Transcription factor HNF1ß controls a transcriptional network regulating kidney cell structure and tight junction integrity.

Mutations in the hepatocyte nuclear factor (HNF)1ß gene (HNF1B) cause autosomal dominant tubulointerstitial kidney disease, a rare and heterogeneous disease characterized by renal cysts and/or malformation, maturity-onset diabetes of the young, hypomagnesemia, and hypokalemia. The electrolyte disturbances may develop in the distal part of the nephron, which is important for fine-tuning of Mg2+ and Ca2+ reabsorption. Therefore, we aimed to study the transcriptional network directed by HNF1ß in the distal part of the nephron. We combined HNF1ß chromatin immunoprecipitation-sequencing and mRNA expression data to identify direct targets of HNF1ß in a renal distal convoluted tubule cell line (mpkDCT). Gene Ontology term pathway analysis demonstrated enrichment of cell polarity, cell-cell junction, and cytoskeleton pathways in the dataset. Genes directly and indirectly regulated by HNF1ß within these pathways included members of the apical and basolateral polarity complexes including Crumbs protein homolog 3 (Crb3), partitioning defective 6 homolog-ß (Pard6b), and LLGL Scribble cell polarity complex component 2 (Llgl2). In monolayers of mouse inner medullary collecting duct 3 cells expressing dominant negative Hnf1b, tight junction integrity was compromised, as observed by reduced transepithelial electrical resistance values and increased permeability for fluorescein (0.4 kDa) compared with wild-type cells. Expression of dominant negative Hnf1b also led to a decrease in height (30%) and an increase in surface (58.5%) of cells grown on membranes. Moreover, three-dimensional spheroids formed by cells expressing dominant negative Hnf1b were reduced in size compared with wild-type spheroids (30%). Together, these findings demonstrate that HNF1ß directs a transcriptional network regulating tight junction integrity and cell structure in the distal part of the nephron.NEW & NOTEWORTHY Genetic defects in transcription factor hepatocyte nuclear factor (HNF)1ß cause a heterogeneous disease characterized by electrolyte disturbances, kidney cysts, and diabetes. By combining RNA-sequencing and HNF1ß chromatin immunoprecipitation-sequencing data, we identified new HNF1ß targets that were enriched for cell polarity pathways. Newly discovered targets included members of polarity complexes Crb3, Pard6b, and Llgl2. Functional assays in kidney epithelial cells demonstrated decreased tight junction integrity and a loss of typical cuboidal morphology in mutant Hnf1b cells.

HNF1ß-associated cyst development and electrolyte disturbances are not explained by BAIAP2L2 expression.

Mutations or deletions in transcription factor hepatocyte nuclear factor 1 homeobox ß (HNF1ß) cause renal cysts and/or malformation, maturity-onset diabetes of the young and electrolyte disturbances. Here, we applied a comprehensive bioinformatic approach on ChIP-seq, RNA-seq, and gene expression array studies to identify novel transcriptional targets of HNF1ß explaining the kidney phenotype of HNF1ß patients. We identified BAR/IMD Domain Containing Adaptor Protein 2 Like 2 (BAIAP2L2), as a novel transcriptional target of HNF1ß and validated direct transcriptional activation of the BAIAP2L2 promoter by a reporter luciferase assay. Using mass spectrometry analysis, we show that BAIAP2L2 binds to other members of the I-BAR domain-containing family: BAIAP2 and BAIAP2L1. Subsequently, the role of BAIAP2L2 in maintaining epithelial cell integrity in the kidney was assessed using Baiap2l2 knockout cell and mouse models. Kidney epithelial cells lacking functional BAIAP2L2 displayed normal F-actin distribution at cell-cell contacts and formed polarized three-dimensional spheroids with a lumen. In vivo, Baiap2l2 knockout mice displayed normal kidney and colon tissue morphology and serum and urine electrolyte concentrations were not affected. Altogether, our study is the first to characterize the function of BAIAP2L2 in the kidney in vivo and we report that mice lacking BAIAP2L2 exhibit normal electrolyte homeostasis and tissue morphology under physiological conditions.

Framework From a Multidisciplinary Approach for Transitioning Variants of Unknown Significance From Clinical Genetic Testing in Kidney Disease to a Definitive Classification.

Introduction: Monogenic causes in over 300 kidney-associated genes account for approximately 12% of end stage kidney disease (ESKD) cases. Advances in sequencing and large customized panels enable the noninvasive diagnosis of monogenic kidney disease at relatively low cost, thereby allowing for more precise management for patients and their families. A major challenge is interpreting rare variants, many of which are classified as variants of unknown significance (VUS). We present a framework in which we thoroughly evaluated and provided evidence of pathogenicity for HNF1B-p.Arg303His, a VUS returned from clinical diagnostic testing for a kidney transplant candidate. Methods: A blueprint was designed by a multidisciplinary team of clinicians, molecular biologists, and diagnostic geneticists. The blueprint included using a health system-based cohort with genetic and clinical information to perform deep phenotyping of VUS heterozygotes to identify the candidate VUS and rule out other VUS, examination of existing genetic databases, as well as functional testing. Results: Our approach demonstrated evidence for pathogenicity for HNF1B-p.Arg303His by showing similar burden of kidney manifestations in this variant to known HNF1B pathogenic variants, and greater burden compared to noncarriers. Conclusion: Determination of a molecular diagnosis for the example family allows for proper surveillance and management of HNF1B-related manifestations such as kidney disease, diabetes, and hypomagnesemia with important implications for safe living-related kidney donation. The candidate gene-variant pair also allows for clinical biomarker testing for aberrations of linked pathways. This working model may be applicable to other diseases of genetic etiology.

Mechanisms of ion transport regulation by HNF1ß in the kidney: beyond transcriptional regulation of channels and transporters.

Hepatocyte nuclear factor 1ß (HNF1ß) is a transcription factor essential for the development and function of the kidney. Mutations in and deletions of HNF1ß cause autosomal dominant tubule interstitial kidney disease (ADTKD) subtype HNF1ß, which is characterized by renal cysts, diabetes, genital tract malformations, and neurodevelopmental disorders. Electrolyte disturbances including hypomagnesemia, hyperuricemia, and hypocalciuria are common in patients with ADTKD-HNF1ß. Traditionally, these electrolyte disturbances have been attributed to HNF1ß-mediated transcriptional regulation of gene networks involved in ion transport in the distal part of the nephron including FXYD2, CASR, KCNJ16, and FXR. In this review, we propose additional mechanisms that may contribute to the electrolyte disturbances observed in ADTKD-HNF1ß patients. Firstly, kidney development is severely affected in Hnf1b-deficient mice. HNF1ß is required for nephron segmentation, and the absence of the transcription factor results in rudimentary nephrons lacking mature proximal tubule, loop of Henle, and distal convoluted tubule cluster. In addition, HNF1ß is proposed to be important for apical-basolateral polarity and tight junction integrity in the kidney. Interestingly, cilia formation is unaffected by Hnf1b defects in several models, despite the HNF1ß-mediated transcriptional regulation of many ciliary genes. To what extent impaired nephron segmentation, apical-basolateral polarity, and cilia function contribute to electrolyte disturbances in HNF1ß patients remains elusive. Systematic phenotyping of Hnf1b mouse models and the development of patient-specific kidney organoid models will be essential to advance future HNF1ß research.

Bifunctional protein PCBD2 operates as a co-factor for hepatocyte nuclear factor 1ß and modulates gene transcription.

Hepatocyte nuclear factor 1ß (HNF1ß) is an essential transcription factor in development of the kidney, liver, and pancreas. HNF1ß-mediated transcription of target genes is dependent on the cell type and the development stage. Nevertheless, the regulation of HNF1ß function by enhancers and co-factors that allow this cell-specific transcription is largely unknown. To map the HNF1ß interactome we performed mass spectrometry in a mouse kidney inner medullary collecting duct cell line. Pterin-4a-carbinolamine dehydratase 2 (PCBD2) was identified as a novel interaction partner of HNF1ß. PCBD2 and its close homolog PCBD1 shuttle between the cytoplasm and nucleus to exert their enzymatic and transcriptional activities. Although both PCBD proteins share high sequence identity (48% and 88% in HNF1 recognition helix), their tissue expression patterns are unique. PCBD1 is most abundant in kidney and liver while PCBD2 is also abundant in lung, spleen, and adipose tissue. Using immunolocalization studies and biochemical analysis we show that in presence of HNF1ß the nuclear localization of PCBD1 and PCBD2 increases significantly. Promoter luciferase assays demonstrate that co-factors PCBD1 and PCBD2 differentially regulate the ability of HNF1ß to activate the promoters of transcriptional targets important in renal electrolyte homeostasis. Deleting the N-terminal sequence of PCBD2, not found in PCBD1, diminished the differential effects of the co-factors on HNF1ß activity. All together these results indicate that PCBD1 and PCBD2 can exert different effects on HNF1ß-mediated transcription. Future studies should confirm whether these unique co-factor activities also apply to HNF1ß-target genes involved in additional processes besides ion transport in the kidney.

Deletion of Cytoplasmic Double-Stranded RNA Sensors Does Not Uncover Viral Small Interfering RNA Production in Human Cells.

Antiviral immunity in insects and plants is mediated by the RNA interference (RNAi) pathway in which viral long double-stranded RNA (dsRNA) is processed into small interfering RNAs (siRNAs) by Dicer enzymes. Although this pathway is evolutionarily conserved, its involvement in antiviral defense in mammals is the subject of debate. In vertebrates, recognition of viral RNA induces a sophisticated type I interferon (IFN)-based immune response, and it has been proposed that this response masks or inhibits antiviral RNAi. To test this hypothesis, we analyzed viral small RNA production in differentiated cells deficient in the cytoplasmic RNA sensors RIG-I and MDA5. We did not detect 22-nucleotide (nt) viral siRNAs upon infection with three different positive-sense RNA viruses. Our data suggest that the depletion of cytoplasmic RIG-I-like sensors is not sufficient to uncover viral siRNAs in differentiated cells. IMPORTANCE The contribution of the RNA interference (RNAi) pathway in antiviral immunity in vertebrates has been widely debated. It has been proposed that RNAi possesses antiviral activity in mammalian systems but that its antiviral effect is masked by the potent antiviral interferon response in differentiated mammalian cells. In this study, we show that inactivation of the interferon response is not sufficient to uncover antiviral activity of RNAi in human epithelial cells infected with three wild-type positive-sense RNA viruses.