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A nonclassical vitamin D receptor pathway suppresses renal fibrosis.

ABSTRACT: The TGF-? superfamily comprises pleiotropic cytokines that regulate SMAD and non-SMAD signaling. TGF-?-SMAD signal transduction is known to be involved in tissue fibrosis, including renal fibrosis. Here, we found that 1,25-dihydroxyvitamin D3-bound [1,25(OH)2D3-bound] vitamin D receptor (VDR) specifically inhibits TGF-?-SMAD signal transduction through direct interaction with SMAD3. In mouse models of tissue fibrosis, 1,25(OH)2D3 treatment prevented renal fibrosis through the suppression of TGF-?-SMAD signal transduction. Based on the structure of the VDR-ligand complex, we generated 2 synthetic ligands. These ligands selectively inhibited TGF-?-SMAD signal transduction without activating VDR-mediated transcription and significantly attenuated renal fibrosis in mice. These results indicate that 1,25(OH)2D3-dependent suppression of TGF-?-SMAD signal transduction is independent of VDR-mediated transcriptional activity. In addition, these ligands did not cause hypercalcemia resulting from stimulation of the transcriptional activity of the VDR. Thus, our study provides a new strategy for generating chemical compounds that specifically inhibit TGF-?-SMAD signal transduction. Since TGF-?-SMAD signal transduction is reportedly involved in several disorders, our results will aid in the development of new drugs that do not cause detectable adverse effects, such as hypercalcemia.

SUBMITTER: Ito I

PROVIDER: S-EPMC3809783 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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A nonclassical vitamin D receptor pathway suppresses renal fibrosis.

Ito Ichiaki I Waku Tsuyoshi T Aoki Masato M Abe Rumi R Nagai Yu Y Watanabe Tatsuya T Nakajima Yuka Y Ohkido Ichiro I Yokoyama Keitaro K Miyachi Hiroyuki H Shimizu Toshiyuki T Murayama Akiko A Kishimoto Hiroyuki H Nagasawa Kazuo K Yanagisawa Junn J

The Journal of clinical investigation 20131025 11

The TGF-β superfamily comprises pleiotropic cytokines that regulate SMAD and non-SMAD signaling. TGF-β-SMAD signal transduction is known to be involved in tissue fibrosis, including renal fibrosis. Here, we found that 1,25-dihydroxyvitamin D3-bound [1,25(OH)2D3-bound] vitamin D receptor (VDR) specifically inhibits TGF-β-SMAD signal transduction through direct interaction with SMAD3. In mouse models of tissue fibrosis, 1,25(OH)2D3 treatment prevented renal fibrosis through the suppression of TGF- ...[more]

PMID: 24135137

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Project description:The nicotinic acetylcholine receptor alpha1 (nAChRalpha1) was investigated as a potential fibrogenic molecule in the kidney, given reports that it may be an alternative urokinase (urokinase plasminogen activator; uPA) receptor in addition to the classical receptor uPAR. In a mouse obstructive uropathy model of chronic kidney disease, interstitial fibroblasts were identified as the primary cell type that bears nAChRalpha1 during fibrogenesis. Silencing of the nAChRalpha1 gene led to significantly fewer interstitial alphaSMA(+) myofibroblasts (2.8 times decreased), reduced interstitial cell proliferation (2.6 times decreased), better tubular cell preservation (E-cadherin 14 times increased), and reduced fibrosis severity (24% decrease in total collagen). The myofibroblast-inhibiting effect of nAChRalpha1 silencing in uPA-sufficient mice disappeared in uPA-null mice, suggesting that a uPA-dependent fibroblastic nAChRalpha1 pathway promotes renal fibrosis. To further establish this possible ligand-receptor relationship and to identify downstream signaling pathways, in vitro studies were performed using primary cultures of renal fibroblasts. (35)S-Labeled uPA bound to nAChRalpha1 with a K(d) of 1.6 x 10(-8) m, which was displaced by the specific nAChRalpha1 inhibitor d-tubocurarine in a dose-dependent manner. Pre-exposure of uPA to the fibroblasts inhibited [(3)H]nicotine binding. The uPA binding induced a cellular calcium influx and an inward membrane current that was entirely prevented by d-tubocurarine preincubation or nAChRalpha1 silencing. By mass spectrometry phosphoproteome analyses, uPA stimulation phosphorylated nAChRalpha1 and a complex of signaling proteins, including calcium-binding proteins, cytoskeletal proteins, and a nucleoprotein. This signaling pathway appears to regulate the expression of a group of genes that transform renal fibroblasts into more active myofibroblasts characterized by enhanced proliferation and contractility. This new fibrosis-promoting pathway may also be relevant to disorders that extend beyond chronic kidney disease.

Project description:Renal fibrosis denotes a common complication of diabetic nephropathy and is a predominant cause of end-stage renal disease. Despite the association between microRNAs (miRNAs or miRs) and renal fibrosis, miRNAs have been reported to play a vital role in the development of chronic renal fibrosis. Therefore, the aim of the present study was to investigate the possible function of miR-101a in chronic renal fibrosis. Initially, microarray-based gene expression profiling of renal fibrosis was employed to screen the differentially expressed genes. An in vivo mouse model of chronic renal fibrosis induced by a unilateral ureteral obstruction (UUO) and an in vitro cell model induced by aristolochic acid (AA) were constructed. miR-101a expression was examined using a fluorescence in situ hybridization (FISH) assay and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Then, the interaction between miR-101a and KDM3A was identified using an online website combined with a dual-luciferase reporter assay. Finally, gain- and loss-of-function experiments were conducted to elucidate the effect of miR-101a on the expression of Col1a1, fibronectin, ?-smooth muscle actin (?-SMA), and YAP-TGF-? (transforming growth factor ?)-Smad signaling pathway-related genes, as well as the degree of renal fibrosis. miR-101a was poorly expressed while KDM3A was robustly induced in chronic renal fibrosis tissues and cells. In addition, miR-101a could target and downregulate KDM3A expression, which led to elevated TGIF1, inhibited expression of Collagen I (Col1a1), fibronectin, ?-SMA, YAP1, and TGF-?2 along with the extent of Smad2/3 phosphorylation, as well as delayed renal fibrosis degree. Besides, overexpressed YAP/TGF-?2 or inhibited TGIF1 partially restored the inhibitory effect of miR-101a on chronic renal fibrosis. Taken together, miR-101a could potentially slow down chronic renal fibrosis by the inactivation of the YAP-TGF-?-Smad signaling pathway via KDM3A, highlighting the potential of miR-101a as a therapeutic target for chronic renal fibrosis treatment.

Project description:Diabetic nephropathy (DN) has become a major cause of end-stage renal disease, and autophagy disorder is implicated in the pathogenesis of DN. Our previous studies found that vitamin D (VD) and VDR (vitamin D receptor) played a renoprotective role by inhibiting inflammation and fibrosis. However, whether VD-VDR regulates autophagy disorders in DN remains unclear. In this study, we established a streptozotocin (STZ)-induced diabetic model in vdr knockout (vdr-KO) mice and VDR specifically overexpressed in renal proximal tubular epithelial cells (Vdr-OE) mice. Our results showed that paricalcitol (an activated vitamin D analog) or Vdr-OE could alleviate STZ-induced ALB (albumin) excretion, renal tubule injury and inflammation, while these were worsened in vdr-KO mice. Defective autophagy was observed in the kidneys of STZ mice, which was more pronounced in vdr-KO mice and could be partially restored by paricalcitol or Vdr-OE. In high glucose-induced HK-2 cells, defective autophagy and decreased PRKAA1/AMPK phosphorylation was observed, which could be partially restored by paricalcitol in a VDR-dependent manner. AMPK inhibitor abolished paricalcitol-induced autophagy activation, and AMPK activator restored the defective autophagy in high glucose-induced HK-2 cells. Furthermore, paricalcitol-mediated AMPK activation was abrogated by CAMKK2/CaMKKβ inhibition, but not by STK11/LKB1 knockout. Meanwhile, paricalcitol rescued the decreased Ca2+ concentration induced by high glucose. In conclusion, VD-VDR can restore defective autophagy in the kidney of STZ-induced diabetic mice, which could be attributed to the activation of the Ca2+-CAMKK2-AMPK pathway in renal tubular epithelial cells.Abbreviations: ACTB/β-actin: actin beta;AGE: advanced glycation end-products;AMPK: AMP-activated protein kinase;CAMKK2/CaMKKβ: calcium-calmodulin dependent protein kinase kinase 2;CQ: chloroquine;DN: diabetic nephropathy;HG: high levels of glucose;KO: knockout;LG: low levels of glucose;MAP1LC3/LC3: microtubule associated protein 1 light chain 3;NOD2: nucleotide binding oligomerization domain containing 2;OE: overexpression;PAS: periodic acid Schiff; Pari: paricalcitol;PTECs: proximal renal tubule epithelial cells;RT: room temperature;SQSTM1/p62: sequestosome 1;STK11/LKB1: serine/threonine kinase 11;STZ: streptozotocin;TEM: transmission electron microscopy;VD: vitamin D;VDR: vitamin D receptor;WT: wild-type.

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A nonclassical vitamin D receptor pathway suppresses renal fibrosis.

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A nonclassical vitamin D receptor pathway suppresses renal fibrosis.

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