Multiomics analysis reveals that hepatocyte nuclear factor 1ß regulates axon guidance genes in the developing mouse kidney.

The transcription factor hepatocyte nuclear factor 1ß (HNF-1ß) is essential for normal development of the kidney and other epithelial organs. In the developing mouse kidney, HNF-1ß is required for the differentiation and patterning of immature nephrons and branching morphogenesis of the ureteric bud (UB). Here, we used ChIP-sequencing (ChIP-seq) and RNA sequencing (RNA-seq) to identify genes that are regulated by HNF-1ß in embryonic mouse kidneys. ChIP-seq revealed that HNF-1ß binds to 8284 sites in chromatin from E14.5 mouse kidneys. Comparison with previous ATAC-seq and histone modification studies showed that HNF-1ß binding peaks colocalized with open chromatin and epigenetic marks of transcriptional activation (H3K27 acetylation, H3K4 trimethylation, H3K4 monomethylation), indicating that the binding sites were functional. To investigate the relationship between HNF-1ß binding and HNF-1ß-dependent gene regulation, RNA-seq was performed on UB cells purified from wild-type and HNF-1ß mutant embryonic kidneys. A total of 1632 genes showed reduced expression in HNF-1ß-deficient UB cells, and 485 genes contained nearby HNF-1ß binding sites indicating that they were directly activated by HNF-1ß. Conversely, HNF-1ß directly repressed the expression of 526 genes in the UB. Comparison with snATAC-seq analysis of UB-derived cells showed that both HNF-1ß-dependent activation and repression correlated with chromatin accessibility. Pathway analysis revealed that HNF-1ß binds near 68 axon guidance genes in the developing kidney. RNA-seq analysis showed that Nrp1, Sema3c, Sema3d, Sema6a, and Slit2 were activated by HNF-1ß, whereas Efna1, Epha3, Epha4, Epha7, Ntn4, Plxna2, Sema3a, Sema4b, Slit3, Srgap1, Unc5c and Unc5d were repressed by HNF-1ß. RNAscope in situ hybridization showed that Nrp1, Sema3c, Sema3d, Sema6a, and Slit2 were expressed in wild-type UB and were dysregulated in HNF-1ß mutant UB. These studies show that HNF-1ß directly regulates the expression of multiple axon guidance genes in the developing mouse kidney. Dysregulation of axon guidance genes may underlie kidney defects in HNF-1ß mutant mice.

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.

Small hairpin inhibitory RNA delivery in the metanephric organ culture identifies long noncoding RNA Pvt1 as a modulator of cyst growth.

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic disorder characterized by the formation of kidney cysts that originate from the epithelial tubules of the nephron and primarily results from mutations in polycystin-1 (PKD1) and polycystin-2 (PKD2). The metanephric organ culture (MOC) is an ex vivo system in which explanted embryonic kidneys undergo tubular differentiation and kidney development. MOC has been previously used to study polycystic kidney disease as treatment with 8-bromo-cAMP induces the formation of kidney cysts. However, the inefficiency of manipulating gene expression in MOC has limited its utility for identifying genes and pathways that are involved in cystogenesis. Here, we used a lentivirus and three serotypes of self-complementary adeno-associated viral (scAAV) plasmids that express green fluorescent protein and found that scAAV serotype D/J transduces the epithelial compartment of MOC at an efficiency of 68%. We used scAAV/DJ to deliver shRNA to knockdown Pvt1, a long noncoding RNA, which was upregulated in kidneys from Pkd1 and Pkd2 mutant mice and humans with ADPKD. shRNA delivery by scAAV/DJ downregulated expression of Pvt1 by 45% and reduced the cyst index by 53% in wild-type MOCs and 32% in Pkd1-null MOCs. Knockdown of Pvt1 decreased the level of c-MYC protein by 60% without affecting Myc mRNA, indicating that Pvt1 regulation of c-MYC was posttranscriptional. These results identify Pvt1 as a long noncoding RNA that modulates cyst progression in MOC.NEW & NOTEWORTHY This study identified scAAV/DJ as effective in transducing epithelial cells of the metanephric organ culture (MOC). We used scAAV/DJ shRNA to knockdown Pvt1 in cystic MOCs derived from Pkd1-null embryos. Downregulation of Pvt1 reduced cyst growth and decreased levels of c-MYC protein. These data suggest that suppression of Pvt1 activity in autosomal dominant polycystic kidney disease might reduce cyst growth.

Advancing Nephrology: Division Leaders Advise ASN.

New treatments, new understanding, and new approaches to translational research are transforming the outlook for patients with kidney diseases. A number of new initiatives dedicated to advancing the field of nephrology-from value-based care to prize competitions-will further improve outcomes of patients with kidney disease. Because of individual nephrologists and kidney organizations in the United States, such as the American Society of Nephrology, the National Kidney Foundation, and the Renal Physicians Association, and international nephrologists and organizations, such as the International Society of Nephrology and the European Renal Association-European Dialysis and Transplant Association, we are beginning to gain traction to invigorate nephrology to meet the pandemic of global kidney diseases. Recognizing the timeliness of this opportunity, the American Society of Nephrology convened a Division Chief Retreat in Dallas, Texas, in June 2019 to address five key issues: (1) asserting the value of nephrology to the health system; (2) productivity and compensation; (3) financial support of faculty's and divisions' educational efforts; (4) faculty recruitment, retention, diversity, and inclusion; and (5) ensuring that fellowship programs prepare trainees to provide high-value nephrology care and enhance attraction of trainees to nephrology. Herein, we highlight the outcomes of these discussions and recommendations to the American Society of Nephrology.

Interstitial microRNA miR-214 attenuates inflammation and polycystic kidney disease progression.

Renal cysts are the defining feature of autosomal dominant polycystic kidney disease (ADPKD); however, the substantial interstitial inflammation is an often-overlooked aspect of this disorder. Recent studies suggest that immune cells in the cyst microenvironment affect ADPKD progression. Here we report that microRNAs (miRNAs) are new molecular signals in this crosstalk. We found that miR-214 and its host long noncoding RNA Dnm3os are upregulated in orthologous ADPKD mouse models and cystic kidneys from humans with ADPKD. In situ hybridization revealed that interstitial cells in the cyst microenvironment are the primary source of miR-214. While genetic deletion of miR-214 does not affect kidney development or homeostasis, surprisingly, its inhibition in Pkd2- and Pkd1-mutant mice aggravates cyst growth. Mechanistically, the proinflammatory TLR4/IFN-γ/STAT1 pathways transactivate the miR-214 host gene. miR-214, in turn as a negative feedback loop, directly inhibits Tlr4. Accordingly, miR-214 deletion is associated with increased Tlr4 expression and enhanced pericystic macrophage accumulation. Thus, miR-214 upregulation is a compensatory protective response in the cyst microenvironment that restrains inflammation and cyst growth.

Role of transcription factor hepatocyte nuclear factor-1ß in polycystic kidney disease.

Hepatocyte nuclear factor-1ß (HNF-1ß) is a DNA-binding transcription factor that is essential for normal kidney development. Mutations of HNF1B in humans produce cystic kidney diseases, including renal cysts and diabetes, multicystic dysplastic kidneys, glomerulocystic kidney disease, and autosomal dominant tubulointerstitial kidney disease. Expression of HNF1B is reduced in cystic kidneys from humans with ADPKD, and HNF1B has been identified as a modifier gene in PKD. Genome-wide analysis of chromatin binding has revealed that HNF-1ß directly regulates the expression of known PKD genes, such as PKHD1 and PKD2, as well as genes involved in PKD pathogenesis, including cAMP-dependent signaling, renal fibrosis, and Wnt signaling. In addition, a role of HNF-1ß in regulating the expression of noncoding RNAs (microRNAs and long noncoding RNAs) has been identified. These findings indicate that HNF-1ß regulates a transcriptional and post-transcriptional network that plays a central role in renal cystogenesis.

Hepatocyte nuclear factor 1ß suppresses canonical Wnt signaling through transcriptional repression of lymphoid enhancer-binding factor 1.

Hepatocyte nuclear factor-1ß (HNF-1ß) is a tissue-specific transcription factor that is required for normal kidney development and renal epithelial differentiation. Mutations of HNF-1ß produce congenital kidney abnormalities and inherited renal tubulopathies. Here, we show that ablation of HNF-1ß in mIMCD3 renal epithelial cells results in activation of ß-catenin and increased expression of lymphoid enhancer-binding factor 1 (LEF1), a downstream effector in the canonical Wnt signaling pathway. Increased expression and nuclear localization of LEF1 are also observed in cystic kidneys from Hnf1b mutant mice. Expression of dominant-negative mutant HNF-1ß in mIMCD3 cells produces hyperresponsiveness to exogenous Wnt ligands, which is inhibited by siRNA-mediated knockdown of Lef1. WT HNF-1ß binds to two evolutionarily conserved sites located 94 and 30 kb from the mouse Lef1 promoter. Ablation of HNF-1ß decreases H3K27 trimethylation repressive marks and increases ß-catenin occupancy at a site 4 kb upstream to Lef1. Mechanistically, WT HNF-1ß recruits the polycomb-repressive complex 2 that catalyzes H3K27 trimethylation. Deletion of the ß-catenin-binding domain of LEF1 in HNF-1ß-deficient cells abolishes the increase in Lef1 transcription and decreases the expression of downstream Wnt target genes. The canonical Wnt target gene, Axin2, is also a direct transcriptional target of HNF-1ß through binding to negative regulatory elements in the gene promoter. These findings demonstrate that HNF-1ß regulates canonical Wnt target genes through long-range effects on histone methylation at Wnt enhancers and reveal a new mode of active transcriptional repression by HNF-1ß.

Innate Immune Signaling Contributes to Tubular Cell Senescence in the Glis2 Knockout Mouse Model of Nephronophthisis.

Nephronophthisis (NPHP), the leading genetic cause of end-stage renal failure in children and young adults, is a group of autosomal recessive diseases characterized by kidney-cyst degeneration and fibrosis for which no therapy is currently available. To date, mutations in >25 genes have been identified as causes of this disease that, in several cases, result in chronic DNA damage in kidney tubular cells. Among such mutations, those in the transcription factor-encoding GLIS2 cause NPHP type 7. Loss of function of mouse Glis2 causes senescence of kidney tubular cells. Senescent cells secrete proinflammatory molecules that induce progressive organ damage through several pathways, among which NF-κB signaling is prevalent. Herein, we show that the NF-κB signaling is active in Glis2 knockout kidney epithelial cells and that genetic inactivation of the toll-like receptor (TLR)/IL-1 receptor or pharmacologic elimination of senescent cells (senolytic therapy) reduces tubule damage, fibrosis, and apoptosis in the Glis2 mouse model of NPHP. Notably, in Glis2, Tlr2 double knockouts, senescence was also reduced and proliferation was increased, suggesting that loss of TLR2 activity improves the regenerative potential of tubular cells in Glis2 knockout kidneys. Our results further suggest that a combination of TLR/IL-1 receptor inhibition and senolytic therapy may delay the progression of kidney disease in NPHP type 7 and other forms of this disease.

Hepatocyte nuclear factor-1ß regulates Wnt signaling through genome-wide competition with ß-catenin/lymphoid enhancer binding factor.

Hepatocyte nuclear factor-1ß (HNF-1ß) is a tissue-specific transcription factor that is essential for normal kidney development and renal tubular function. Mutations of HNF-1ß produce cystic kidney disease, a phenotype associated with deregulation of canonical (ß-catenin-dependent) Wnt signaling. Here, we show that ablation of HNF-1ß in mIMCD3 renal epithelial cells produces hyperresponsiveness to Wnt ligands and increases expression of Wnt target genes, including Axin2, Ccdc80, and Rnf43 Levels of ß-catenin and expression of Wnt target genes are also increased in HNF-1ß mutant mouse kidneys. Genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) in wild-type and mutant cells showed that ablation of HNF-1ß increases by 6-fold the number of sites on chromatin that are occupied by ß-catenin. Remarkably, 50% of the sites that are occupied by ß-catenin in HNF-1ß mutant cells colocalize with HNF-1ß-occupied sites in wild-type cells, indicating widespread reciprocal binding. We found that the Wnt target genes Ccdc80 and Rnf43 contain a composite DNA element comprising a ß-catenin/lymphoid enhancer binding factor (LEF) site overlapping with an HNF-1ß half-site. HNF-1ß and ß-catenin/LEF compete for binding to this element, and thereby HNF-1ß inhibits ß-catenin-dependent transcription. Collectively, these studies reveal a mechanism whereby a transcription factor constrains canonical Wnt signaling through direct inhibition of ß-catenin/LEF chromatin binding.

Renal tubular cell spliced X-box binding protein 1 (Xbp1s) has a unique role in sepsis-induced acute kidney injury and inflammation.

Sepsis is a systemic inflammatory state in response to infection, and concomitant acute kidney injury (AKI) increases mortality significantly. Endoplasmic reticulum stress is activated in many cell types upon microbial infection and modulates inflammation. The role of endoplasmic reticulum signaling in the kidney during septic AKI is unknown. Here we tested the role of the spliced X-box binding protein 1 (Xbp1s), a key component of the endoplasmic reticulum stress-activated pathways, in the renal response to sepsis in the lipopolysaccharide (LPS) model. Xbp1s was increased in the kidneys of mice treated with LPS but not in other models of AKI, or several chronic kidney disease models. The functional significance of Xbp1s induction was examined by genetic manipulation in renal tubules. Renal tubule-specific overexpression of Xbp1s caused severe tubule dilation and vacuolation with expression of the injury markers Kim1 and Ngal, the pro-inflammatory molecules interleukin-6 (Il6) and Toll-like receptor 4 (Tlr4), decreased kidney function and 50% mortality in five days. Renal tubule-specific genetic ablation of Xbp1 had no phenotype at baseline. However, after LPS, Xbp1 knockdown mice displayed lower renal NGAL, pro-apoptotic factor CHOP, serum creatinine levels, and a tendency towards lower Tlr4 compared to LPS-treated mice with intact Xbp1s. LPS treatment in Xbp1s-overexpressing mice caused a mild increase in NGAL and CHOP compared to LPS-treated mice without genetic Xbp1s overexpression. Thus, increased Xbp1s signaling in renal tubules is unique to sepsis-induced AKI and contributes to renal inflammation and injury. Inhibition of this pathway may be a potential portal to alleviate injury.

New insights into the role of HNF-1ß in kidney (patho)physiology.

Hepatocyte nuclear factor-1ß (HNF-1ß) is an essential transcription factor that regulates the development and function of epithelia in the kidney, liver, pancreas, and genitourinary tract. Humans who carry HNF1B mutations develop heterogeneous renal abnormalities, including multicystic dysplastic kidneys, glomerulocystic kidney disease, renal agenesis, renal hypoplasia, and renal interstitial fibrosis. In the embryonic kidney, HNF-1ß is required for ureteric bud branching, initiation of nephrogenesis, and nephron segmentation. Ablation of mouse Hnf1b in nephron progenitors causes defective tubulogenesis, whereas later inactivation in elongating tubules leads to cyst formation due to downregulation of cystic disease genes, including Umod, Pkhd1, and Pkd2. In the adult kidney, HNF-1ß controls the expression of genes required for intrarenal metabolism and solute transport by tubular epithelial cells. Tubular abnormalities observed in HNF-1ß nephropathy include hyperuricemia with or without gout, hypokalemia, hypomagnesemia, and polyuria. Recent studies have identified novel post-transcriptional and post-translational regulatory mechanisms that control HNF-1ß expression and activity, including the miRNA cluster miR17 ∼ 92 and the interacting proteins PCBD1 and zyxin. Further understanding of the molecular mechanisms upstream and downstream of HNF-1ß may lead to the development of new therapeutic approaches in cystic kidney disease and other HNF1B-related renal diseases.

Mechanism of Fibrosis in HNF1B-Related Autosomal Dominant Tubulointerstitial Kidney Disease.

BACKGROUND: Mutation of HNF1B, the gene encoding transcription factor HNF-1ß, is one cause of autosomal dominant tubulointerstitial kidney disease, a syndrome characterized by tubular cysts, renal fibrosis, and progressive decline in renal function. HNF-1ß 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-1ß-deficient mIMCD3 renal epithelial cells, used RNA-sequencing analysis to reveal differentially expressed genes in wild-type and HNF-1ß-deficient mIMCD3 cells, and performed cell lineage analysis in HNF-1ß mutant mice. RESULTS: The HNF-1ß-deficient cells exhibited properties characteristic of mesenchymal cells such as fibroblasts, 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-1ß directly represses Twist2, and ablation of Twist2 partially rescued the fibroblastic phenotype of HNF-1ß mutant cells. Kidneys from HNF-1ß mutant mice showed increased expression of Twist2 and its downstream target Snai2. Cell lineage analysis indicated that HNF-1ß mutant epithelial cells do not transdifferentiate into kidney myofibroblasts. Rather, HNF-1ß mutant epithelial cells secrete high levels of TGF-ß ligands that activate downstream Smad transcription factors in renal interstitial cells. CONCLUSIONS: Ablation of HNF-1ß in renal epithelial cells leads to the activation of a Twist2-dependent transcriptional network that induces EMT and aberrant TGF-ß signaling, resulting in renal fibrosis through a cell-nonautonomous mechanism.

Long noncoding RNA Hoxb3os is dysregulated in autosomal dominant polycystic kidney disease and regulates mTOR signaling.

Autosomal dominant polycystic kidney disease (ADPKD) is a debilitating disease that is characterized by the accumulation of numerous fluid-filled cysts in the kidney. ADPKD is primarily caused by mutations in two genes, PKD1 and PKD2 Long noncoding RNAs (lncRNA), defined by a length >200 nucleotides and absence of a long ORF, have recently emerged as epigenetic regulators of development and disease; however, their involvement in PKD has not been explored previously. Here, we performed deep RNA-Seq to identify lncRNAs that are dysregulated in two orthologous mouse models of ADPKD (kidney-specific Pkd1 and Pkd2 mutant mice). We identified a kidney-specific, evolutionarily conserved lncRNA called Hoxb3os that was down-regulated in cystic kidneys from Pkd1 and Pkd2 mutant mice. The human ortholog HOXB3-AS1 was down-regulated in cystic kidneys from ADPKD patients. Hoxb3os was highly expressed in renal tubules in adult WT mice, whereas its expression was lost in the cyst epithelium of mutant mice. To investigate the function of Hoxb3os, we utilized CRISPR/Cas9 to knock out its expression in mIMCD3 cells. Deletion of Hoxb3os resulted in increased phosphorylation of mTOR and its downstream targets, including p70 S6 kinase, ribosomal protein S6, and the translation repressor 4E-BP1. Consistent with activation of mTORC1 signaling, Hoxb3os mutant cells displayed increased mitochondrial respiration. The Hoxb3os mutant phenotype was partially rescued upon re-expression of Hoxb3os in knockout cells. These findings identify Hoxb3os as a novel lncRNA that is down-regulated in ADPKD and regulates mTOR signaling and mitochondrial respiration.

Activated renal tubular Wnt/ß-catenin signaling triggers renal inflammation during overload proteinuria.

Imbalance of Wnt/ß-catenin signaling in renal cells is associated with renal dysfunction, yet the precise mechanism is poorly understood. Previously we observed activated Wnt/ß-catenin signaling in renal tubules during proteinuric nephropathy with an unknown net effect. Therefore, to identify the definitive role of tubular Wnt/ß-catenin, we generated a novel transgenic "Tubcat" mouse conditionally expressing stabilized ß-catenin specifically in renal tubules following tamoxifen administration. Four weeks after tamoxifen injection, uninephrectomized Tubcat mice displayed proteinuria and elevated blood urea nitrogen levels compared to non-transgenic mice, implying a detrimental effect of the activated signaling. This was associated with infiltration of the tubulointerstitium predominantly by M1 macrophages and overexpression of the inflammatory chemocytokines CCL-2 and RANTES. Induction of overload proteinuria by intraperitoneal injection of low-endotoxin bovine serum albumin following uninephrectomy for four weeks aggravated proteinuria and increased blood urea nitrogen levels to a significantly greater extent in Tubcat mice. Renal dysfunction correlated with the degree of M1 macrophage infiltration in the tubulointerstitium and renal cortical up-regulation of CCL-2, IL-17A, IL-1ß, CXCL1, and ICAM-1. There was overexpression of cortical TLR-4 and NLRP-3 in Tubcat mice, independent of bovine serum albumin injection. Finally, there was no fibrosis, activation of epithelial-mesenchymal transition or non-canonical Wnt pathways observed in the kidneys of Tubcat mice. Thus, conditional activation of renal tubular Wnt/ß-catenin signaling in a novel transgenic mouse model demonstrates that this pathway enhances intrarenal inflammation via the TLR-4/NLRP-3 inflammasome axis in overload proteinuria.

Adenylyl cyclase 5 deficiency reduces renal cyclic AMP and cyst growth in an orthologous mouse model of polycystic kidney disease.

Cyclic AMP promotes cyst growth in polycystic kidney disease (PKD) by stimulating cell proliferation and fluid secretion. Previously, we showed that the primary cilium of renal epithelial cells contains a cAMP regulatory complex comprising adenylyl cyclases 5 and 6 (AC5/6), polycystin-2, A-kinase anchoring protein 150, protein kinase A, and phosphodiesterase 4C. In Kif3a mutant cells that lack primary cilia, the formation of this regulatory complex is disrupted and cAMP levels are increased. Inhibition of AC5 reduces cAMP levels in Kif3a mutant cells, suggesting that AC5 may mediate the increase in cAMP in PKD. Here, we examined the role of AC5 in an orthologous mouse model of PKD caused by kidney-specific ablation of Pkd2. Knockdown of AC5 with siRNA attenuated the increase in cAMP levels in Pkd2-deficient renal epithelial cells. Levels of cAMP and AC5 mRNA transcripts were elevated in the kidneys of mice with collecting duct-specific ablation of Pkd2. Compared with Pkd2 single mutant mice, AC5/Pkd2 double mutant mice had less kidney enlargement, lower cyst index, reduced kidney injury, and improved kidney function. Importantly, cAMP levels and cAMP-dependent signaling were reduced in the kidneys of AC5/Pkd2 double mutant compared to the kidneys of Pkd2 single mutant mice. Additionally, we localized endogenous AC5 in the primary cilium of renal epithelial cells and showed that ablation of AC5 reduced ciliary elongation in the kidneys of Pkd2 mutant mice. Thus, AC5 contributes importantly to increased renal cAMP levels and cyst growth in Pkd2 mutant mice, and inhibition of AC5 may be beneficial in the treatment of PKD.

Loss of transcriptional activation of the potassium channel Kir5.1 by HNF1ß drives autosomal dominant tubulointerstitial kidney disease.

Hepatocyte nuclear factor 1 homeobox B (HNF1ß) is an essential transcription factor for the development and functioning of the kidney. Mutations in HNF1ß cause autosomal dominant tubulointerstitial kidney disease characterized by renal cysts and maturity-onset diabetes of the young (MODY). Moreover, these patients suffer from a severe electrolyte phenotype consisting of hypomagnesemia and hypokalemia. Until now, genes that are regulated by HNF1ß are only partially known and do not fully explain the phenotype of the patients. Therefore, we performed chIP-seq in the immortalized mouse kidney cell line mpkDCT to identify HNF1ß binding sites on a genome-wide scale. In total 7,421 HNF1ß-binding sites were identified, including several genes involved in electrolyte transport and diabetes. A highly specific and conserved HNF1ß site was identified in the promoter of Kcnj16 that encodes the potassium channel Kir5.1. Luciferase-promoter assays showed a 2.2-fold increase in Kcnj16 expression when HNF1ß was present. Expression of the Hnf1ß p.Lys156Glu mutant, previously identified in a patient with autosomal dominant tubulointerstitial kidney disease, did not activate Kcnj16 expression. Knockdown of Hnf1ß in mpkDCT cells significantly reduced the appearance of Kcnj16 (Kir5.1) and Kcnj10 (Kir4.1) by 38% and 37%, respectively. These results were confirmed in a HNF1ß renal knockout mouse which exhibited downregulation of Kcnj16, Kcnj10 and Slc12a3 transcripts in the kidney by 78%, 83% and 76%, respectively, compared to HNF1ß wild-type mice. Thus, HNF1ß is a transcriptional activator of Kcnj16. Hence, patients with HNF1ß mutations may have reduced Kir5.1 activity in the kidney, resulting in hypokalemia and hypomagnesemia.

Hepatocyte Nuclear Factor-1ß Regulates Urinary Concentration and Response to Hypertonicity.

The transcription factor hepatocyte nuclear factor-1ß (HNF-1ß) is essential for normal kidney development and function. Inactivation of HNF-1ß in mouse kidney tubules leads to early-onset cyst formation and postnatal lethality. Here, we used Pkhd1/Cre mice to delete HNF-1ß specifically in renal collecting ducts (CDs). CD-specific HNF-1ß mutant mice survived long term and developed slowly progressive cystic kidney disease, renal fibrosis, and hydronephrosis. Compared with wild-type littermates, HNF-1ß mutant mice exhibited polyuria and polydipsia. Before the development of significant renal structural abnormalities, mutant mice exhibited low urine osmolality at baseline and after water restriction and administration of desmopressin. However, mutant and wild-type mice had similar plasma vasopressin and solute excretion levels. HNF-1ß mutant kidneys showed increased expression of aquaporin-2 mRNA but mislocalized expression of aquaporin-2 protein in the cytoplasm of CD cells. Mutant kidneys also had decreased expression of the UT-A urea transporter and collectrin, which is involved in apical membrane vesicle trafficking. Treatment of HNF-1ß mutant mIMCD3 cells with hypertonic NaCl inhibited the induction of osmoregulated genes, including Nr1h4, which encodes the transcription factor FXR that is required for maximal urinary concentration. Chromatin immunoprecipitation and sequencing experiments revealed HNF-1ß binding to the Nr1h4 promoter in wild-type kidneys, and immunoblot analysis revealed downregulated expression of FXR in HNF-1ß mutant kidneys. These findings reveal a novel role of HNF-1ß in osmoregulation and identify multiple mechanisms, whereby mutations of HNF-1ß produce defects in urinary concentration.

microRNA-17 family promotes polycystic kidney disease progression through modulation of mitochondrial metabolism.

Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent genetic cause of renal failure. Here we identify miR-17 as a target for the treatment of ADPKD. We report that miR-17 is induced in kidney cysts of mouse and human ADPKD. Genetic deletion of the miR-17∼92 cluster inhibits cyst proliferation and PKD progression in four orthologous, including two long-lived, mouse models of ADPKD. Anti-miR-17 treatment attenuates cyst growth in short-term and long-term PKD mouse models. miR-17 inhibition also suppresses proliferation and cyst growth of primary ADPKD cysts cultures derived from multiple human donors. Mechanistically, c-Myc upregulates miR-17∼92 in cystic kidneys, which in turn aggravates cyst growth by inhibiting oxidative phosphorylation and stimulating proliferation through direct repression of Pparα. Thus, miR-17 family is a promising drug target for ADPKD, and miR-17-mediated inhibition of mitochondrial metabolism represents a potential new mechanism for ADPKD progression.

Planar cell polarity genes Celsr1 and Vangl2 are necessary for kidney growth, differentiation, and rostrocaudal patterning.

The mammalian kidney contains nephrons comprising glomeruli and tubules joined to ureteric bud-derived collecting ducts. It has a characteristic bean-like shape, with near-complete rostrocaudal symmetry around the hilum. Here we show that Celsr1, a planar cell polarity (PCP) gene implicated in neural tube morphogenesis, is required for ureteric tree growth in early development and later in gestation prevents tubule overgrowth. We also found an interaction between Celsr1 and Vangl2 (another PCP gene) in ureteric tree growth, most marked in the caudal compartment of the kidneys from compound heterozygous mutant mice with a stunted rump. Furthermore, these genes together are required for the maturation of glomeruli. Interestingly, we demonstrated patients with CELSR1 mutations and spina bifida can have significant renal malformations. Thus, PCP genes are important in mammalian kidney development and have an unexpected role in rostrocaudal patterning during organogenesis.

Loss of Glis2/NPHP7 causes kidney epithelial cell senescence and suppresses cyst growth in the Kif3a mouse model of cystic kidney disease.

Enlargement of kidney tubules is a common feature of multiple cystic kidney diseases in humans and mice. However, while some of these pathologies are characterized by cyst expansion and organ enlargement, in others, progressive interstitial fibrosis and kidney atrophy prevail. The Kif3a knockout mouse is an established non-orthologous mouse model of cystic kidney disease. Conditional inactivation of Kif3a in kidney tubular cells results in loss of primary cilia and rapid cyst growth. Conversely, loss of function of the gene GLIS2/NPHP7 causes progressive kidney atrophy, interstitial inflammatory infiltration, and fibrosis. Kif3a null tubular cells have unrestrained proliferation and reduced stabilization of p53 resulting in a loss of cell cycle arrest in the presence of DNA damage. In contrast, loss of Glis2 is associated with activation of checkpoint kinase 1, stabilization of p53, and induction of cell senescence. Interestingly, the cystic phenotype of Kif3a knockout mice is partially rescued by genetic ablation of Glis2 and pharmacological stabilization of p53. Thus, Kif3a is required for cell cycle regulation and the DNA damage response, whereas cell senescence is significantly enhanced in Glis2 null cells. Hence, cell senescence is a central feature in nephronophthisis type 7 and Kif3a is unexpectedly required for efficient DNA damage response and cell cycle arrest.