mIMCD3 Cells Research Articles

3D spheroid model of mIMCD3 cells for studying ciliopathies and renal epithelial disorders.

We have developed a novel 3D cell culture model that uses mouse inner-medullary collecting duct (mIMCD3) cells to generate epithelial spheroids. This model is amenable to efficient siRNA knockdown and subsequent rescue with human patient-derived alleles. Spheroids develop apicobasal polarity and complete lumens, and they are consequently an ideal model for polarity defects seen in renal ciliopathies such as nephronophthisis. Briefly, mIMCD3 cells are transfected and subsequently passaged to a Matrigel mixture, which is seeded in chamber slides and covered in growth medium. Once the spheroids are formed, Matrigel is dissolved and immunocytochemistry is performed in the chamber slides. The technique is amenable to semiautomatic imaging analysis, and it can test multiple genes simultaneously, gene-dosing effects and a variety of therapeutic interventions. The spheroid technique is a unique and simple 6-d in vitro method of interrogating ex vivo tissue organization.

Leishmania infantum-chagasi activates SHP-1 and reduces NFAT5/TonEBP activity in the mouse kidney inner medulla.

Visceral leishmaniasis patients have been reported to have a urine concentration defect. Concentration of urine by the renal inner medulla is essentially dependent on a transcription factor, NFAT5/TonEBP, because it activates expression of osmoprotective genes betaine/glycine transporter 1 (BGT1) and sodium/myo-inositol transporter (SMIT), and water channel aquaporin-2, all of which are imperative for concentrating urine. Leishmania parasites evade macrophage immune defenses by activating protein tyrosine phosphatases, among which SHP-1 is critical. We previously demonstrated that SHP-1 inhibits tonicity-dependent activation of NFAT5/TonEBP in HEK293 cells through screening a genome-wide small interfering (si) RNA library against phosphatases (Zhou X, Gallazzini M, Burg MB, Ferraris JD. Proc Natl Acad Sci USA 107: 7072-7077, 2010). We sought to examine whether Leishmania can activate SHP-1 and inhibit NFAT5/TonEBP activity in the renal inner medulla in a murine model of visceral leishmaniasis by injection of female BALB/c mice with a single intravenous dose of 5 × 10(5) L. chagasi metacyclic promastigotes. We found that SHP-1 is expressed in the kidney inner medulla. L. chagasi activates SHP-1 with an increase in stimulatory phosphorylation of SHP-1-Y536 in the region. L. chagasi reduces expression of NFAT5/TonEBP mRNA and protein as well as expression of its targeted genes: BGT1, SMIT, and aquaporin-2. The culture supernatant from L. chagasi metacyclic promastigotes increases SHP-1 protein abundance and potently inhibits NFAT5 transcriptional activity in mIMCD3 cells. However, L. chagasi in our animal model has no significant effect on urinary concentration. We conclude that L. chagasi, most likely through its secreted virulence factors, activates SHP-1 and reduces NFAT5/TonEBP gene expression, which leads to reduced NFAT5/TonEBP transcriptional activity in the kidney inner medulla.

Regulation of (pro)renin receptor expression in mIMCD via the GSK-3β-NFAT5-SIRT-1 signaling pathway.

The localization and regulation of (pro)renin receptor (PRR) expression in kidney collecting duct cells are not well established. We hypothesized that low salt (LS) contributes to the regulation of PRR expression in these cells via the GSK-3β-NFAT5-sirtuin1 (SIRT-1) signaling pathway. Mouse inner medullary collecting duct (mIMCD) cells were treated with NaCl at 130 (normal salt; NS), 63 (LS), or 209 mM (high salt; HS) alone or in combination with NFAT5 scrambled small interfering (si) RNA, NFAT5 siRNA, or the SIRT-1 inhibitor EX-527. Compared with NS, LS increased the mRNA and protein expression of PRR by 71% and 69% (P < 0.05), and reduced phosphorylation of GSK-3β by 62% (P < 0.01), mRNA and protein expressions of NFAT5 by 65% and 45% (P < 0.05), and SIRT-1 by 44% and 50% (P < 0.01), respectively. LS also enhanced p65 NF-κB by 102% (P < 0.01). Treatment with HS significantly reduced the mRNA and protein expression of PRR by 32% and 23% (P < 0.05), and increased the mRNA and protein expression of NFAT5 by 39% and 45% (P < 0.05) and SIRT-1 by 51% and 56% (P < 0.05), respectively. HS+NFAT5 siRNA reduced the mRNA and protein expression of NFAT5 by 51% and 35% (P < 0.01) and increased the mRNA and protein expression of PRR by 148% and 70% (P < 0.01), respectively. HS+EX-527 significantly increased the mRNA and protein expression of PRR by 96% and 58% (P < 0.05), respectively. We conclude that expression of PRR in mIMCD cells is regulated by the GSK-3β-NFAT5- SIRT-1 signaling pathway.

Integrin-linked kinase regulates tubular aquaporin-2 content and intracellular location: a link between the extracellular matrix and water reabsorption.

One of the clinical alterations observed in chronic renal disease (CRD) is the impaired urine concentration, known as diabetes insipidus (DI). Tubulointerstitial fibrosis of the kidney is also a pathological finding observed in CRD and involves composition of extracellular matrix (ECM). However, an association between these two events has not been elucidated. In this study, we showed that the extracellular-to-intracellular scaffold protein integrin-linked kinase (ILK) regulates expression of tubular water channel aquaporin-2 (AQP2) and its apical membrane presence in the renal tubule. Basally, polyuria and decreased urine osmolality were present in ILK conditional-knockdown (cKD-ILK) adult mice compared with nondepleted ILK littermates. No changes were observed in arginine-vasopressin (AVP) blood levels, renal receptor (V2R), or AQP3 expression. However, tubular AQP2 was decreased in expression and apical membrane presence in cKD-ILK mice, where the canonical V2R/cAMP axis activation is still functional, but independent of the absence of ILK. Thus, cKD-ILK constitutes a nephrogenic diabetes insipidus (NDI) model. AQP2 and ILK colocalize in cultured inner medullary collecting duct (mIMCD3) cells. Specific ILK siRNAs and collagen I (Col) decrease ILK and AQP2 levels and AQP2 presence on the membrane of tubular mIMCD3 cells, which impairs the capacity of the cells to transport water under hypotonic stress. The present work points to ILK as a therapeutic target in NDI.

Identification of hepatocyte nuclear factor‐1β gene regulatory networks in mouse kidney cells (690.9)

Hepatocyte nuclear factor‐1β (HNF‐1β) is a transcription factor that is expressed in renal tubules in the mammalian kidney. Mutations of HNF‐1β produce kidney cysts and electrolyte disorders. HNF‐1β has been shown to regulate the expression of cystic disease genes and solute transporters. The aim of this study was to identify the complete spectrum of genes that are regulated by HNF‐1β in the kidney. We performed genome‐wide chromatin immunoprecipitation and massively parallel sequencing (ChIP‐seq) in combination with microarray analysis to identify direct mRNA and miRNA targets in mouse mIMCD3 cells. ChIP‐seq analysis identified 4,725 mRNAs and 85 miRNAs as potential targets of HNF‐1β. Microarray analysis showed altered expression of 4,878 mRNAs and 51 miRNAs in cells expressing dominant‐negative mutant HNF‐1β. Combining the ChIP‐seq and microarray data identified 1,545 mRNAs and 22 miRNAs that were directly regulated by HNF‐1β. Pathway analysis revealed that HNF‐1β directly regulated the expression of multiple genes involved in cholesterol and sphingolipid metabolism. Liquid chromatography‐mass spectrometry showed corresponding alterations in the levels of sterols and sphingolipids in cells expressing mutant HNF‐1β. These findings provide new insights into the HNF‐1β transcriptional network in kidney cells and identify a novel role for HNF‐1β in regulating lipid metabolism in the kidney.

MicroRNA regulation of endothelin-1 mRNA in renal collecting duct cells

AimsRecently, microRNAs (miRNAs) have been implicated in control of Edn1 mRNA in several tissues. Here we examined the role of miRNA action on Edn1 mRNA expression in renal distal collecting duct cells. Main methodsA microarray study was conducted to provide a comprehensive assessment of miRNAs present in a murine inner medullary collecting duct (mIMCD-3) cell line. The experiment was designed as a comparison between mIMCD-3 cells grown in the presence and absence of aldosterone. Argonaute (Ago) immunoprecipitation experiments were used to investigate binding of the RNA induced silencing complex (RISC) to Edn1 mRNA. Key findingsThirty-four miRNAs were detected in very high abundance in mIMCD-3 cells, and a large number of others were present at lower levels. The microarray experiments were validated by quantitative PCR analysis of selected miRNAs. The microarray data, in combination with in silico examination of the Edn1 3′ UTR provided a panel of candidate miRNAs that could act upon the Edn1 expression. Edn1 mRNA was co-immunoprecipitated with an Argonaute protein antibody, and this interaction was blocked by anti-miR-709 oligonucleotides. SignificanceThese results define the miRNA landscape of the mIMCD-3 cell line. Moreover, Edn1 was shown to interact with Argonaute protein suggesting that it is a target of the RNA induced silencing complex (RISC).

Controlled tubulogenesis from dispersed ureteric bud-derived cells using a micropatterned gel

Developmental engineering is a potential option for neo-organogenesis of complex organs such as the kidney. The application of this principle requires the ability to construct a tubular structure from dispersed renal progenitor cells with defined size and geometry. In this present study we report the generation of tubular structures from dispersed ureteric bud cells in vitro by using a micropatterned gel. Dispersed CMUB-1 cells, a mouse ureteric bud-derived cell line, or mIMCD cells, a mouse collecting duct-derived cell line, were suspended in collagen I and seeded into an agarose-based micropatterned gel. We found that within 24-36 h of incubation, the cells developed a tubular structure that conformed to the geometry of the micropattern of the gel. The lumen formation of the tubular structure was confirmed by immunohistochemical staining and observed by confocal microscopy. We found that higher concentrations of collagen I negatively influenced the efficiency of tubular formation. Tubule formation in CMUB-1, but not mIMCD, cells was positively influenced by the addition of aldosterone (10, 50 and 200 µg/ml), FGF (50 and 100 µg/ml) and fibronectin (10 and 50 µg/ml) to the growth medium. We further demonstrated the functionality of the generated tubes by in vitro budding, which was induced by growth factors, such as glial cell-derived neurotrophic factor (GDNF) or fibroblast growth factor 7 (FGF7), in the presence of beads soaked with the activin A inhibitor follistatin. Our current study thus demonstrates the possibility of constructing a functional tubular structure from dispersed ureteric bud cells in vitro in a controlled manner. Copyright © 2014 John Wiley & Sons, Ltd.

Sfmbt2 10th intron-hosted miR-466(a/e)-3p are important epigenetic regulators of Nfat5 signaling, osmoregulation and urine concentration in mice

Sfmbt2-hosted miR-466a-3p and its close relatives are often among the most significantly up-regulated or down-regulated miRNAs in responses to numerous deleterious environmental stimuli. The exact roles of these miRNAs in cellular stress responses, however, are not clear. Here we showed that many Sfmbt2-hosted miRNAs were highly hypertonic stress responsive in vitro and in vivo. In renal medulla, water deprivation induced alterations in the expression of miR-466(a/b/c/e/p)-3p in a pattern similar to that of miR-200b-3p, a known regulator of osmoresponsive transcription factor Nfat5. Remarkably, exposure of mIMCD3 cells to an arginine vasopressin analog time-dependently down-regulated the expression of miR-466(a/b/c/e/p)-3p and miR-200b-3p, which provides a novel regulatory mechanism for these osmoresponsive miRNAs. In cultured mIMCD3 cells we further demonstrated that miR-466a-3p and miR-466g were capable of targeting Nfat5 by interacting with its 3′UTR. In transgenic mice overexpressing miR-466a-3p, significant down-regulation of Nfat5 and many other osmoregulation-related genes was observed in both the renal cortex and medulla. Moreover, sustained transgenic over-expression of miR-466a-3p was found to be associated with polydipsia, polyuria and disturbed ion homeostasis and kidney morphology. Since the mature sequence of miR-466a-3p is completely equivalent to that of miR-466e-3p and that the seed sequence of miR-466a-3p is completely equivalent to that of miR-297(a/b/c)-3p, miR-466d-3p, miR-467g and miR-669d-3p, and that miR-466a-3p differs from miR-466(b/c/p)-3p only in a 5′ nucleotide, we propose that miR-466a-3p and many of its close relatives are important epigenetic regulators of renal Nfat5 signaling, osmoregulation and urine concentration in mice.

Effects of cadmium chloride on mouse inner medullary collecting duct cells

Cadmium is a known renal toxin. The cytotoxic effect of cadmium chloride (CdCl2) was evaluated on renal inner medullary collecting duct cells (mIMCD3). The 24 hr LC50 value for CdCl2 in mIMCD3 cells was 40 µM. The present study showed that mIMCD3 cells were sensitive to CdCl2 exposure.

Gamma-melanocyte stimulating hormone regulates the expression and cellular localization of epithelial sodium channel in inner medullary collecting duct cells

Gamma2-melanocyte-stimulating hormone (γ2MSH) is a peptide hormone released by the pituitary gland which is thought to act directly on the renal inner medulla to promote increased sodium excretion into urine (natriuresis). The aim of this study was to determine if a stable analog, [Nle3, D-Phe6]-γ2MSH (NDP-γ2MSH), of the native peptide regulated the activity, expression and cellular localization of epithelial sodium channel (ENaC) in a murine inner medullary collecting duct (mIMCD-3) cell line. Our results indicate that expression of the γ2MSH receptor, melanocortin receptor 3 receptor (MC3R), is up-regulated by culturing the cells in media with an increased osmolality (∼400mOsm/kg). Furthermore, stimulation of cAMP signaling and sodium transport by 1nM NDP-γ2MSH occurs only in cells cultured in the high osmolality media. Finally, treatment of mIMCD-3 cells cultured in high osmolality medium for 1h with 1nM NDP-γ2MSH causes a reduction in expression of serum- and glucocorticoid-induced kinase (sgk1) and a reduction in expression and cell surface abundance of the alpha subunit of ENaC. Collectively, this data suggest that γ2MSH directly regulates both ENaC expression and cellular localization in the inner medulla to exert its natriuretic effect.

Sp1 trans-activates and is required for maximal aldosterone induction of the αENaC gene in collecting duct cells

The epithelial Na+ channel (ENaC) in the distal nephron constitutes the rate-limiting step for renal sodium reabsorption. Aldosterone increases tubular sodium absorption in large part by increasing αENaC transcription in collecting duct principal cells. We previously reported that Af9 binds to +78/+92 of αENaC and recruits Dot1a to repress basal and aldosterone-sensitive αENaC transcription in mouse inner medullary collecting duct (mIMCD)3 cells. Despite this epigenetic repression, basal αENaC transcription is still evident and physiologically necessary, indicating basal operation of positive regulators. In the present study, we identified Sp1 as one such regulator. Gel shift and antibody competition assays using a +208/+240 probe revealed DNA-Sp1-containing complexes in mIMCD3 cells. Mutation of the +222/+229 element abrogated Sp1 binding in vitro and in promoter-reporter constructs stably expressed in mIMCD3 cells. Compared with the wild-type promoter, an αENaC promoter-luciferase construct with +222/+229 mutations exhibited much lower activity and impaired trans-activation in Sp1 overexpression experiments. Conversely, Sp1 knockdown inhibited endogenous αENaC mRNA and the activity of the wild-type αENaC promoter but not the mutated construct. Aldosterone triggered Sp1 recruitment to the αENaC promoter, which was required for maximal induction of αENaC promoter activity and was blocked by spironolactone. Sequential chromatin immunoprecipitation assays and functional tests of +78/+92 and +222/+229 αENaC promoter mutants indicated that while Sp1, Dot1a, and Af9 co-occupy the αENaC promoter, the Sp1 effects are functionally independent from Dot1a and Af9. In summary, Sp1 binding to a cis-element at +222/+229 represents the first identified constitutive driver of αENaC transcription, and it contributes to maximal aldosterone trans-activation of αENaC.

Post-Translational Loss of Renal TRPV5 Calcium Channel Expression, Ca2+ Wasting, and Bone Loss in Experimental Colitis

Dysregulated Ca(2+) homeostasis likely contributes to the etiology of inflammatory bowel disease-associated loss of bone mineral density. Experimental colitis leads to decreased expression of Klotho, a protein that supports renal Ca(2+) reabsorption by stabilizing the transient receptor potential vanilloid 5 (TRPV5) channel on the apical membrane of distal tubule epithelial cells. Colitis was induced in mice via administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) or transfer of CD4(+)interleukin-10(-/-) and CD4(+), CD45RB(hi) T cells. We investigated changes in bone metabolism, renal processing of Ca(2+), and expression of TRPV5. Mice with colitis had normal serum levels of Ca(2+) and parathormone. Computed tomography analysis showed a decreased density of cortical and trabecular bone, and there was biochemical evidence for reduced bone formation and increased bone resorption. Increased fractional urinary excretion of Ca(2+) was accompanied by reduced levels of TRPV5 protein in distal convoluted tubules, with a concomitant increase in TRPV5 sialylation. In mouse renal intermedullary collecting duct epithelial (mIMCD3) cells transduced with TRPV5 adenovirus, the inflammatory cytokines tumor necrosis factor, interferon-γ, and interleukin-1β reduced levels of TRPV5 on the cell surface, leading to its degradation. Cytomix induced interaction between TRPV5 and UBR4 (Ubiquitin recoginition 4), an E3 ubiquitin ligase; knockdown of UBR4 with small interfering RNAs prevented cytomix-induced degradation of TRPV5. The effects of cytokines on TRPV5 were not observed in cells stably transfected with membrane-bound Klotho; TRPV5 expression was preserved when colitis was induced with TNBS in transgenic mice that overexpressed Klotho or in mice with T-cell transfer colitis injected with soluble recombinant Klotho. After induction of colitis in mice via TNBS administration or T-cell transfer, tumor necrosis factor and interferon-γ reduced the expression and activity of Klotho, which otherwise would protect TRPV5 from hypersialylation andcytokine-induced TRPV5 endocytosis, UBR4-dependent ubiquitination, degradation, and urinary wasting of Ca(2+).

Mechanisms of shear stress mediated nitric oxide production by inner medullary collecting duct cells

We previously reported that increased shear stress (SS) stimulates nitric oxide (NO) production in mouse inner medullary collecting duct‐3 (mIMCD3) cells. Recent work in mpk‐cortical collecting duct cells determined that SS‐induced endothelin‐1 (ET1) expression was mediated via activation of the epithelial sodium channel (ENaC). We have shown that ET1 via ETB receptor stimulation of NOS1 mediates NO production in mIMCD3 cells. Thus, we hypothesized that SS‐induced NO production in mIMCD3 cells is dependent on an ENaC‐ET1‐ETB‐NOS1 mechanism. mIMCD3 cells were grown to confluency on glass slides, subjected to SS (30 dynes/cm2 for 1 h), and nitrite production measured by HPLC and calculated as fold change from static conditions. Cells were pretreated with ENaC inhibitors, 1 μM amiloride or 0.2 μM benzamil, ETB antagonist 1000 nM BQ‐788, or 100 nM 1400W (NOS2‐selective inhibitor) or 1 μM VNIO (NOS1‐selective inhibitor), and nitrite production determined. Both ENaC inhibitors significantly blunted the SS‐induced increase in nitrite (control = 1.8 ± 0.3 vs 1.3 ± 0.2 and 0.9 ± 0.3 respectively N = 4, P &lt; 0.05). BQ‐788 attenuated the SS mediated increase in NO (control = 3.9 ± 1.0 vs 1.7 ±0.1, N =5, P &lt; 0.05). NOS2 inhibition had no significant effect on SS‐induced nitrite production (p&gt;;0.05), while NOS1 inhibition lead to an ~50% reduction in SS‐mediated nitrite (control = 2.8 ± 0.5 vs 1.5 ± 0.4 N= 5–10, P &lt; 0.05). In conclusion, mIMCD3 SS induced NO production is mediated via activation of ENaC, ETB receptor, and NOS1. Future studies will determine whether ENaC via ET1/ETB/NOS1 directly regulates mIMCD3 NO production. Funding support from AHA to KAH and NIH to JSP.

The Effect of Aldosterone on the miRNA Content of a Murine Inner Medullary Collecting Duct Cell

MicroRNAs (miRNAs) are a family of small noncoding RNAs that have been shown to regulate gene expression by inhibiting translation or targeting specific mRNAs for degradation. miRNAs play important roles in the regulation of metabolism in the healthy cell and in the pathogenesis of many diseases including diabetic nephropathy and hypertension‐related renal disease.Aldosterone is the major known hormone responsible for sodium retention in the kidney collecting duct. Our working hypothesis is that aldosterone action alters the miRNA content in the inner medullary collecting duct resulting in increased translation and stabilization of mRNAs involved in sodium reabsorption.Here we report the miRNA landscape in mIMCD‐3 cells using Toray 3D‐GeneTM miRNA microarray analysis to evaluate 1080 miRNAs. Microarray analysis identified 35 highly expressed miRNAs, as well as several miRNAs whose levels appear to be affected by the presence of aldosterone. Two aldosterone responsive miRNAs affected levels of serum/glucocorticoid regulated kinase 1and sodium/potassium‐transporting ATPase subunit beta‐1 mRNA. Argonaute pull‐down experiments also demonstrated that endothelin‐1 mRNA is a target of the RNA‐induced silencing complex indicating miRNA action on this transcript.This work was supported by NIH NIDDK 1RO1DK082680 to B.D.C and C.S.W. and a 5T32DK076541–04 pre‐doctoral fellowship to M.E.J. Additional funding support and miRNA microarrays were provided by Toray Industries, Inc.

Common genetic variants of the human uromodulin gene regulate transcription and predict plasma uric acid levels

Uromodulin (UMOD) genetic variants cause familial juvenile hyperuricemic nephropathy, characterized by hyperuricemia with decreased renal excretion of UMOD and uric acid, suggesting a role for UMOD in the regulation of plasma uric acid. To determine this, we screened common variants across the UMOD locus in one community-based Chinese population of 1000 individuals and the other population from 642 American twins and siblings of European and Hispanic ancestry. Transcriptional activity of promoter variants was estimated in luciferase reporter plasmids transfected into HEK-293 cells and mIMCD3 cells. In the primary Chinese population, we found that carriers of the GCC haplotype had higher plasma uric acid, and three promoter variants were associated with plasma uric acid. UMOD promoter variants displayed reciprocal effects on urine uric acid excretion and plasma uric acid concentration, suggesting a primary effect on renal tubular handling of urate. These UMOD genetic marker-on-trait associations for uric acid were replicated in the independent American cohort. Site-directed mutagenesis at trait-associated UMOD promoter variants altered promoter activity in transfected luciferase reporter plasmids. Thus, UMOD promoter variants seem to initiate a cascade of transcriptional and biochemical changes influencing UMOD secretion, leading to altered plasma uric acid levels.

Effect of Poria cocos on hypertonic stress‐induced water channel expression and apoptosis in renal collecting duct cells

Aquaporin‐2 (AQP2), a family of water channels, plays an important role in the urinary concentrating process and regulation of water balance in the kidney. The dried sclerotia of Poria cocos Wolf (WPC) has been used for chronic edema and nephrosis. This study was conducted to evaluate the effects of WPC on hypertonic stress (175 mM NaCl)‐induced AQP2 expression and apoptosis in murine inner medullary collecting duct cell line (mIMCD‐3). WPC significantly attenuated the hypertonicity‐induced AQP2 protein and mRNA levels and blocked the apical membrane insertion of AQP2 in a concentration‐dependent manner. Tonicity‐responsive enhancer binding protein (TonEBP) mRNA was also recovered by WPC under hypertonic stress. Furthermore, pretreatment with WPC presented the similar effect with KT5720 which was decreased hypertonic stress‐induced AQP2 expression. On the other hand, WPC significantly recovered hypertonic stress‐injured mIMCD‐3 viability and cell number. WPC markedly inhibited hypertonic stress‐induced Bax or caspase‐3 expression, whereas Bcl‐2 expression was increased. Besides, WPC attenuated the hypertonicity‐induced morphological changes in nuclei, resulting in anti‐apoptotic effect. Thus, these data suggests that WPC has beneficial effects on the therapeutic approach to renal disorder.

Flow stimulates inner medullary collecting duct endothelin‐1 production

Collecting duct (CD)‐derived endothelin‐1 (ET‐1) inhibits Na and water reabsorption; its deficiency causes marked hypertension. CD ET‐1 synthesis is enhanced by extracellular fluid volume expansion. In cultured cortical CD (CCD) cells, increased flow (as would occur in tubule fluid during volume expansion) stimulates ET‐1 production; this is ENaC‐dependent. Since the inner medullary CD (IMCD) is the major site of renal ET‐1 synthesis, we examined the effect of flow on IMCD ET‐1 production. Mouse IMCD3 cells were subjected to static conditions or flow (2 dyne/cm2 for 2 hr), followed by determination of ET‐1 mRNA. Flow increased ET‐1 mRNA by 2.2‐fold. Absence of perfusate Ca prevented the flow response. BAPTA, W‐7, calmidazolium, KN‐93, calphostin C and cyclosporin A reduced the ET‐1 flow response, indicating that PKC and Ca/calmodulin/calmodulin kinase/calcineurin pathways are essential for the flow response. Amiloride or benzamil did not affect the ET‐1 response to flow. Increasing perfusate osmolality to 450 mOsm with NaCl, mannitol or urea elicited a marked flow response (4.4‐fold increase in ET‐1 mRNA). Removal of cilia with chloral hydrate reduced the flow response, while flow failed to stimulate ET‐1 mRNA in mIMCD3 cells deficient in polycystin‐2. These data suggest that IMCD ET‐1 synthesis is stimulated by tubule fluid flow via increased solute delivery and possibly cilia deformation which in turn activates PKC‐ and Ca‐dependent pathways. We propose that Na delivery stimulates CCD ET‐1 which, in turn, inhibits CCD Na reabsorption, while solute and water delivery stimulate IMCD ET‐1 which, in turn, inhibits IMCD water and urea reabsorption.

An Af9 cis-element directly targets Dot1a to mediate transcriptional repression of the αENaC gene.

The epithelial Na(+) channel subunit-α (αENaC) of the distal nephron is essential for salt balance. We previously demonstrated that the histone methyltransferase Dot1a and its protein partner Af9 basally repress αENaC transcription in mouse inner medullary collecting duct type 3 (mIMCD3) cells and link aldosterone-elicited chromatin modifications to αENaC transcriptional activation. Af9 DNA-binding activity has never been demonstrated, and whether and where Af9 binds to the αENaC promoter to target Dot1a are unknown. The present study sought to identify functional Af9 cis-element(s) in the -57/+439 "R3" subregion of αENaC, the principal site for Dot1a-Af9 interaction, in mIMCD3 cells. We also exploited connecting tubule/collecting duct-specific Dot1l-deficient mice (Dot1l(AC)) to determine the impact of Dot1l inactivation on renal αENaC expression in vivo. mIMCD3 cell lines expressing αENaC promoter-reporter constructs harboring deletion of +74/+107 demonstrated greatly reduced association of Af9 and Dot1a by ChIP/qPCR. Aldosterone treatment resulted in further decrements in Af9 and Dot1a association with the αENaC promoter. Gel shift and antibody competition assays using wild-type and mutant oligomers revealed Af9-containing +78/+92 αENaC DNA-protein complexes in nuclear extracts of mIMCD3 cells. Mutation of the +78/+92 element resulted in higher basal αENaC promoter activity and impaired Dot1a-mediated inhibition in trans-repression assays. In agreement, mice with connecting tubule/collecting duct-specific knockout of Dot1l exhibited greater αENaC mRNA levels in kidney compared with control. Thus, we conclude that +78/+92 of αENaC represents the primary Af9 binding site involved in recruiting Dot1a to repress basal and aldosterone-sensitive αENaC transcription and that Dot1l inactivation promotes αENaC mRNA expression by eliminating Dot1a-mediated repression.

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To determine if endothelin-1 (ET-1) stimulates the phosphorylation of ERK1/2 in the mouse inner medullary collecting duct (IMCD), and if this in turn upregulates nitric oxide (NO) production.Confluent mouse IMCD segment-3 cells (mIMCD-3) were stimulated with 50 nM ET-1 for 24 h with and without various doses of ET receptor antagonists, BQ123 (ETA antagonist,) or BQ788 (ETB antagonist) and phosphorylation of ERK1/2 determined by immunoblots. As well, NOS isoform expression and nitrite production were assessed. Finally, increasing doses of the MEK inhibitors, PD98,059 or U0126, were incubated with mIMCD-3 cells and the ET-1 dependent nitrite production determined.ET-1 via the ETB receptor significantly increased ERK1/2 phosphorylation, and was prevented by MEK inhibition. ET-1 also stimulates nitrite production by mIMCD-3 cells (basal: 54.5 ± 26 pmol/mg pr/h vs ET-1: 221 ± 28 pmol/mg pr/h; N = 4) via the ETB receptor (BQ788 + ET-1: 83.7 ± 27 pmol/mg pr/h); however, ET-1 does not regulate NOS1 or NOS3 expression. MEK inhibition did not prevent the ET-1 stimulated nitrite production contrary to our initial hypothesis (vehicle + ET-1: 157 ± 13 pmol/mg pr/hr vs PD98,059 + ET-1: 305.7 ± 24 pmol/mg pr/h, N = 4, P > 0.05).Although the mouse IMCD-3 cells only express the NOS1β splice variant, ET-1 did regulate mouse IMCD nitrite production. ET-1 stimulates ERK1/2 phosphorylation in the mouse IMCD, but ERK1/2 signaling is not involved in the ET-1 dependent increase in NO production by IMCD cells. Thus, we propose that ET-1 regulates protein–protein interactions that are necessary for NO production, that are independent of MAPK signaling cascades.

High throughput high content reverse genetics visual screens of ciliogenesis and cilia maintenance

Cilia are small, hair-like structures occurring on the apical surface of most of vertebrate cells. Defects in cilia cause a range of developmental phenotypes grouped into conditions called ciliopathies. Our aim is to dissect the structure and function of cilia and signalling pathways mediated by this organelle. To evaluate this, we are performing a high-throughput siRNA screen using siRNA pools (from the Dharmacon mouse genome siRNA library) targeting over 19,000 separate transcripts and immunofluorescence staining of ciliated mIMCD3 (transformed mouse inner medullary collecting duct) cells to determine cilia number, length and morphology. Secondary datasets from the screen will include measurements of cell size and morphology, nuclear morphology and cell cycle profiles. We have successfully set up a facility for high-throughput high-content imaging, optimized a reverse transfection protocol and validated a series of positive and negative controls. We are currently completing the analysis of candidate hits and expect to obtain several hundred positive hits from the whole screen. We will present the first dataset from this screen with a discussion of prioritization strategies for the validation of the most relevant and interesting candidate hits.