Enhancer and super-enhancer landscape in polycystic kidney disease.

Widespread aberrant gene expression is a pathological hallmark of polycystic kidney disease (PKD). Numerous pathogenic signaling cascades, including c-Myc, Fos, and Jun, are transactivated. However, the underlying epigenetic regulators are poorly defined. Here we show that H3K27ac, an acetylated modification of DNA packing protein histone H3 that marks active enhancers, is elevated in mouse and human samples of autosomal dominant PKD. Using comparative H3K27ac ChIP-Seq analysis, we mapped over 16000 active intronic and intergenic enhancer elements in Pkd1-mutant mouse kidneys. We found that the cystic kidney epigenetic landscape resembles that of a developing kidney, and over 90% of upregulated genes in Pkd1-mutant kidneys are co-housed with activated enhancers in the same topologically associated domains. Furthermore, we identified an evolutionarily conserved enhancer cluster downstream of the c-Myc gene and super-enhancers flanking both Jun and Fos loci in mouse and human models of autosomal dominant PKD. Deleting these regulatory elements reduced c-Myc, Jun, or Fos abundance and suppressed proliferation and 3D cyst growth of Pkd1-mutant cells. Finally, inhibiting glycolysis and glutaminolysis or activating Ppara in Pkd1-mutant cells lowerd global H3K27ac levels and its abundance on c-Myc enhancers. Thus, our work suggests that epigenetic rewiring mediates the transcriptomic dysregulation in PKD, and the regulatory elements can be targeted to slow cyst growth.

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.

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.

Transcription Factor Hepatocyte Nuclear Factor-1ß (HNF-1ß) Regulates MicroRNA-200 Expression through a Long Noncoding RNA.

The transcription factor hepatocyte nuclear factor-1ß (HNF-1ß) regulates tissue-specific gene expression in the kidney and other epithelial organs. Mutations of HNF-1ß produce kidney cysts, and previous studies have shown that HNF-1ß regulates the transcription of cystic disease genes, including Pkd2 and Pkhd1. Here, we combined chromatin immunoprecipitation and next-generation sequencing (ChIP-Seq) with microarray analysis to identify microRNAs (miRNAs) that are directly regulated by HNF-1ß in renal epithelial cells. These studies identified members of the epithelial-specific miR-200 family (miR-200b/200a/429) as novel transcriptional targets of HNF-1ß. HNF-1ß binds to two evolutionarily conserved sites located 28 kb upstream to miR-200b. Luciferase reporter assays showed that the HNF-1ß binding sites were located within a promoter that was active in renal epithelial cells. Mutations of the HNF-1ß binding sites abolished promoter activity. RT-PCR analysis revealed that a long noncoding RNA (lncRNA) is transcribed from the promoter and encodes the miR-200 cluster. Inhibition of the lncRNA with siRNAs decreased the levels of miR-200 but did not affect expression of the Ttll10 host gene. The expression of the lncRNA and miR-200 was decreased in kidneys from HNF-1ß knock-out mice and renal epithelial cells expressing dominant-negative mutant HNF-1ß. The expression of miR-200 targets, Zeb2 and Pkd1, was increased in HNF-1ß knock-out kidneys and in cells expressing mutant HNF-1ß. Overexpression of miR-200 decreased the expression of Zeb2 and Pkd1 in HNF-1ß mutant cells. These studies reveal a novel pathway whereby HNF-1ß directly contributes to the control of miRNAs that are involved in epithelial-mesenchymal transition and cystic kidney disease.

Tissue-specific regulation of the mouse Pkhd1 (ARPKD) gene promoter.

Autosomal recessive polycystic kidney disease, an inherited disorder characterized by the formation of cysts in renal collecting ducts and biliary dysgenesis, is caused by mutations of the polycystic kidney and hepatic disease 1 (PKHD1) gene. Expression of PKHD1 is tissue specific and developmentally regulated. Here, we show that a 2.0-kb genomic fragment containing the proximal promoter of mouse Pkhd1 directs tissue-specific expression of a lacZ reporter gene in transgenic mice. LacZ is expressed in renal collecting ducts beginning during embryonic development but is not expressed in extrarenal tissues. The Pkhd1 promoter contains a binding site for the transcription factor hepatocyte nuclear factor (HNF)-1ß, which is required for activity in transfected cells. Mutation of the HNF-1ß-binding site abolishes the expression of the lacZ reporter gene in renal collecting ducts. Transgenes containing the 2.0-kb promoter and 2.7 kb of additional genomic sequence extending downstream to the second exon are expressed in the kidney, intrahepatic bile ducts, and male reproductive tract. This pattern overlaps with the endogenous expression of Pkhd1 and coincides with sites of expression of HNF-1ß. We conclude that the proximal 2.0-kb promoter is sufficient for tissue-specific expression of Pkhd1 in renal collecting ducts in vivo and that HNF-1ß is required for Pkhd1 promoter activity in collecting ducts. Additional genomic sequences located from exons 1-2 or elsewhere in the gene locus are required for expression in extrarenal tissues.

PKD1 and PKD2 mRNA cis-inhibition drives polycystic kidney disease progression.

Autosomal dominant polycystic kidney disease (ADPKD), among the most common human genetic conditions and a frequent etiology of kidney failure, is primarily caused by heterozygous PKD1 mutations. Kidney cyst formation occurs when PKD1 dosage falls below a critical threshold. However, no framework exists to harness the remaining allele or reverse PKD1 decline. Here, we show that mRNAs produced by the noninactivated PKD1 allele are repressed via their 3'-UTR miR-17 binding element. Eliminating this motif (Pkd1∆17) improves mRNA stability, raises Polycystin-1 levels, and alleviates cyst growth in cellular, ex vivo, and mouse PKD models. Remarkably, Pkd2 is also inhibited via its 3'-UTR miR-17 motif, and Pkd2∆17-induced Polycystin-2 derepression retards cyst growth in Pkd1-mutant models. Moreover, acutely blocking Pkd1/2 cis-inhibition, including after cyst onset, attenuates murine PKD. Finally, modeling PKD1∆17 or PKD2∆17 alleles in patient-derived primary ADPKD cultures leads to smaller cysts, reduced proliferation, lower pCreb1 expression, and improved mitochondrial membrane potential. Thus, evading 3'-UTR cis-interference and enhancing PKD1/2 mRNA translation is a potentially mutation-agnostic ADPKD-arresting approach.

A methionine-Mettl3-N6-methyladenosine axis promotes polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenic disorder marked by numerous progressively enlarging kidney cysts. Mettl3, a methyltransferase that catalyzes the abundant N6-methyladenosine (m6A) RNA modification, is implicated in development, but its role in most diseases is unknown. Here, we show that Mettl3 and m6A levels are increased in mouse and human ADPKD samples and that kidney-specific transgenic Mettl3 expression produces tubular cysts. Conversely, Mettl3 deletion in three orthologous ADPKD mouse models slows cyst growth. Interestingly, methionine and S-adenosylmethionine (SAM) levels are also elevated in ADPKD models. Moreover, methionine and SAM induce Mettl3 expression and aggravate ex vivo cyst growth, whereas dietary methionine restriction attenuates mouse ADPKD. Finally, Mettl3 activates the cyst-promoting c-Myc and cAMP pathways through enhanced c-Myc and Avpr2 mRNA m6A modification and translation. Thus, Mettl3 promotes ADPKD and links methionine utilization to epitranscriptomic activation of proliferation and cyst growth.

Acute kidney injury and aberrant planar cell polarity induce cyst formation in mice lacking renal cilia.

Polycystic kidney disease (PKD) is an inherited disorder that is characterized by the accumulation of cysts in the renal parenchyma and progressive decline in renal function. Recent studies suggest that PKD arises from abnormalities of the primary cilium. We have previously shown that kidney-specific inactivation of the ciliogenic gene Kif3a during embryonic development produces kidney cysts and renal failure. Here, we used tamoxifen-inducible, kidney-specific gene targeting to inactivate Kif3a in the postnatal mouse kidney. Kidney-specific inactivation of Kif3a in newborn mice resulted in the loss of primary cilia and produced kidney cysts primarily in the loops of Henle, whereas inactivation in adult mice did not lead to the rapid development of cysts despite a comparable loss of primary cilia. The age-dependence and locations of the cysts suggested that cyst formation required increased rates of cell proliferation. To test this possibility, we stimulated cell proliferation in the adult kidney by inducing acute kidney injury and tubular regeneration. Acute kidney injury induced cyst formation in adult Kif3a mutant mice. Analysis of pre-cystic tubules in Kif3a mutant mice showed that the loss of cilia did not stimulate cell proliferation but instead resulted in aberrant planar cell polarity as manifested by abnormalities in the orientation of cell division. We conclude that primary cilia are required for the maintenance of planar cell polarity in the mammalian kidney and that acute kidney injury exacerbates cystic disease.

Anti-microRNA screen uncovers miR-17 family within miR-17~92 cluster as the primary driver of kidney cyst growth.

Autosomal dominant polycystic kidney disease (ADPKD) is the leading genetic cause of renal failure. We have recently shown that inhibiting miR-17~92 is a potential novel therapeutic approach for ADPKD. However, miR-17~92 is a polycistronic cluster that encodes microRNAs (miRNAs) belonging to the miR-17, miR-18, miR-19 and miR-25 families, and the relative pathogenic contribution of these miRNA families to ADPKD progression is unknown. Here we performed an in vivo anti-miR screen to identify the miRNA drug targets within the miR-17~92 miRNA cluster. We designed anti-miRs to individually inhibit miR-17, miR-18, miR-19 or miR-25 families in an orthologous ADPKD model. Treatment with anti-miRs against the miR-17 family reduced cyst proliferation, kidney-weight-to-body-weight ratio and cyst index. In contrast, treatment with anti-miRs against the miR-18, 19, or 25 families did not affect cyst growth. Anti-miR-17 treatment recapitulated the gene expression pattern observed after miR-17~92 genetic deletion and was associated with upregulation of mitochondrial metabolism, suppression of the mTOR pathway, and inhibition of cyst-associated inflammation. Our results argue against functional cooperation between the various miR-17~92 cluster families in promoting cyst growth, and instead point to miR-17 family as the primary therapeutic target for ADPKD.

Kidney cysts, pancreatic cysts, and biliary disease in a mouse model of autosomal recessive polycystic kidney disease.

Mutations in PKHD1 cause autosomal recessive polycystic kidney disease (ARPKD). We produced a mouse model of ARPKD by replacing exons 1-3 of Pkhd1 with a lacZ reporter gene utilizing homologous recombination. This approach yielded heterozygous Pkhd1 (lacZ/+) mice, that expressed beta-galactosidase in tissues where Pkhd1 is normally expressed, and homozygous Pkhd1 (lacZ/lacZ) knockout mice. Heterozygous Pkhd1 (lacZ/+) mice expressed beta-galactosidase in the kidney, liver, and pancreas. Homozygous Pkhd1 (lacZ/lacZ) mice lacked Pkhd1 expression and developed progressive renal cystic disease involving the proximal tubules, collecting ducts, and glomeruli. In the liver, inactivation of Pkhd1 resulted in dilatation of the bile ducts and periportal fibrosis. Dilatation of pancreatic exocrine ducts was uniformly seen in Pkhd1 (lacZ/lacZ ) mice, with pancreatic cysts arising less frequently. The expression of beta-galactosidase, Pkd1, and Pkd2 was reduced in the kidneys of Pkhd1 (lacZ/lacZ ) mice compared with wild-type littermates, but no changes in blood urea nitrogen (BUN) or liver function tests were observed. Collectively, these results indicate that deletion of exons 1-3 leads to loss of Pkhd1 expression and results in kidney cysts, pancreatic cysts, and biliary ductal plate malformations. The Pkhd1 (lacZ/lacZ ) mouse represents a new orthologous animal model for studying the pathogenesis of kidney cysts and biliary dysgenesis that characterize human ARPKD.

Multiple renal cysts, urinary concentration defects, and pulmonary emphysematous changes in mice lacking TAZ.

TAZ (transcriptional coactivator with PDZ-binding motif), also called WWTR1 (WW domain containing transcription regulator 1), is a 14-3-3-binding molecule homologous to Yes-associated protein. TAZ acts as a coactivator for several transcription factors as well as a modulator of membrane-associated PDZ domain-containing proteins, but its (patho)physiological roles remain unknown. Here we show that gene inactivation of TAZ in mice resulted in pathological changes in the kidney and lung that resemble the common human diseases polycystic kidney disease and pulmonary emphysema. Taz-null/lacZ knockin mutant homozygotes demonstrated renal cyst formation as early as embryonic day 15.5 with dilatation of Bowman's capsules and proximal tubules, followed by pelvic dilatation and hydronephrosis. After birth, only one-fifth of TAZ-deficient homozygotes grew to adulthood and demonstrated multicystic kidneys with severe urinary concentrating defects and polyuria. Furthermore, adult TAZ-deficient homozygotes exhibited diffuse emphysematous changes in the lung. Thus TAZ is essential for developmental mechanisms involved in kidney and lung organogenesis, whose disturbance may lead to the pathogenesis of common human diseases.