BASIC RESEARCH www.jasn.org Mechanism of Fibrosis in HNF1B-Related Autosomal Dominant Tubulointerstitial Kidney Disease Siu Chiu Chan,1 Ying Zhang,2 Annie Shao,1 Svetlana Avdulov,1 Jeremy Herrera,1 Karam Aboudehen,1 Marco Pontoglio,3 and Peter Igarashi 1 1Department of Medicine and 2Minnesota Supercomputing Institute, University of Minnesota, Minneapolis, Minnesota; and 3Department of Development, Reproduction and Cancer, Institut Cochin, Institut National de la Santé et de la Recherche Médicale U1016/Centre National de la Recherche Scientifique Unité Mixte de Recherche 8104, Université Paris-Descartes, Paris, France ABSTRACT Background Mutation of HNF1B, the gene encoding transcription factor HNF-1b, is one cause of auto- somal dominant tubulointerstitial kidney disease, a syndrome characterized by tubular cysts, renal fibrosis, and progressive decline in renal function. HNF-1b has also been implicated in epithelial–mesenchymal transition (EMT) pathways, and sustained EMT is associated with tissue fibrosis. The mechanism whereby mutated HNF1B leads to tubulointerstitial fibrosis is not known. Methods To explore the mechanism of fibrosis, we created HNF-1b–deficient mIMCD3 renal epithelial cells, used RNA-sequencing analysis to reveal differentially expressed genes in wild-type and HNF-1b–deficient mIMCD3 cells, and performed cell lineage analysis in HNF-1b mutant mice. Results The HNF-1b–deficient cells exhibited properties characteristic of mesenchymal cells such as fi- broblasts, including spindle-shaped morphology, loss of contact inhibition, and increased cell migration. These cells also showed upregulation of fibrosis and EMT pathways, including upregulation of Twist2, Snail1, Snail2, and Zeb2, which are key EMT transcription factors. Mechanistically, HNF-1b directly re- presses Twist2, and ablation of Twist2 partially rescued the fibroblastic phenotype of HNF-1b mutant cells. Kidneys from HNF-1b mutant mice showed increased expression of Twist2 and its downstream target Snai2. Cell lineage analysis indicated that HNF-1b mutant epithelial cells do not transdifferentiate into kidney myofibroblasts. Rather, HNF-1b mutant epithelial cells secrete high levels of TGF-b ligands that activate downstream Smad transcription factors in renal interstitial cells. Conclusions Ablation of HNF-1b in renal epithelial cells leads to the activation of a Twist2-dependent transcriptional network that induces EMT and aberrant TGF-b signaling, resulting in renal fibrosis through a cell-nonautonomous mechanism. J Am Soc Nephrol 29: 2493–2509, 2018. doi: https://doi.org/10.1681/ASN.2018040437 Autosomal dominant tubulointerstitial kidney that cause interstitial nephritis. Gout, hyperurice- disease (ADTKD) is a recently recognized clinico- mia, and hypomagnesemia may be present. Renal pathological entity characterized by autosomal pathologic features comprise interstitial fibrosis, dominant inheritance and progressive decline in renal function.1 Clinical and laboratory features in- Received April 26, 2018. Accepted July 12, 2018. clude normal or small kidney size, elevated serum Published online ahead of print. Publication date available at creatinine, bland urinary sediment, absent or low- www.jasn.org. grade proteinuria, and impaired urinary concentra- Correspondence: Dr. Peter Igarashi, Department of Medicine, tion.2 Systemic BP is generally not elevated during University of Minnesota Medical School, 420 Delaware Street SE, early stages of the disease, and there is no prior MMC 194, Minneapolis, MN 55455. Email: [email protected] history of exposure to drugs, metals, or toxins Copyright © 2018 by the American Society of Nephrology J Am Soc Nephrol 29: 2493–2509, 2018 ISSN : 1046-6673/2910-2493 2493 BASIC RESEARCH www.jasn.org tubular atrophy, thickening and lamellation of tubular base- Significance Statement ment membranes, kidney microcysts or macrocysts, and absence of immune complex deposits. ADTKD encompasses Mutation of HNF1B, which encodes the transcription factor HNF-1b,isa disorders previously known as medullary cystic kidney disease cause of autosomal dominant tubulointerstitial kidney disease (ADTKD), b fi and familial juvenile hyperuricemic nephropathy. ADTKD is but the mechanism whereby mutated HNF-1 leads to renal brosis is not known. The authors demonstrated that ablation of HNF-1b is suf- genetically heterogeneous and can arise from mutations in at ficient to induce epithelial–mesenchymal transition (EMT) in vitro and in – least five genes3 7: UMOD, which encodes uromodulin/ vivo through derepression of the HNF-1b–regulated gene Twist2 and Tamm–Horsfall protein, the major urinary protein produced its downstream target Snai2. Lineage tracing revealed that ablation of in the loop of Henle; MUC1, which encodes the apical mem- HNF-1b induces renal fibrosis via a cell-nonautonomous process in- brane protein mucin-1; REN, which encodes the enzyme renin volving epithelial/mesenchymal crosstalk. Further, they showed that epithelial cells with mutant HNF-1b secrete high levels of profibrotic that catalyzes the conversion of angiotensinogen to angioten- TGF-b ligands that activate downstream Smad transcription factors sin-1; SEC61A1, which is involved in protein translocation in in renal interstitial cells. Collectively, these findings reveal a novel the endoplasmic reticulum; and HNF1B, which encodes the Twist2-dependent EMT pathway that underlies renal fibrosis in HNF1B- transcription factor hepatocyte NF-1b (HNF-1b). Causative related ADTKD. genes have not yet been identified in a significant portion of affected individuals. and the mechanisms whereby human mutations lead to a b HNF-1 is a homeodomain-containing transcription fac- broad spectrum of clinical phenotypes remain to be fully fi tor that regulates tissue-speci c gene expression in the kidney, elucidated. Constitutive ablation of Hnf1b in mice results in 8 b liver, pancreas, and other epithelial organs. HNF-1 is essen- embryonic lethality due to failure of endoderm development,23 b tial for normal kidney development; ablation of HNF-1 in and kidney-specificdeletionofHnf1b using the Ksp/Cre deleter nephron progenitors leads to disturbances in Notch-depen- strain results in postnatal kidney failure.14,18 We recently used dent nephron patterning, and ablation in the ureteric bud Pkhd1/Cre24 mice to ablate Hnf1b specifically in renal collecting inhibits branching morphogenesis and Wnt9b-dependent ducts.21 Collecting duct-specific deletion of Hnf1b results in 9,10 nephron induction and GDNF/Ret signaling. In humans, longer survival and slower progression of cystic disease, renal mutations of HNF1B have been linked to congenital abnor- fibrosis, and hydronephrosis. Mutant mice also exhibit polyuria, malities of the kidney and urinary tract, including renal agen- polydipsia, and impaired urinary concentration recapitulating esis/hypoplasia, multicystic dysplastic kidneys, horseshoe clinical features of ADTKD in humans with mutations in 11 kidneys, and glomerulocystic kidney disease. Mutations HNF1B. of HNF1B can also produce ADTKD, often associated with To investigate the pathogenesis of renal interstitial fibrosis in hyperuricemia, hypomagnesemia, hypokalemia, diabetes ADTKD, three ADTKD-UMOD mouse models had been 12,13 mellitus, and Müllerian duct abnormalities. Previous generated.25–27 Characterization of the mutant mice suggests b studies suggested that HNF-1 regulates the transcription of that renal fibrosis arises from endoplasmic reticulum (ER) UMOD, raising the possibility that disturbances in a common stress and secondary mitochondrial dysfunction. However, transcriptional network may underlie the pathogenesis of the mechanism whereby mutations of HNF1B produce 14 ADTKD. Abnormalities in unfolded protein response tubulointerstitial fibrosis has not been explored. Using HNF-1b (UPR) and mitochondrial metabolism have also been mutant cell lines and mouse models, we found that loss of 15 implicated. In addition to inherited kidney diseases, muta- HNF-1b induces epithelial–mesenchymal transition (EMT) via tions of HNF1B have been detected in sporadic cases of derepression of the transcription factor Twist2. As a consequence, 16 renal hypoplasia/dysplasia. Expression of HNF1B is also the expression of TGF-b ligands is upregulated in renal tubules, 17 downregulated in polycystic kidney disease. which leads to renal fibrosis via a cell-nonautonomous process. Several genetically modified mouse models have been de- veloped to unravel the pathogenesis of human HNF1B-related kidney disease.14,18 Common features of the HNF-1b mutant METHODS mouse models are enlarged kidneys with fluid-filled cysts, multilayered cyst epithelium, and hydronephrosis. Molecular Transgenic Mice characterization of HNF-1b mutant mice has shown that Ksp/Cre mice that express Cre recombinase under the control HNF-1b plays a significant role in cystic kidney diseases of the Ksp-cadherin (Cdh16)promoterandHnf1bf/f mice through the regulation of cystogenes such as Pkhd1, Pkd2, containing loxP sites flanking exon 1 of Hnf1b have been de- Umod,andKif12.14,18,19 Recent chromatin immunoprecipita- scribed previously.14 R26R-EYFP mice that express EYFP after tion sequencing (ChIP-seq) experiments have shown that Cre/loxP recombination were provided by Dr. Frank Costantini HNF-1b regulates cholesterol metabolism through transcrip- (Columbia University).28 Ksp/Cre mice were crossed with tional activation of Srebp2 and Pcsk9,20 urinary concentration Hnf1bf/+ mice, and the bitransgenic progeny were intercrossed through activation of Fxr,21 and potassium