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Post-translational loss of renal TRPV5 calcium channel expression, Ca(2+) wasting, and bone loss in experimental colitis.

ABSTRACT: 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-?, and interleukin-1? 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-? reduced the expression and activity of Klotho, which otherwise would protect TRPV5 from hypersialylation and cytokine-induced TRPV5 endocytosis, UBR4-dependent ubiquitination, degradation, and urinary wasting of Ca(2+).

SUBMITTER: Radhakrishnan VM

PROVIDER: S-EPMC3755094 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Post-translational loss of renal TRPV5 calcium channel expression, Ca(2+) wasting, and bone loss in experimental colitis.

Radhakrishnan Vijayababu M VM Ramalingam Rajalakshmy R Larmonier Claire B CB Thurston Robert D RD Laubitz Daniel D Midura-Kiela Monica T MT McFadden Rita-Marie T RM Kuro-O Makoto M Kiela Pawel R PR Ghishan Fayez K FK

Gastroenterology 20130605 3

<h4>Background & aims</h4>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.<h4>Methods</h4>Colitis was induced in mice via administration of 2,4,6-trinitrobenzenesulf ...[more]

PMID: 23747339

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Project description:BACKGROUND AND PURPOSE:Epithelial surface hydration is critical for proper gut function. However, colonic tissues from individuals with inflammatory bowel disease or animals with colitis are hyporesponsive to Cl(-) secretagogues. The Cl(-) secretory responses to the muscarinic receptor agonist bethanechol are virtually absent in colons of mice with dextran sodium sulphate (DSS)-induced colitis. Our aim was to define the mechanism underlying this cholinergic hyporesponsiveness. EXPERIMENTAL APPROACH:Colitis was induced by 4% DSS water, given orally. Epithelial ion transport was measured in Ussing chambers. Colonic crypts were isolated and processed for mRNA expression via RT-PCR and protein expression via immunoblotting and immunolocalization. KEY RESULTS:Expression of muscarinic M(3) receptors in colonic epithelium was not decreased during colitis. Short-circuit current (I(SC)) responses to other Ca(2+)-dependent secretagogues (histamine, thapsigargin, cyclopiazonic acid and calcium ionophore) were either absent or severely attenuated in colonic tissue from DSS-treated mice. mRNA levels of several ion transport molecules (a Ca(2+)-regulated Cl(-) channel, the intermediate-conductance Ca(2+)-activated K(+) channel, the cystic fibrosis transmembrane conductance regulator, the Na(+)/K(+)-ATPase pump or the Na(+)/K(+)/2Cl(-) co-transporter) were not reduced in colonic crypts from DSS-treated mice. However, protein expression of Na(+)/K(+)-ATPase alpha1 subunits was decreased twofold during colitis. Activation of Ca(2+)-activated K(+) channels increased I(SC) significantly less in DSS colons compared with control, as did the protein kinase C activator, phorbol 12-myristate 13-acetate. CONCLUSIONS AND IMPLICATIONS:Decreased Na(+)/K(+)-ATPase expression probably contributes to overall epithelial hyporesponsiveness during colitis, while dysfunctional K(+) channels may account, at least partially, for lack of epithelial secretory responses to Ca(2+)-mediated secretagogues.

Project description:Hypercalciuria is a major cause of nephrolithiasis, and is a common and complex disorder involving genetic and environmental factors. Identification of genetic factors for monogenic forms of hypercalciuria is hampered by the limited availability of large families, and to facilitate such studies, we screened for hypercalciuria in mice from an N-ethyl-N-nitrosourea mutagenesis programme. We identified a mouse with autosomal dominant hypercalciuria (HCALC1). Linkage studies mapped the Hcalc1 locus to a 11.94 Mb region on chromosome 6 containing the transient receptor potential cation channel, subfamily V, members 5 (Trpv5) and 6 (Trpv6) genes. DNA sequence analysis of coding regions, intron-exon boundaries and promoters of Trpv5 and Trpv6 identified a novel T to C transition in codon 682 of TRPV5, mutating a conserved serine to a proline (S682P). Compared to wild-type littermates, heterozygous (Trpv5(682P/+)) and homozygous (Trpv5(682P/682P)) mutant mice had hypercalciuria, polyuria, hyperphosphaturia and a more acidic urine, and ∼10% of males developed tubulointerstitial nephritis. Trpv5(682P/682P) mice also had normal plasma parathyroid hormone but increased 1,25-dihydroxyvitamin D(3) concentrations without increased bone resorption, consistent with a renal defect for the hypercalciuria. Expression of the S682P mutation in human embryonic kidney cells revealed that TRPV5-S682P-expressing cells had a lower baseline intracellular calcium concentration than wild-type TRPV5-expressing cells, suggesting an altered calcium permeability. Immunohistological studies revealed a selective decrease in TRPV5-expression from the renal distal convoluted tubules of Trpv5(682P/+) and Trpv5(682P/682P) mice consistent with a trafficking defect. In addition, Trpv5(682P/682P) mice had a reduction in renal expression of the intracellular calcium-binding protein, calbindin-D(28K), consistent with a specific defect in TRPV5-mediated renal calcium reabsorption. Thus, our findings indicate that the TRPV5 S682P mutant is functionally significant and study of HCALC1, a novel model for autosomal dominant hypercalciuria, may help further our understanding of renal calcium reabsorption and hypercalciuria.

Dataset Information

Post-translational loss of renal TRPV5 calcium channel expression, Ca(2+) wasting, and bone loss in experimental colitis.

Publications

Post-translational loss of renal TRPV5 calcium channel expression, Ca(2+) wasting, and bone loss in experimental colitis.

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