstitial osmolality in the kidney inner medullae of HK rats but did not affect urinary cAMP levels. HDAC inhibitors prevented the downregulation of AQP2 induced by potassium deprivation, probably by enhancing H3K27 acetylation.

Unexpected localization of AQP3 and AQP4 induced by migration of primary cultured IMCD cells

Aquaporin-2–4 (AQP) are expressed in the principal cells of the renal collecting duct (CD). Beside their role in water transport across membranes, several studies showed that AQPs can influence the migration of cells. It is unknown whether this also applies for renal CD cells. Another fact is that the expression of these AQPs is highly modulated by the external osmolality. Here we analyzed the localization of AQP2–4 in primary cultured renal inner medullary CD (IMCD) cells and how osmolality influences the migration behavior of these cells. The primary IMCD cells showed a collective migration behavior and there were no differences in the migration speed between cells cultivated either at 300 or 600 mosmol/kg. Acute increase from 300 to 600 mosmol/kg led to a marked reduction and vice versa an acute decrease from 600 to 300 mosmol/kg to a marked increase in migration speed. Interestingly, none of the analyzed AQPs were localized at the leading edge. While AQP3 disappeared within the first 2–3 rows of cells, AQP4 was enriched at the rear end. Further analysis indicated that migration induced lysosomal degradation of AQP3. This could be prevented by activation of the protein kinase A, inducing localization of AQP3 and AQP2 at the leading edge and increasing the migration speed.

Psychotropic drugs upregulate aquaporin-2 via vasopressin-2 receptor/cAMP/protein kinase A signaling in inner medullary collecting duct cells.

Psychotropic drugs may be associated with hyponatremia, but an understanding of how they induce water retention in the kidney remains elusive. Previous studies have postulated that they may increase vasopressin production in the hypothalamus without supporting evidence. In this study, we investigated the possibility of drug-induced nephrogenic syndrome of inappropriate antidiuresis using haloperidol, sertraline, and carbamazepine. Haloperidol, sertraline, or carbamazepine were treated in inner medullary collecting duct (IMCD) suspensions and primary cultured IMCD cells prepared from male Sprague-Dawley rats. The responses of intracellular cAMP production, aquaporin-2 (AQP2) protein expression and localization, vasopressin-2 receptor (V2R) and AQP2 mRNA, and cAMP-responsive element-binding protein (CREB) were tested with and without tolvaptan and the protein kinase A (PKA) inhibitors H89 and Rp-cAMPS. In IMCD suspensions, cAMP production was increased by haloperidol, sertraline, or carbamazepine and was relieved by tolvaptan cotreatment. In primary cultured IMCD cells, haloperidol, sertraline, or carbamazepine treatment increased total AQP2 and decreased phosphorylated Ser261-AQP2 protein expression. Notably, these responses were reversed by cotreatment with tolvaptan or a PKA inhibitor. AQP2 membrane trafficking was induced by haloperidol, sertraline, or carbamazepine and was also blocked by cotreatment with tolvaptan or a PKA inhibitor. Furthermore, upregulation of V2R and AQP2 mRNA and phosphorylated CREB was induced by haloperidol, sertraline, or carbamazepine and was blocked by tolvaptan cotreatment. We conclude that, in the rat IMCD, psychotropic drugs upregulate AQP2 via V2R-cAMP-PKA signaling in the absence of vasopressin stimulation. The vasopressin-like action on the kidney appears to accelerate AQP2 transcription and dephosphorylate AQP2 at Ser261.NEW & NOTEWORTHY It is unclear whether antipsychotic drugs can retain water in the kidney in the absence of vasopressin. This study demonstrates that haloperidol, sertraline, and carbamazepine can produce nephrogenic syndrome of inappropriate antidiuresis because they directly upregulate vasopressin-2 receptor and aquaporin-2 (AQP2) via cAMP/PKA signaling. We showed that, in addition to AQP2 trafficking, AQP2 protein abundance was rapidly increased by treatment with antipsychotic drugs in association with dephosphorylation of AQP2 at Ser261 and accelerated AQP2 transcription.

Mechanical Stimulation of Piezo1 Induced Protein Expression and Intracellular Trafficking of Aquaporin‐2 in IMCD Cells

Pressure and shear stress‐induced stretch of tubular epithelial cells is relevant to both physiological and diseased kidney. Piezo1 is a mechanically‐activated cation channel stimulated by membrane tension and stretch; it is highly expressed in the inner medulla of the kidney. Aquaporin‐2 (AQP2) is located in the principal cells of the collecting ducts in the kidney, which is a key aquaporin determining final urine output. The current study aims to investigate whether activation of Piezo1 induces AQP2 expression in the collecting duct principal cells. In human kidney biopsy specimen, Piezo1 was found expressed mainly in the collecting ducts where it co‐localized with AQP2. Upon treating primary cultured rat inner medullary collecting duct (IMCD) cells with Piezo1 activator Yoda1 (0.01μM to 1μM), the protein expression of AQP2 was markedly increased. Activation of Piezo1 by Yoda1 was also associated with intracellular F‐actin depolymerization and dramatically increased accumulation of AQP2 on the apical plasma membrane of IMCD cells. Upregulation of AQP2 protein was also observed when Piezo1 was stimulated with mechanical cyclic tensile strain at 5% elongation, 1Hz for 3hour in IMCD cells, which was markedly inhibited by GsMTx4 (0.1μM to 5μM), a Piezo1 antagonist. Both stretch and Yoda1 treatment induced elevation of intracellular calcium levels and upregulation of calpain2 protein expression (calcium‐activated non‐lysosomal thiol‐proteases) as well as phosphorylation of Akt protein in rat IMCD cells. Dehydration for 24h (which is presumably leading to the swelling of principal cells, Chou CL, Am J Physiol Renal Physiol, 2008) in mice was associated with increased levels of piezo1 mRNA and protein expression in the kidney inner medulla. In conclusion, activation of Piezo1 by mechanical stretch or activators increased protein expression and intracellular trafficking of AQP2 in rat IMCD cells, which was associated with F‐actin depolymerizing and elevated intracellular calcium levels. These studies indicate that piezo1 may play a functional role in the regulation of urinary osmolarity in pressure‐induced stretch of principal cells relevant to both physiological and pathophysiological conditions.

Berberine ameliorates lipopolysaccharide‑induced inflammatory responses in mouse inner medullary collecting duct‑3 cells by downregulation of NF‑κB pathway.

The major role of inner medullary collecting duct (IMCD) cells is to maintain water and sodium homeostasis. In addition to the major role, it also participates in the protection of renal and systemic inflammation. Although IMCD cells could take part in renal and systemic inflammation, investigations on renal inflammation in IMCD cells have rarely been reported. Although berberine (BBR) has been reported to show diverse pharmacological effects, its anti-inflammatory and protective effects on IMCD cells have not been studied. Therefore, in the present study, we examined the anti-inflammatory and protective effects of BBR in mouse IMCD-3 (mIMCD-3) cells against lipopolysaccharide (LPS). An MTT assay was carried out to investigate the toxicity of BBR on mIMCD-3 cells. Reverse transcription quantitative-PCR and western blotting were performed to analysis pro-inflammatory molecules and cytokines. Mechanisms of BBR were examined by western blotting and immunocytochemistry. According to previous studies, pro-inflammatory molecules, such as inducible nitric oxide synthase and cyclooxygenase-2, and pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6 and tumor necrosis factor-α are increased in LPS-exposed mIMCD-3 cells. However, the production of these pro-inflammatory molecules is significantly inhibited by treatment with BBR. In addition, BBR inhibited translocation of nuclear factor (NF)-κB p65 from the cytosol to the nucleus, and degradation of inhibitory κ-Bα in LPS-exposed mIMCD-3 cells. In conclusion, BBR could inhibit renal inflammatory responses via inhibition of NF-κB signaling and ultimately contribute to amelioration of renal injury during systemic inflammation.

Bilateral ureteral obstruction is rapidly accompanied by ER stress and activation of autophagic degradation of IMCD proteins, including AQP2.

After the release of bilateral ureteral obstruction (BUO), postobstructive diuresis from an impaired urine concentration mechanism is associated with reduced aquaporin 2 (AQP2) abundance in the inner medullary collecting duct (IMCD). However, the underlying molecular mechanism of this AQP2 reduction is incompletely understood. To elucidate the mechanisms responsible for this phenomenon, we studied molecular changes in IMCDs isolated from rats with 4-h BUO or sham operation at the early onset of AQP2 downregulation using mass spectrometry-based proteomic analysis. Two-hundred fifteen proteins had significant changes in abundances, with 65% of them downregulated in the IMCD of 4-h BUO rats compared with sham rats. Bioinformatic analysis revealed that significantly changed proteins were associated with functional Gene Ontology terms, including “cell-cell adhesion,” “cell-cell adherens junction,” “mitochondrial inner membrane,” “endoplasmic reticulum chaperone complex,” and the KEGG pathway of glycolysis/gluconeogenesis. Targeted liquid chromatography-tandem mass spectrometry or immunoblot analysis confirmed the changes in 19 proteins representative of each predominant cluster, including AQP2. Electron microscopy demonstrated disrupted tight junctions, disorganized adherens junctions, swollen mitochondria, enlargement of the endoplasmic reticulum lumen, and numerous autophagosomes/lysosomes in the IMCD of rats with 4-h BUO. AQP2 and seven proteins chosen as representative of the significantly altered clusters had a significant increase in immunofluorescence-based colocalization with autophagosomes/lysosomes. Immunogold electron microscopy confirmed colocalization of AQP2 with the autophagosome marker microtubule-associated protein 1A/1B-light chain 3 and the lysosomal marker cathepsin D in IMCD cells of rats with 4-h BUO. We conclude that enhanced autophagic degradation of AQP2 and other critical proteins, as well as endoplasmic reticulum stress in the IMCD, are initiated shortly after BUO.

Claudin 19 Is Regulated by Extracellular Osmolality in Rat Kidney Inner Medullary Collecting Duct Cells.

The inner medullary collecting duct (IMCD) is subject to severe changes in ambient osmolality and must either allow water transport or be able to seal the lumen against a very high osmotic pressure. We postulate that the tight junction protein claudin-19 is expressed in IMCD and that it takes part in epithelial adaptation to changing osmolality at different functional states. Presence of claudin-19 in rat IMCD was investigated by Western blotting and immunofluorescence. Primary cell culture of rat IMCD cells on permeable filter supports was performed under different osmotic culture conditions and after stimulation by antidiuretic hormone (AVP). Electrogenic transepithelial transport properties were measured in Ussing chambers. IMCD cells cultivated at 300 mosm/kg showed high transepithelial resistance, a cation selective paracellular pathway and claudin-19 was mainly located in the tight junction. Treatment by AVP increased cation selectivity but did not alter transepithelial resistance or claudin-19 subcellular localization. In contrast, IMCD cells cultivated at 900 mosm/kg had low transepithelial resistance, anion selectivity, and claudin-19 was relocated from the tight junctions to intracellular vesicles. The data shows osmolality-dependent transformation of IMCD epithelium from tight and sodium-transporting to leaky, with claudin-19 expression in the tight junction associated to tightness and cation selectivity under low osmolality.

Abstract 082: Histone Deacetylase 1 Activates Renal Inner-Medullary Nitric Oxide Production

Fluid flow along the renal nephron, particularly within the inner-medullary collecting duct (IMCD), increases in response to high salt intake. We previously showed in a series of reports that high salt intake increases nitric oxide synthase 1 (NOS1) activity in the CD mediating inhibition of the epithelial sodium channel (ENaC) to promote natriuresis. However, the mechanism of how high salt increases NOS1 activity in the CD is not known. Histone deacetylase 1 (HDAC1) is known to mediate post-translational deacetylation in response to flow in other cell types. Hence, we hypothesized that HDAC1 activates renal medullary NOS1-dependent NO production in response to high salt intake. Renal medullas were isolated from C57bl/6J mice after one week of normal (NS, 0.4% NaCl) or high salt (HS, 4% NaCl) diet. Inner-medullary HDAC1 expression (NS: 1.00±0.1, HS: 1.61±0.1 RDU; n=5/group, p=0.001), but not activity (NS: 0.07±0.02, HS: 0.05±0.01μM/μg, n=5/group, p>0.05), was significantly elevated in mice on HS compared to NS. HDAC1-selective inhibitor, MS-275 (300 nM), was utilized in in vitro and ex vivo experiments to determine the contribution of HDAC1 to NOS activity. Nitrite production, analyzed by HPLC, increased >5-fold (static: 231±31.1, flow: 1291±183 pmol/mg pr/hr; n=7/group, p<0.001) in mouse IMCD cells in response to acute 10 dynes/cm 2 fluid-flow. MS-275 significantly blunted flow-responsive NO production (MS-275: 342±44.0 pmol/mg pr/hr; n=9/group, p<0.001). Likewise, ENaC activity, which is negatively regulated by NOS1, was significantly increased in isolated collecting ducts post ex vivo MS-275 treatment from mice on HS (HS: 0.34±0.11 NP o , HS+MS-275: 0.97±0.16 NP o ; n=22/group, p<0.05). Moreover, acute DETA-NONOate treatment, an NO donor, reversed this finding (HS+MS-275: 0.77±0.14 NP o , HS+MS-275+DETA: 0.46±0.14 NP o ; n=10/group, p<0.05). Lastly, acute MS-275 treatment in vitro did not increase NOS1 acetylation status as determined by co-immunoprecipitation, suggesting that HDAC1 may not directly deacetylate NOS1. Taken together, these data indicate that HS intake increases renal medullary HDAC1 to positively regulate NOS1-derived NO production.

Vasopressin-Independent Regulation of Aquaporin-2 by Tamoxifen in Kidney Collecting Ducts.

Arginine vasopressin (AVP) mediates water reabsorption in the kidney collecting ducts through regulation of aquaporin-2 (AQP2). Also, estrogen has been known to regulate AQP2. Consistently, we previously demonstrated that tamoxifen (TAM), a selective estrogen receptor modulator, attenuates the downregulation of AQP2 in lithium-induced nephrogenic diabetes insipidus (NDI). In this study, we investigated the AVP-independent regulation of AQP2 by TAM and the therapeutic effect of TAM on the dysregulation of AQP2 and impaired urinary concentration in a unilateral ureteral obstruction (UUO) model. Primary cultured inner medullary collecting duct (IMCD) cells from kidneys of male Sprague-Dawley rats were treated with TAM. Rats subjected to 7 days of UUO were treated with TAM by oral gavage. Changes of intracellular trafficking and expression of AQP2 were evaluated by quantitative PCR, Western blotting, and immunohistochemistry. TAM induced AQP2 protein expression and intracellular trafficking in primary cultured IMCD cells, which were independent of the vasopressin V2 receptor (V2R) and cAMP activation, the critical pathways involved in AVP-stimulated regulation of AQP2. TAM attenuated the downregulation of AQP2 in TGF-β treated IMCD cells and IMCD suspensions prepared from UUO rats. TAM administration in vivo attenuated the downregulation of AQP2, associated with an improvement of urinary concentration in UUO rats. In addition, TAM increased CaMKII expression, suggesting that calmodulin signaling pathway is likely to be involved in the TAM-mediated AQP2 regulation. In conclusion, TAM is involved in AQP2 regulation in a vasopressin-independent manner and improves urinary concentration by attenuating the downregulation of AQP2 and maintaining intracellular trafficking in UUO.

Vasopressin Increases Urinary Acidification via V1a Receptors in Collecting Duct Intercalated Cells.

Antagonists of the V1a vasopressin receptor (V1aR) are emerging as a strategy for slowing progression of CKD. Physiologically, V1aR signaling has been linked with acid-base homeostasis, but more detailed information is needed about renal V1aR distribution and function. We used a new anti-V1aR antibody and high-resolution microscopy to investigate Va1R distribution in rodent and human kidneys. To investigate whether V1aR activation promotes urinary H+ secretion, we used a V1aR agonist or antagonist to evaluate V1aR function in vasopressin-deficient Brattleboro rats, bladder-catheterized mice, isolated collecting ducts, and cultured inner medullary collecting duct (IMCD) cells. Localization of V1aR in rodent and human kidneys produced a basolateral signal in type A intercalated cells (A-ICs) and a perinuclear to subapical signal in type B intercalated cells of connecting tubules and collecting ducts. Treating vasopressin-deficient Brattleboro rats with a V1aR agonist decreased urinary pH and tripled net acid excretion; we observed a similar response in C57BL/6J mice. In contrast, V1aR antagonist did not affect urinary pH in normal or acid-loaded mice. In ex vivo settings, basolateral treatment of isolated perfused medullary collecting ducts with the V1aR agonist or vasopressin increased intracellular calcium levels in ICs and decreased luminal pH, suggesting V1aR-dependent calcium release and stimulation of proton-secreting proteins. Basolateral treatment of IMCD cells with the V1aR agonist increased apical abundance of vacuolar H+-ATPase in A-ICs. Our results show that activation of V1aR contributes to urinary acidification via H+ secretion by A-ICs, which may have clinical implications for pharmacologic targeting of V1aR.

Atg7-dependent canonical autophagy regulates the degradation of aquaporin 2 in prolonged hypokalemia

Prolonged hypokalemia induces a decrease of urinary concentrating ability via down-regulation of aquaporin 2 (AQP2); however, the precise mechanisms remain unknown. To investigate the role of autophagy in the degradation of AQP2, we generated the principal cell-specific Atg7 deletion (Atg7Δpc) mice. In hypokalemic Atg7-floxed (Atg7f/f) mice, huge irregular shaped LC3-positive autophagic vacuoles accumulated mainly in inner medullary collecting duct (IMCD) cells. Total- and pS261-AQP2 were redistributed from apical and subapical domains into these vacuoles, which were not co-localized with RAB9. However, in the IMCD cells of hypokalemic Atg7Δpc mice, these canonical autophagic vacuoles were markedly reduced, whereas numerous small regular shaped LC3-negative/RAB9-positive non-canonical autophagic vacuoles were observed along with diffusely distributed total- and pS261-AQP2 in the cytoplasm. The immunoreactivity of pS256-AQP2 in the apical membrane of IMCD cells was markedly decreased, and no redistribution was observed in both hypokalemic Atg7f/f and Atg7Δpc mice. These findings suggest that AQP2 down regulation in hypokalemia was induced by reduced phosphorylation of AQP2, resulting in a reduction of apical plasma labeling of pS256-AQP2 and degradation of total- and pS261-AQP2 via an LC3/ATG7-dependent canonical autophagy pathway.

Identification of NFAT5 as a transcriptional regulator of the EDN1 gene in collecting duct.

The inner medullary collecting duct (IMCD) produces very high levels of endothelin-1 (ET-1) that acts as an autocrine inhibitor of IMCD Na+ and water reabsorption. Recent studies suggest that IMCD ET-1 production is enhanced by extracellular hypertonicity as can occur during high salt intake. Although NFAT5 has been implicated in the IMCD ET-1 hypertonicity response, no studies in any cell type have identified NFAT5 as a transcriptional regulator of the EDN1 gene; the current study examined this using a mouse IMCD cell line (IMCD3). Media hypertonicity increased IMCD3 ET-1 mRNA in a dose- and time-dependent manner associated with increased NFAT5 nuclear localization. Knockdown of NFAT5 using small-interfering RNA or by CRISPR/Cas9-mediated targeting of exon 4 of the NFAT5 gene reduced the ET-1 hypertonicity response. Chromatin immunoprecipitation using an NFAT5 antibody pulled down ET-1 promoter regions containing NFAT5 consensus binding sequences. Transfected ET-1 promoter reporter constructs revealed maximal hypertonicity-induced reporter activity in the proximal 1-kb region; mutation of the two NFAT5 consensus-binding sites in this region abolished hypertonicity-induced reporter activity. The 1-kb ET-1 promoter-reporter construct lost hypertonicity responsiveness when transfected in CRISPR/Cas9-induced NFAT5-deficient cells. In summary, these findings represent the first description that NFAT5 is a direct transcriptional regulator of the EDN1 gene in IMCD cells and point to a potentially important mechanism by which body Na+ homeostasis is maintained.

NFAT5 up-regulates expression of the kidney-specific ubiquitin ligase gene Rnf183 under hypertonic conditions in inner-medullary collecting duct cells

We previously reported that among the 37 RING finger protein (RNF) family members, RNF183 mRNA is specifically expressed in the kidney under normal conditions. However, the mechanism supporting its kidney-specific expression pattern remains unclear. In this study, we elucidated the mechanism of the transcriptional activation of murine Rnf183 in inner-medullary collecting duct cells. Experiments with anti-RNF183 antibody revealed that RNF183 is predominantly expressed in the renal medulla. Among the 37 RNF family members, Rnf183 mRNA expression was specifically increased in hypertonic conditions, a hallmark of the renal medulla. RNF183 up-regulation was consistent with the activation of nuclear factor of activated T cells 5 (NFAT5), a transcription factor essential for adaptation to hypertonic conditions. Accordingly, siRNA-mediated knockdown of NFAT5 down-regulated RNF183 expression. Furthermore, the -3,466 to -3,136-bp region upstream of the mouse Rnf183 promoter containing the NFAT5-binding motif is conserved among mammals. A luciferase-based reporter vector containing the NFAT5-binding site was activated in response to hypertonic stress, but was inhibited by a mutation at the NFAT5-binding site. ChIP assays revealed that the binding of NFAT5 to this DNA site is enhanced by hypertonic stress. Of note, siRNA-mediated RNF183 knockdown increased hypertonicity-induced caspase-3 activation and decreased viability of mIMCD-3 cells. These results indicate that (i) RNF183 is predominantly expressed in the normal renal medulla, (ii) NFAT5 stimulates transcriptional activation of Rnf183 by binding to its cognate binding motif in the Rnf183 promoter, and (iii) RNF183 protects renal medullary cells from hypertonicity-induced apoptosis.

Tonicity inversely modulates lipocalin-2 (Lcn2/24p3/NGAL) receptor (SLC22A17) and Lcn2 expression via Wnt/\u03b2-catenin signaling in renal inner medullary collecting duct cells: implications for cell fate and bacterial infection

BackgroundWe have previously evidenced apical expression of the 24p3/NGAL/lipocalin-2 receptor (Lcn2-R; SLC22A17) in inner medullary collecting duct (IMCD) cells, which are present in vivo in a hyperosmotic/-tonic environment that activates canonical Wnt/β-catenin signaling. The localization of Lcn2-R in the inner medulla is intriguing considering local bacterial infections trigger toll-like receptor-4 (TLR-4)-mediated secretion of the bacteriostatic Fe3+-free (apo-)Lcn2.AimTo determine the effects of osmolarity/tonicity changes, Wnt/β-catenin and TLR-4 activation on Lcn2-R and Lcn2 expression and cell viability in rat primary IMCD and mouse (m)IMCD3 cells.MethodsNormosmolarity/-tonicity was 300 mosmol/l whereas hyperosmolarity/-tonicity was induced by adding 100 mmol/l NaCl + 100 mmol/l urea (600 mosmol/l, 1-7 days). Lcn2-R and Lcn2 expression were determined by qPCR, immunoblotting, flow cytometry and immunofluorescence microscopy. β-catenin was silenced by RNAi. Cell viability/death was determined with MTT and LDH release assays. TLR-4 was activated by bacterial lipopolysaccharides (LPS).ResultsHyperosmotic/-tonic media upregulated Lcn2-R by ~4-fold and decreased Lcn2 expression/secretion, along with Wnt/β-catenin activation, in IMCD cells. These effects of hyperosmotic/-tonic media on Lcn2-R/Lcn2 expression were reverted by normosmolarity/-tonicity, β-catenin silencing and/or LPS. Exposure of cells with endogenous or stably overexpressing Lcn2-R to apo-Lcn2 or LPS decreased cell viability.ConclusionsLcn2-R upregulation and Lcn2 downregulation via Wnt/β-catenin may promote adaptive osmotolerant survival of IMCD cells in response to hyperosmolarity/-tonicity whereas Lcn2 upregulation and Lcn2-R downregulation via TLR-4 and/or normosmolarity/-tonicity may protect IMCD cells against bacterial infections and prevent autocrine death induction by Lcn2.

Bile Acid G Protein-Coupled Membrane Receptor TGR5 Modulates Aquaporin 2-Mediated Water Homeostasis.

The bile acid-activated receptors, including the membrane G protein-coupled receptor TGR5 and nuclear farnesoid X receptor (FXR), have roles in kidney diseases. In this study, we investigated the role of TGR5 in renal water handling and the underlying molecular mechanisms. We used tubule suspensions of inner medullary collecting duct (IMCD) cells from rat kidneys to investigate the effect of TGR5 signaling on aquaporin-2 (AQP2) expression, and examined the in vivo effects of TGR5 in mice with lithium-induced nephrogenic diabetes insipidus (NDI) and Tgr5 knockout (Tgr5-/-) mice. Activation of TGR5 by lithocholic acid (LCA), an endogenous TGR5 ligand, or INT-777, a synthetic TGR5-specific agonist, induced AQP2 expression and intracellular trafficking in rat IMCD cells via a cAMP-protein kinase A signaling pathway. In mice with NDI, dietary supplementation with LCA markedly decreased urine output and increased urine osmolality, which was associated with significantly upregulated AQP2 expression in the kidney inner medulla. Supplementation with endogenous FXR agonist had no effect. In primary IMCD suspensions from lithium-treated rats, treatment with INT-767 (FXR and TGR5 dual agonist) or INT-777, but not INT-747 (FXR agonist), increased AQP2 expression. Tgr5-/- mice exhibited an attenuated ability to concentrate urine in response to dehydration, which was associated with decreased AQP2 expression in the kidney inner medulla. In lithium-treated Tgr5-/- mice, LCA treatment failed to prevent reduction of AQP2 expression. TGR5 stimulation increases renal AQP2 expression and improves impaired urinary concentration in lithium-induced NDI. TGR5 is thus involved in regulating water metabolism in the kidney.