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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.

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.

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.

The transcription factor hepatocyte nuclear factor-1β (HNF-1β) is essential for normal kidney development and function. Inactivation of HNF-1β in mouse kidney tubules leads to early-onset cyst formation and postnatal lethality. Here, we used Pkhd1/Cre mice to delete HNF-1β specifically in renal collecting ducts (CDs). CD-specific HNF-1β mutant mice survived long term and developed slowly progressive cystic kidney disease, renal fibrosis, and hydronephrosis. Compared with wild-type littermates, HNF-1β 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-1β 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-1β 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-1β binding to the Nr1h4 promoter in wild-type kidneys, and immunoblot analysis revealed downregulated expression of FXR in HNF-1β mutant kidneys. These findings reveal a novel role of HNF-1β in osmoregulation and identify multiple mechanisms, whereby mutations of HNF-1β produce defects in urinary concentration.

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., Autosomal dominant polycystic kidney disease (ADPKD) is a life-threatening genetic disease that leads to renal failure. Here Hajarnis et al. show that miR-17 modulates cyst progression in ADPKD through metabolic reprogramming of mitochondria and its inhibition slows cyst development and improves renal functions.

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.

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

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 1 homeobox B (HNF1β) is an essential transcription factor for the development and functioning of the kidney. Mutations in HNF1β 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 HNF1β 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 HNF1β binding sites on a genome-wide scale. In total 7,421 HNF1β-binding sites were identified, including several genes involved in electrolyte transport and diabetes. A highly specific and conserved HNF1β 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 HNF1β was present. Expression of the Hnf1β p.Lys156Glu mutant, previously identified in a patient with autosomal dominant tubulointerstitial kidney disease, did not activate Kcnj16 expression. Knockdown of Hnf1 β 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 HNF1β renal knockout mouse which exhibited downregulation of Kcnj16 , Kcnj10 and Slc12a3 transcripts in the kidney by 78%, 83% and 76%, respectively, compared to HNF1β wild-type mice. Thus, HNF1β is a transcriptional activator of Kcnj16 . Hence, patients with HNF1 β mutations may have reduced Kir5.1 activity in the kidney, resulting in hypokalemia and hypomagnesemia.

Hepatocyte nuclear factor-1β (HNF-1β) is an epithelial tissue-specific transcription factor that regulates gene expression in the kidney, liver, pancreas, intestine, and other organs. Mutations of HNF-1β in humans produce renal cysts and congenital kidney anomalies. Here, we identify the LIM-domain protein zyxin as a novel binding partner of HNF-1β in renal epithelial cells. Zyxin shuttles to the nucleus where it colocalizes with HNF-1β. Immunoprecipitation of zyxin in leptomycin B-treated cells results in coprecipitation of HNF-1β. The protein interaction requires the second LIM domain of zyxin and two distinct domains of HNF-1β. Overexpression of zyxin stimulates the transcriptional activity of HNF-1β, whereas small interfering RNA silencing of zyxin inhibits HNF-1β-dependent transcription. Epidermal growth factor (EGF) induces translocation of zyxin into the nucleus and stimulates HNF-1β-dependent promoter activity. The EGF-mediated nuclear translocation of zyxin requires activation of Akt. Expression of dominant-negative mutant HNF-1β, knockdown of zyxin, or inhibition of Akt inhibits EGF-stimulated cell migration. These findings reveal a novel pathway by which extracellular signals are transmitted to the nucleus to regulate the activity of a transcription factor that is essential for renal epithelial differentiation.

Polycystic kidney disease (PKD), the most common genetic cause of chronic kidney failure, is characterized by the presence of numerous, progressively enlarging fluid-filled cysts in the renal parenchyma. The cysts arise from renal tubules and are lined by abnormally functioning and hyperproliferative epithelial cells. Despite recent progress, no Food and Drug Administration-approved therapy is available to retard cyst growth. MicroRNAs (miRNAs) are short noncoding RNAs that inhibit posttranscriptional gene expression. Dysregulated miRNA expression is observed in PKD, but whether miRNAs are directly involved in kidney cyst formation and growth is not known. Here, we show that miR-17∼92, an oncogenic miRNA cluster, is up-regulated in mouse models of PKD. Kidney-specific transgenic overexpression of miR-17∼92 produces kidney cysts in mice. Conversely, kidney-specific inactivation of miR-17∼92 in a mouse model of PKD retards kidney cyst growth, improves renal function, and prolongs survival. miR-17∼92 may mediate these effects by promoting proliferation and through posttranscriptional repression of PKD genes Pkd1 , Pkd2 , and hepatocyte nuclear factor- 1 β. These studies demonstrate a pathogenic role of miRNAs in mouse models of PKD and identify miR-17∼92 as a therapeutic target in PKD. Our results also provide a unique hypothesis for disease progression in PKD involving miRNAs and regulation of PKD gene dosage.

The advent of technologies that allow tissue specific expression or ablation of genes has contributed enormously to our knowledge of the mechanism regulating organ development and maintenance in mice. The tetracycline inducible system allows reversible regulation of gene products upon administration of doxycycline. Here we describe the generation and activity of two transgenic lines expressing the cDNAs for the Tet responsive transcription factors rtTA and tTA (Tet-on and off) respectively under the control of an element that drives expression in the epithelium of the developing and adult kidney. Both lines show inducible and reversible activity in the embryonic and adult organ.

Germline mutations in the fumarate hydratase (FH) tumor suppressor gene predispose to leiomyomatosis, renal cysts, and renal cell cancer (HLRCC). HLRCC tumors overexpress HIF1alpha and hypoxia pathway genes. We conditionally inactivated mouse Fh1 in the kidney. Fh1 mutants developed multiple clonal renal cysts that overexpressed Hif1alpha and Hif2alpha. Hif targets, such as Glut1 and Vegf, were upregulated. We found that Fh1-deficient murine embryonic stem cells and renal carcinomas from HLRCC showed similar overexpression of HIF and hypoxia pathway components to the mouse cysts. Our data have shown in vivo that pseudohypoxic drive, resulting from HIF1alpha (and HIF2alpha) overexpression, is a direct consequence of Fh1 inactivation. Our mouse may be useful for testing therapeutic interventions that target angiogenesis and HIF-prolyl hydroxylation.

β-Catenin is a unique intracellular protein functioning as an integral component of the cell–cell adherens complex and a principal signaling protein mediating canonical Wnt signaling. Little is known about its function in adult kidneys in the normal physiologic state or after acute kidney injury (AKI). To study this, we generated conditional knockout mice in which the β-catenin gene was specifically disrupted in renal tubules (Ksp-β-cat-/-). These mice were phenotypically normal with no appreciable defects in kidney morphology and function. In the absence of β-catenin, γ-catenin functionally substituted for it in E-cadherin binding, thereby sustaining the integrity of epithelial adherens junctions in the kidneys. In AKI induced by ischemia reperfusion or folic acid, the loss of tubular β-catenin substantially aggravated renal lesions. Compared with controls, Ksp-β-cat-/- mice displayed higher mortality, elevated serum creatinine, and more severe morphologic injury. Consistently, apoptosis was more prevalent in kidneys of the knockout mice, which was accompanied by increased expression of p53 and Bax, and decreased phosphorylated Akt and survivin. In vitro activation of β-catenin by Wnt1 or stabilization of β-catenin protected tubular epithelial cells from apoptosis, activated Akt, induced survivin, and repressed p53 and Bax expression. Hence, endogenous β-catenin is pivotal for renal tubular protection after AKI by promoting cell survival through multiple mechanisms.

Hedgehog (Hh) is a core signaling pathway implicated in fundamental processes during embryonic kidney development. We previously found that loss-of-function mutations in the transcription factor GLIS2, a putative vertebrate ortholog of Drosophila Ci, cause nephronophthisis type 7 in humans and mice. Kidney tubular cells in Glis2-knockout mice acquire mesenchymal phenotype, but the cellular mechanisms of this transition are unknown. Here, we demonstrate that Glis2 is a functional component of Hh signaling and is necessary to suppress this pathway in the postnatal kidney. In the epithelial compartment, Glis2 opposes Gli1 activity by binding cis-acting regulatory sequences in the 5′ flanking regions of Snai1 and Wnt4, thereby inhibiting de-differentiation of tubular cells. We conclude that Glis2 is necessary to inhibit Hh signaling and to maintain the mature tubular epithelial phenotype in the adult kidney. This is the first description of a molecular mechanism that links the Hh signaling pathway to cystic kidney diseases and can open new avenues for the treatment of diverse ciliopathies.

Hepatocyte nuclear factor-1beta (HNF-1beta) is a transcription factor required for the expression of several renal cystic genes and whose prenatal deletion leads to polycystic kidney disease (PKD). We show here that inactivation of Hnf1b from postnatal day 10 onward does not elicit cystic dilations in tubules after their proliferative morphogenetic elongation is over. Cystogenic resistance is intrinsically linked to the quiescent state of cells. In fact, when Hnf1b deficient quiescent cells are forced to proliferate by an ischemia-reperfusion injury, they give rise to cysts, owing to loss of oriented cell division. Remarkably, in quiescent cells, the transcription of crucial cystogenic target genes is maintained even in the absence of HNF-1beta. However, their expression is lost as soon as cells proliferate and the chromatin of target genes acquires heterochromatin marks. These results unveil a previously undescribed aspect of gene regulation. It is well established that transcription is shut off during the mitotic condensation of chromatin. We propose that transcription factors such as HNF-1beta might be involved in reprogramming gene expression after transcriptional silencing is induced by mitotic chromatin condensation. Notably, HNF-1beta remains associated with the mitotically condensed chromosomal barrels. This association suggests that HNF-1beta is a bookmarking factor that is necessary for reopening the chromatin of target genes after mitotic silencing.

NH3movement across plasma membranes has traditionally been ascribed to passive, lipid-phase diffusion. However, ammonia-specific transporters, Mep/Amt proteins, are present in primitive organisms and mammals express orthologs of Mep/Amt proteins, the Rh glycoproteins. These findings suggest that the mechanisms of NH3movement in mammalian tissues should be reexamined. Rh C glycoprotein (Rhcg) is expressed in the collecting duct, where NH3secretion is necessary for both basal and acidosis-stimulated ammonia transport. To determine whether the collecting duct secretes NH3via Rhcg or via lipid-phase diffusion, we generated mice with collecting duct-specific Rhcg deletion (CD-KO). CD-KO mice had loxP sites flanking exons 5 and 9 of the Rhcg gene (Rhcgfl/fl) and expressed Cre-recombinase under control of the Ksp-cadherin promoter (Ksp-Cre). Control (C) mice were Rhcgfl/flbut Ksp-Cre negative. We confirmed kidney-specific genomic recombination using PCR analysis and collecting duct-specific Rhcg deletion using immunohistochemistry. Under basal conditions, urinary ammonia excretion was less in KO vs. C mice; urine pH was unchanged. After acid-loading for 7 days, CD-KO mice developed more severe metabolic acidosis than did C mice. Urinary ammonia excretion did not increase significantly on the first day of acidosis in CD-KO mice, despite an intact ability to increase urine acidification, whereas it increased significantly in C mice. On subsequent days, urinary ammonia excretion slowly increased in CD-KO mice, but was always significantly less than in C mice. We conclude that collecting duct Rhcg expression contributes to both basal and acidosis-stimulated renal ammonia excretion, indicating that collecting duct ammonia secretion is, at least in part, mediated by Rhcg and not solely by lipid diffusion.

Ammonia metabolism and transport are critical for acid-base homeostasis. The ammonia transporter family member Rh C glycoprotein (Rhcg) is expressed in distal renal tubular segments, and its expression is regulated in parallel with renal ammonia metabolism. However, there are inconsistencies in its reported subcellular distribution, with both apical and basolateral Rhcg reported in rat and human kidney and only apical expression in mouse kidney. Because the membrane location of Rhcg is critical for understanding its physiological role, we reassessed mouse Rhcg localization using refined immunolocalization methods. Two antibodies directed against different Rhcg-specific epitopes identified both apical and basolateral Rhcg immunolabel in mouse kidney. Immunogold electron microscopy both confirmed basolateral plasma membrane Rhcg expression and showed that apical immunolabel represented expression in both the apical plasma membrane and in subapical cytoplasmic vesicles. Immunoblots and Northern blots identified similar bands in Balb/c and C57BL/6 kidneys, suggesting basolateral Rhcg may result from alternative trafficking. Basolateral Rhcg intensity was strain dependent, with less basolateral Rhcg expression in the Balb/c mouse compared with the C57BL/6 mouse. In mice with collecting duct-specific Rhcg gene deletion, generated using Cre-loxP techniques, neither apical nor basolateral Rhcg immunolabel was identified in the collecting duct, confirming that basolateral Rhcg was the product of the same gene product as apical Rhcg. Although basolateral Rhcg expression differed between C57BL/6 and Balb/c mice, Rh B glycoprotein, which is exclusively basolateral, was expressed at similar levels in the two strains. We conclude that Rhcg is present in both the apical and basolateral plasma membrane in the mouse kidney, where it is likely to contribute to renal ammonia metabolism.

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.

Hepatocyte nuclear factor-1β (HNF-1β) is a Pit-1, Oct-1/2, Unc-86 (POU) homeodomain-containing transcription factor expressed in the kidney, liver, pancreas, and other epithelial organs. Mutations of HNF-1β cause maturity-onset diabetes of the young, type 5 (MODY5), which is characterized by early-onset diabetes mellitus and congenital malformations of the kidney, pancreas, and genital tract. Knockout of HNF-1β in the mouse kidney results in cyst formation. However, the signaling pathways and transcriptional programs controlled by HNF-1β are poorly understood. Using genome-wide chromatin immunoprecipitation and DNA microarray (ChIP-chip) and microarray analysis of mRNA expression, we identified SOCS3 (suppressor of cytokine signaling-3) as a previously unrecognized target gene of HNF-1β in the kidney. HNF-1β binds to the SOCS3 promoter and represses SOCS3 transcription. The expression of SOCS3 is increased in HNF-1β knockout mice and in renal epithelial cells expressing dominant-negative mutant HNF-1β. Increased levels of SOCS-3 inhibit HGF-induced tubulogenesis by decreasing phosphorylation of Erk and STAT-3. Conversely, knockdown of SOCS-3 in renal epithelial cells expressing dominant-negative mutant HNF-1β rescues the defect in HGF-induced tubulogenesis by restoring phosphorylation of Erk and STAT-3. Thus, HNF-1β regulates tubulogenesis by controlling the levels of SOCS-3 expression. Manipulating the levels of SOCS-3 may be a useful therapeutic approach for human diseases induced by HNF-1β mutations.

HNF-1β is a tissue-specific transcription factor that is expressed in the kidney and other epithelial organs. Humans with mutations in HNF-1β develop kidney cysts, and HNF-1β regulates the transcription of several cystic disease genes. However, the complete spectrum of HNF-1β-regulated genes and pathways is not known. Here, using chromatin immunoprecipitation/next generation sequencing and gene expression profiling, we identified 1545 protein-coding genes that are directly regulated by HNF-1β in murine kidney epithelial cells. Pathway analysis predicted that HNF-1β regulates cholesterol metabolism. Expression of dominant negative mutant HNF-1β or kidney-specific inactivation of HNF-1β decreased the expression of genes that are essential for cholesterol synthesis, including sterol regulatory element binding factor 2 (Srebf2) and 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr). HNF-1β mutant cells also expressed lower levels of cholesterol biosynthetic intermediates and had a lower rate of cholesterol synthesis than control cells. Additionally, depletion of cholesterol in the culture medium mitigated the inhibitory effects of mutant HNF-1β on the proteins encoded by Srebf2 and Hmgcr, and HNF-1β directly controlled the renal epithelial expression of proprotein convertase subtilisin-like kexin type 9, a key regulator of cholesterol uptake. These findings reveal a novel role of HNF-1β in a transcriptional network that regulates intrarenal cholesterol metabolism.

The transcription factor hepatocyte nuclear factor-1β (HNF-1β) regulates tissue-specific gene expression in the kidney and other epithelial organs. Mutations of HNF-1β produce kidney cysts, and previous studies have shown that HNF-1β 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-1β 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-1β. HNF-1β binds to two evolutionarily conserved sites located 28 kb upstream to miR-200b. Luciferase reporter assays showed that the HNF-1β binding sites were located within a promoter that was active in renal epithelial cells. Mutations of the HNF-1β 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-1β knock-out mice and renal epithelial cells expressing dominant-negative mutant HNF-1β. The expression of miR-200 targets, Zeb2 and Pkd1, was increased in HNF-1β knock-out kidneys and in cells expressing mutant HNF-1β. Overexpression of miR-200 decreased the expression of Zeb2 and Pkd1 in HNF-1β mutant cells. These studies reveal a novel pathway whereby HNF-1β directly contributes to the control of miRNAs that are involved in epithelial-mesenchymal transition and cystic kidney disease.

Fibrocystin, a type I membrane protein of unknown function, is the protein affected in the autosomal recessive form of polycystic kidney disease. Here we show that fibrocystin undergoes regulated proteolysis. Several proteolytic cleavages occur within the predicted ectodomain, whereas at least one cleavage occurs within the cytoplasmic portion. The latter generates a C-terminal intracellular fragment that harbors the nuclear localization signal KRKVSRLAVTGERTATPAPKIPRIT and translocates to the nucleus. Proteolytic cleavage of fibrocystin occurs constitutively in long term cultures of polarized inner medullary collecting duct cells (mIMCD-3). Activation of protein kinase C and release of intracellular Ca2+ are required for proteolysis under these conditions. In short term cultures of human embryonic kidney 293 cells (HEK-293), proteolytic cleavage of fibrocystin can be elicited by stimulation of intracellular Ca2+ release or activation of protein kinase C. These results identify a novel Ca2+-dependent pathway that signals from fibrocystin located in the cell membrane to the nucleus.

We examined the expression of Slc12a2 (NKCC1) transcripts in the developing mouse by Northern blot analysis and in situ hybridization (ISH) using riboprobes transcribed from a cDNA encoding the transmembrane domain of human Slc12a2. In developing kidney, the 7.5-kb Slc12a2 transcript was expressed at all stages examined (13.5 d.p.c. to adult) but was more abundant in immature metanephroi. ISH revealed that NKCC1 was expressed in both mesenchymal cells and early nephric structures, but not branching ureteric buds, of developing metanephroi. A marked increase in expression was observed in the endocapillary cells of capillary loop stage glomeruli, and high expression was observed in the glomeruli of more mature nephrons. This was in contrast to Slc12a1 (NKCC2), where expression was excluded from the glomerulus. Extra-renal expression of Slc12a2 was examined in 13.5, 15.5, and 16.5 d.p.c. mouse embryos. Slc12a2 was highly expressed in the developing lung, gut, submandibular gland, tooth bud, and nasal epithelium. Slc12a2 expression was also observed in the developing central and peripheral nervous systems, including choroid plexus and trigeminal and dorsal root ganglia.

Ischemic injury to the kidney produces acute tubular necrosis and apoptosis followed by tubular regeneration and recovery of renal function. Although mitotic cells are present in the tubules of postischemic kidneys, the origins of the proliferating cells are not known. Bone marrow cells (BMCs) can differentiate across lineages to repair injured organs, including the kidney. However, the relative contribution of intrarenal cells and extrarenal cells to kidney regeneration is not clear. We produced transgenic mice that expressed enhanced GFP (EGFP) specifically and permanently in mature renal tubular epithelial cells. Following ischemia/reperfusion injury (IRI), EGFP-positive cells showed incorporation of BrdU and expression of vimentin, which provides direct evidence that the cells composing regenerating tubules are derived from renal tubular epithelial cells. In BMC-transplanted mice, 89% of proliferating epithelial cells originated from host cells, and 11% originated from donor BMCs. Twenty-eight days after IRI, the kidneys contained 8% donor-derived cells, of which 8.4% were epithelial cells, 10.6% were glomerular cells, and 81% were interstitial cells. No renal functional improvement was observed in mice that were transplanted with exogenous BMCs. These results show that intrarenal cells are the main source of renal repair, and a single injection of BMCs does not make a significant contribution to renal functional or structural recovery.

Alterations in Wnt/beta-catenin signaling have been linked to abnormal kidney development and tumorigenesis. To gain more insights into the effects of these alterations, we created mice carrying a conditional deletion of the Apc tumor suppressor gene specifically in the renal epithelium. As expected, the loss of Apc leads to increased levels of beta-catenin protein in renal epithelium. Most of these mice die shortly after birth, and multiple kidney cysts were found upon histological examination. Only rarely did these animals survive to adulthood. Analysis of these adults revealed severely cystic kidneys associated with the presence of renal adenomas. Our results confirm an important role for proper regulation of Wnt/beta-catenin signaling in renal development and provide evidence that dysregulation of the pathway can initiate tumorigenesis in the kidney.

The transcription factor NFAT5/TonEBP, a member of the NFAT/Rel family of transcription factors, has been implicated in diverse cellular responses, including the response to osmotic stress, integrin-dependent cell migration, T cell activation, and the Ras pathway in Drosophila . To clarify the in vivo role of NFAT5, we generated NFAT5-null mice. Homozygous mutants were genetically underrepresented after embryonic day 14.5. Surviving mice manifested a progressive and profound atrophy of the kidney medulla with impaired activation of several osmoprotective genes, including those encoding aldose reductase, Na + /Cl – -coupled betaine/γ-aminobutyric acid transporter, and the Na + /myo-inositol cotransporter. The aldose reductase gene is controlled by a tonicity-responsive enhancer, which was refractory to hypertonic stress in fibroblasts lacking NFAT5, establishing this enhancer as a direct transcriptional target of NFAT5. Our findings demonstrate a central role for NFAT5 as a tonicity-responsive transcription factor required for kidney homeostasis and function.

Kidney-specific cadherin (Ksp-cadherin) is a tissue-specific member of the cadherin family that is expressed exclusively in the kidney and developing genitourinary tract. Recent studies have shown that the proximal 250 bp of the Ksp-cadherin gene promoter are sufficient to direct tissue-specific gene expression in vivo and in vitro. The proximal 120 bp of the promoter are evolutionarily conserved between mouse and human and contain a DNase I hypersensitive site that is kidney cell specific. At position -55, the promoter contains a consensus recognition site for hepatocyte nuclear factor-1 (HNF-1). Mutations of the consensus HNF-1 site and downstream GC-boxes inhibit promoter activity in transfected cells. HNF-1alpha and HNF-1beta bind specifically to the -55 site, and both proteins transactivate the promoter directly. Expression of Ksp-cadherin is not altered in the kidneys of HNF-1alpha-deficient mice. However, expression of a gain-of-function HNF-1beta mutant stimulates Ksp-cadherin promoter activity in transfected cells, whereas expression of a dominant-negative mutant inhibits activity. These studies identify Ksp-cadherin as the first kidney-specific promoter that has been shown to be regulated by HNF-1beta. Mutations of HNF-1beta, as occur in humans with inherited renal cysts and diabetes, may cause dysregulated Ksp-cadherin promoter activity.

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)-1β, which is required for activity in transfected cells. Mutation of the HNF-1β-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-1β. 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-1β 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.

Autosomal recessive polycystic kidney disease (ARPKD) results from mutations in the human PKHD1 gene. Both this gene, and its mouse ortholog, Pkhd1, are primarily expressed in renal and biliary ductal structures. The mouse protein product, fibrocystin/polyductin complex (FPC), is a 445-kDa protein encoded by a 67-exon transcript that spans >500 kb of genomic DNA. In the current study, we observed multiple alternatively spliced Pkhd1 transcripts that varied in size and exon composition in embryonic mouse kidney, liver, and placenta samples, as well as among adult mouse pancreas, brain, heart, lung, testes, liver, and kidney. Using reverse transcription PCR and RNASeq, we identified 22 novel Pkhd1 kidney transcripts with unique exon junctions. Various mechanisms of alternative splicing were observed, including exon skipping, use of alternate acceptor/donor splice sites, and inclusion of novel exons. Bioinformatic analyses identified, and exon-trapping minigene experiments validated, consensus binding sites for serine/arginine-rich proteins that modulate alternative splicing. Using site-directed mutagenesis, we examined the functional importance of selected splice enhancers. In addition, we demonstrated that many of the novel transcripts were polysome bound, thus likely translated. Finally, we determined that the human PKHD1 R760H missense variant alters a splice enhancer motif that disrupts exon splicing in vitro and is predicted to truncate the protein. Taken together, these data provide evidence of the complex transcriptional regulation of Pkhd1/PKHD1 and identified motifs that regulate its splicing. Our studies indicate that Pkhd1/PKHD1 transcription is modulated, in part by intragenic factors, suggesting that aberrant PKHD1 splicing represents an unappreciated pathogenic mechanism in ARPKD. Key messages: Multiple mRNA transcripts are generated for Pkhd1 in renal tissues Pkhd1 transcription is modulated by standard splice elements and effectors Mutations in splice motifs may alter splicing to generate nonfunctional peptides.

Diethylstilbestrol (DES) was widely used to treat pregnant women through 1971. The reproductive tracts of their female offspring exposed to DES in utero are characterized by anatomic abnormalities. Here we show that DES administered to mice in utero produces changes in the expression pattern of several Hox genes that are involved in patterning of the reproductive tract. DES produces posterior shifts in Hox gene expression and homeotic anterior transformations of the reproductive tract. In human uterine or cervical cell cultures, DES induces HOXA9 or HOXA10 gene expression, respectively, to levels approximately twofold that induced by estradiol. The DES-induced expression is not inhibited by cyclohexamide. Estrogens are novel morphogens that directly regulate the expression pattern of posterior Hox genes in a manner analogous to retinoic acid regulation of anterior Hox genes. Alterations in HOX gene expression are a molecular mechanism by which DES affects reproductive tract development. Changes in Hox gene expression are a potential marker for the effects of in utero drug use that may become apparent only at late stages of development.

Epithelial-mesenchymal interactions are required for the development of all solid organs but few molecular mechanisms that underlie these interactions have been identified. Pod1 is a basic-helix-loop-helix (bHLH) transcription factor that is highly expressed in the mesenchyme of developing organs that include the lung, kidney, gut and heart and in glomerular visceral epithelial cells (podocytes). To determine the function of Pod1 in vivo, we have generated a lacZ-expressing null Pod1 allele. Null mutant mice are born but die in the perinatal period with severely hypoplastic lungs and kidneys that lack alveoli and mature glomeruli. Although Pod1 is exclusively expressed in the mesenchyme and podocytes, major defects are observed in the adjacent epithelia and include abnormalities in epithelial differentiation and branching morphogenesis. Pod1 therefore appears to be essential for regulating properties of the mesenchyme that are critically important for lung and kidney morphogenesis. Defects specific to later specialized cell types where Pod1 is expressed, such as the podocytes, were also observed, suggesting that this transcription factor may play multiple roles in kidney morphogenesis.

Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a novel, kidney-specific member of the cadherin superfamily that is expressed exclusively in the basolateral membrane of renal tubular epithelial cells. To characterize the Ksp-cadherin gene promoter, a λ bacteriophage clone containing 3.7 kb of the proximal 5′ flanking region of the mouse Ksp-cadherin gene was isolated. The transcription initiation site was mapped by RNase protection assays and 5′ rapid amplification of cDNA ends, and a 709-bp intron was identified within the 5′ untranslated region. The proximal 5′ flanking region was “TATA-less” but contained other consensus promoter elements including an initiator (Inr), GC boxes, and a CAAT box. Potential binding sites were identified for transcription factors that are involved in tissue-specific gene expression including activator protein-2 (AP-2), hepatocyte nuclear factor-3 (HNF-3), basic helix-loop-helix (bHLH) proteins, CCAAT/enhancer-binding protein (C/EBP), and GATA factors. Transfection of luciferase reporter plasmids containing 2.6 kb of the 5′ flanking region markedly increased luciferase activity in renal epithelial cells (MDCK and mIMCD-3) but not in mesenchymal cells (NIH 3T3 and MMR1). Deletion analysis identified an 82-bp region from −31 to −113 that was essential for promoter activity in transfected renal epithelial cells. Electrophoretic mobility-shift assays showed that mIMCD-3 cells contain nuclear proteins that bind to this region of the promoter. Mutational analysis showed that sequences within the HNF-3 consensus site and CAAT box were involved in protein binding and promoter activity. We conclude that the proximal 5′ flanking region of the mouse Ksp-cadherin gene contains an orientation-dependent promoter that is kidney epithelial cell specific. The region of the promoter from −113 to −31 is required for transcriptional activity and contains binding sites for nuclear proteins that are specifically expressed in renal epithelial cells.

Under the influence of sex steroids, human endometrium undergoes sequential development in preparation for implantation. Hoxa11 is essential for implantation in the mouse. Here we describe a potential role for HOXA11 in human endometrial development and implantation. Northern analysis demonstrates that HOXA11 is expressed in a menstrual cycle phase-dependent fashion in adult human endometrium. HOXA11 messenger RNA levels dramatically increase at the time of implantation and remain increased in pregnancy. In vitro, HOXA11 expression is increased in response to estrogen or progesterone. There is a dose-responsive increase over the physiologic range of progesterone concentration. Pretreatment with Cyclohexamide does not decrease the response to estrogen. Steroids are novel regulators HOX gene expression. The spatial and temporal pattern of HOXA11 expression in the human endometrium suggests a role in endometrial development, implantation, and maintenance of pregnancy.

Familial amyloidotic polyneuropathy (FAP) is a hereditary generalized amyloidosis that results from mutations in the transthyretin (TTR) gene. More then 100 mutations of TTR have been described. Corresponding to the wide variety of TTR mutations, FAP presents with diverse clinical phenotypes. TTR-Phe64Ser is a rare mutation that has previously only been described once in a Canadian family that presented with oculoleptomeningeal symptoms. We report the clinical and molecular characterization of the first described case of a TTR-Phe64Ser mutation in an African-American family with profound gastrointestinal symptoms.

Hox genes are well-known transcriptional regulators that play an essential role in directing embryonic development. Mice that are homozygous for a targeted disruption of the Hoxa10 gene exhibit uterine factor infertility. We have recently demonstrated that HOXA10 is expressed in the adult human uterus. To examine expression of HOXA10 during the menstrual cycle, Northern blot analysis and in situ hybridization were performed. Expression of HOXA10 dramatically increased during the midsecretory phase of the menstrual cycle, corresponding to the time of implantation and increase in circulating progesterone. Expression of HOXA10 in cultured endometrial cells was stimulated by estrogen or progesterone. Stimulation of HOXA10 by progesterone was concentration-dependent within the physiologic range, and the effect of estrogen was inhibited by cycloheximide. These results identify sex steroids as novel regulators of HOX gene expression. HOXA10 may have an important function in regulating endometrial development during the menstrual cycle and in establishing conditions necessary for implantation in the human.

Basic-helix-loop-helix (bHLH) proteins are transcriptional regulatory proteins that govern cell fate determination and differentiation in a variety of tissues. We have identified a novel bHLH protein, named Pod-1, that belongs to a recently-described subfamily of bHLH proteins that have essential roles in the embryonic development of mesodermal tissues. In the adult human and mouse, Pod-1 was most highly expressed in the kidney, lung and heart. In developing mouse embryos, Pod-1 was selectively expressed in mesenchymal cells at sites of epithelial-mesenchymal interaction in the kidney, lung, intestine and pancreas. Pod-1 was also expressed in visceral glomerular epithelial cells (podocytes) in the kidney, and its expression coincided with the onset of podocyte differentiation. The expression of Pod-1 in embryonic kidney explants was inhibited using antisense oligonucleotides. Inhibition of Pod-1 expression resulted in decreased mesenchymal cell condensation around the ureteric bud and a 40% decrease in ureteric branching. Pod-1 is the first tissue-restricted basic-helix-loop-helix protein that has been identified in the developing kidney where it may play a role in the regulation of morphogenetic events.

All animals must excrete the waste products of metabolism and maintain a constant body composition despite changes in the external environment. In higher animals, these functions are performed by specialized excretory organs. The excretory organs range from a single excretory cell in the nematode

Mammalian homologues of the Drosophila cut homeobox gene encode transcriptional repressors that are involved in tissue-specific and developmental gene regulation. We examined the expression of a murine cut homologue (Cux-1) in the adult mouse. In many somatic tissues, Cux-1 was expressed as a 13-kb transcript. However, the highest expression of Cux-1 was in the testis, where a unique 2.4-kb splice variant was identified. Less abundant transcripts of 5 kb, 6.5 kb, and 8.5 kb were also detected only in the testis. The nucleotide sequence of the 2.4-kb Cux-1 transcript was identical to the 13-kb transcript in the region of overlap, but the testis-specific transcript encoded a truncated protein that contained only one Cut repeat in addition to the Cut-related homeodomain. Studies of mice homozygous for the atrichosis (at/at) mutation suggested that the 2.4-kb transcript was expressed in germ cells in the testis. In situ hybridization verified that Cux-1 was transiently expressed in post-meiotic germ cells at the round spermatid stage. Immunoblot analysis of nuclear extracts showed that the testis-specific Cux-1 transcripts encoded a 55-kDa protein. These results demonstrate that multiple products of a cut-related homeobox gene are expressed in the testis. The highly restricted pattern of expression of Cux-1 in the testis suggests that it may be involved in regulation of postmeiotic gene expression.

The cut locus of Drosophila encodes a diverged homeodomain-containing protein that is required for the development of external sensory (es) organs and other tissues. A homologous gene (Cux-1) that encodes a transcriptional repressor has been identified in the mouse and other mammals. We have identified a second murine homeobox-containing gene (designated Cux-2) that is structurally related to Drosophila cut. The murine Cux-2 homeobox was similar to Drosophila cut and encoded a homeodomain that contained a characteristic histidine residue at position 50. The predicted Cux-2 protein contained 1426 amino acids and included three internal 60-amino acid repeats (Cut repeats) that were previously found in Drosophila Cut and murine Cux-1. Unlike Cux-1, expression of Cux-2 was restricted to neural tissue. In the adult brain, Cux-2 was prominently expressed in neurons in the thalamus and limbic system. In embryos, Cux-2 was expressed in the developing central and peripheral nervous systems, including the telencephalon and peripheral ganglia of the trigeminal and glossopharyngeal nerves. A glutathione S-transferase fusion protein containing the carboxyl-terminal Cut repeat and homeodomain of Cux-2 exhibited sequence-specific binding to oligonucleotides derived from the promoter of the Ncam gene. Using an interspecific backcross panel, Cux-1 and Cux-2 were mapped to distinct loci that were genetically linked on distal chromosome 5. These results demonstrate that a family of homeobox genes related to Drosophila cut is located on chromosome 5 in the mouse. Cux-2 is expressed exclusively in the central and peripheral nervous systems, and the Cux-2 gene product binds to DNA in a sequence-specific manner. Cux-2 may encode a transcription factor that is involved in neural specification in mammals.

The murine Nkcc2/Slcl2a1 gene encodes a bumetanide-sensitive Na-K-Cl cotransporter that is expressed exclusively in the kidney in the thick ascending limb of the loop of Henle. Nuclear run-off assays demonstrated that kidney-specific expression of Nkcc2 was due, at least in part, to kidney-specific gene transcription. To begin study of the gene promoter, a genomic clone that contained 13.5 kilobases of the 5'-flanking region of Nkcc2 was isolated. A single transcription initiation site was located 1330 base pairs (bp) upstream of the start codon. The sequence of the proximal 5'-flanking region contained typical eukaryotic promoter elements including a TATA box, two CCAAT boxes, and an initiator. A (G-A)28.(C-T)28 microsatellite and consensus binding sites for hepatocyte nuclear factor 1, cAMP-response element binding protein, CCAAT/enhancer-binding proteins, and basic helix-loop-helix proteins, were also identified. To functionally express the promoter, 2255 bp of the proximal 5'-flanking region was ligated to a luciferase reporter gene and transfected into thick ascending limb (TAL) cells, a stable cell line derived from microdissected loops of Henle of the Tg(SV40E)Bri7 mouse. TAL cells exhibited furosemide-sensitive Na-K((NH4)+)-Cl cotransport activity and endogenously expressed the 5.0-kilobase Nkcc2 transcript. Luciferase activity was 130-fold greater following transfection into TAL cells compared with transfection into cells that did not express Nkcc2 (NIH 3T3 fibroblasts). Deletion analysis revealed that promoter activity in TAL cells was similar in constructs extending from the transcription initiation site to -1529 to -469, whereas further deletion to -190 resulted in a 76% decrease in activity. We conclude that the Nkcc2 promoter exhibits kidney cell-specific activity. Regulatory elements required for maximal promoter activity are located in a 280-bp DNA segment that contains consensus binding sites for several transcription factors expressed in the kidney.

Gene targeting in mice is a powerful tool for identifying the in vivo functions of proteins and for producing new animal models of human diseases. In the conventional approach, the gene encoding a protein of interest is disrupted by homologous recombination in embryonic stem (ES) cells. Targeted ES

Cadherins are recognized as the principal mediators of homotypic cellular recognition and play a demonstrated role in the morphogenic direction of tissue development. We report here the identification of a structurally unique, kidney-specific member of the cadherin multigene family (Ksp-cadherin). cDNA cloning and molecular analysis of the 130-kDa protein confirmed that it was novel and indicated that it most closely resembled members of the LI-cadherin/HPT-1 cadherin subgroup. The predicted protein possesses the definitive cadherin-specific sequence motifs LDRE, DXND, and DXD in well conserved sequential arrangement, and the characteristic cysteine residues found in the last ectodomains of almost all known cadherins. Like LI-cadherin and HPT-1, Ksp-cadherin lacks the prosequence and HAV adhesion recognition sequence typical of most classical cadherins, and possesses a truncated cytoplasmic domain (18-22 amino acids). When expressed in a transient Vaccinia/T7 expression system, Ksp-cadherin displayed the classic calcium sensitivity to trypsin proteolysis that is observed in all cadherins. Immunolocalization studies and Northern analysis indicated that expression of Ksp-cadherin was kidney-specific and limited to the basolateral membranes of renal tubular epithelial cells. In summary, we have identified and cloned a novel, kidney-specific member of the cadherin multigene family that we propose be designated Ksp-cadherin.

Stem cells are generally defined as clonogenic cells that are capable of both self-renewal and multilineage differentiation ([1–3][1][⇓][2][⇓][3]). The fertilized zygote has the highest developmental potential and gives rise to the cells of the entire organism. More than 20 years ago

MicroRNAs (miRNAs) contribute to the regulation of early kidney development, but their role during later stages of renal tubule maturation is not well understood. Here, we found that ablation of the miRNA-processing enzyme Dicer from maturing renal tubules produces tubular and glomerular cysts in mice. Inactivation of Dicer is associated with downregulation of miR-200, a kidney-enriched miRNA family, and upregulation of the polycystic kidney disease gene Pkd1. Inhibition of miR-200 in cultured renal epithelial cells disrupted tubulogenesis and led to upregulation of Pkd1. Using bioinformatic and in vitro approaches, we found that miR-200b/c/429 induce post-transcriptional repression of Pkd1 through two conserved binding sites in the 3'-Untranslated regions of Pkd1. Overexpression of PKD1 in renal epithelial cells was sufficient to disrupt tubulogenesis and produce cyst-like structures. In conclusion, miRNAs are essential for the maturation of renal tubules, and Pkd1 is a target of miR-200. These results also suggest that miRNAs may modulate PKD1 gene dosage and play a role in the initiation of cystogenesis.

Kidney cysts occur following inactivation of polycystins in otherwise intact cilia or following complete removal of cilia by inactivation of intraflagellar transport-related proteins. We investigated the mechanisms of cyst formation in these two distinct processes by combining conditional inactivation of polycystins with concomitant ablation of cilia in developing and adult kidney and liver. We found that loss of intact cilia suppresses cyst growth following inactivation of polycystins and that the severity of cystic disease was directly related to the length of time between the initial loss of the polycystin proteins and the subsequent involution of cilia. This cilia-dependent cyst growth was not explained by activation of the MAPK/ERK, mTOR or cAMP pathways and is likely to be distinct from the mechanism of cyst growth following complete loss of cilia. The data establish the existence of a novel pathway defined by polycystin-dependent inhibition and cilia-dependent activation that promotes rapid cyst growth.

The study of kidney development at the cellular and molecular levels remains an active area of nephrologic research. This review highlights recent advances in two specific areas: transcriptional control of nephrogenesis and the role of cell death in normal kidney development. The mesenchymal-epithelial conversion that occurs during kidney development requires alterations in gene expression. Some transcription factors involved in early steps in nephrogenesis have recently been identified from the effects of gene "knockout" or dysregulated expression in transgenic mice. These include the product of the Wilms' tumor suppressor gene (WT-1), a mammalian paired homologue (Pax-2), and the N-myc oncoprotein. Other proteins, including Pax-8, hepatocyte nuclear factor-1, hepatocyte nuclear factor-4, Kid-1, and formins, exhibit spatiotemporally restricted patterns of expression, homology to products of developmental control genes in Drosophila, or mutant phenotypes consistent with possible roles in nephrogenesis. During the past year, another important observation was that apoptosis (programmed cell death) occurs during normal kidney development. Studies of knockout mice suggest that Bcl-2, which protects against death in many contexts, is also involved in kidney development.

Although the cause of prune belly syndrome is unknown, familial evidence suggests a genetic component. Recently 2 nonfamilial cases of prune belly syndrome with chromosome 17q12 deletions encompassing the HNF1β gene have made this a candidate gene for prune belly syndrome. To date, there has been no large-scale screening of patients with prune belly syndrome for HNF1β mutations. We assessed the role of HNF1β in prune belly syndrome by screening for genomic mutations with functional characterization of any detected mutations.We studied patients with prune belly syndrome who were prospectively enrolled in our Pediatric Genitourinary DNA Repository since 2001. DNA from patient samples was amplified by polymerase chain reaction, sequenced for coding and splice regions of the HNF1β gene, and compared to control databases. We performed functional assay testing of the ability of mutant HNF1β to activate a luciferase construct with an HNF1β DNA binding site.From 32 prune belly syndrome probands (30 males, 2 females) HNF1β sequencing detected a missense mutation (V61G) in 1 child with prune belly syndrome. Absent in control databases, V61G was previously reported in 2 patients without prune belly syndrome who had congenital genitourinary anomalies. Functional testing showed similar luciferase activity compared to wild-type HNF1β, suggesting the V61G substitution does not disturb HNF1β function.One genomic HNF1β mutation was detected in 3% of patients with prune belly syndrome but found to be functionally normal. Thus, functionally significant HNF1β mutations are uncommon in prune belly syndrome, despite case reports of HNF1β deletions. Further genetic study is necessary, as identification of the genetic basis of prune belly syndrome may ultimately lead to prevention and improved treatments for this rare but severe syndrome.

Polycystic kidney disease (PKD) is a genetic disorder that is characterized by cyst formation in kidney tubules. PKD arises from abnormalities of the primary cilium, a sensory organelle located on the cell surface. Here, we show that the primary cilium of renal epithelial cells contains a protein complex comprising adenylyl cyclase 5/6 (AC5/6), A-kinase anchoring protein 150 (AKAP150), and protein kinase A. Loss of primary cilia caused by deletion of Kif3a results in activation of AC5 and increased cAMP levels. Polycystin-2 (PC2), a ciliary calcium channel that is mutated in human PKD, interacts with AC5/6 through its C terminus. Deletion of PC2 increases cAMP levels, which can be corrected by reexpression of wild-type PC2 but not by a mutant lacking calcium channel activity. Phosphodiesterase 4C (PDE4C), which catabolizes cAMP, is also located in renal primary cilia and interacts with the AKAP150 complex. Expression of PDE4C is regulated by the transcription factor hepatocyte nuclear factor-1β (HNF-1β), mutations of which produce kidney cysts. PDE4C is down-regulated and cAMP levels are increased in HNF-1β mutant kidney cells and mice. Collectively, these findings identify PC2 and PDE4C as unique components of an AKAP complex in primary cilia and reveal a common mechanism for dysregulation of cAMP signaling in cystic kidney diseases arising from different gene mutations.

The mTOR pathway is the central regulator of cell size1. External signals from growth factors and nutrients converge on the mTORC1 multi-protein complex to modulate downstream targets, but how the different inputs are integrated and translated into specific cellular responses is incompletely understood2–4. Deregulation of the mTOR pathway occurs in polycystic kidney disease (PKD)5–7, where cilia (filiform sensory organelles) fail to sense urine flow because of inherited mutations in ciliary proteins8. We therefore investigated if cilia have a role in mTOR regulation. Here, we show that ablation of cilia in transgenic mice results in enlarged cells when compared with control animals. In vitro analysis demonstrated that bending of the cilia by flow is required for mTOR downregulation and cell-size control. Surprisingly, regulation of cell size by cilia is independent of flow-induced calcium transients, or Akt. However, the tumour-suppressor protein Lkb1 localises in the cilium, and flow results in increased AMPK phosphorylation at the basal body. Conversely, knockdown of Lkb1 prevents normal cell-size regulation under flow conditions. Our results demonstrate that the cilium regulates mTOR signalling and cell size, and identify the cilium-basal body compartment as a spatially restricted activation site for Lkb1 signalling.