Role of Transcription Factor Hepatocyte Nuclear Factor-1β in Polycystic Kidney Disease

Abstract

Hepatocyte nuclear factor-1β (HNF-1β) 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-1β 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-1β in regulating the expression of noncoding RNAs (microRNAs and long noncoding RNAs) has been identified. These findings indicate that HNF-1β regulates a transcriptional and post-transcriptional network that plays a central role in renal cystogenesis.

1. Introduction

1.1. Structure and function of HNF-1β

Hepatocyte nuclear factor-1β (HNF-1β) is a DNA-binding transcription factor that regulates tissue-specific gene expression in epithelial organs including the kidney.¹ In the kidney, HNF-1β is expressed in all tubular epithelial cells composing the nephrons and collecting ducts where it controls the expression of genes involved in membrane transport, cell differentiation, and metabolism.^{2; 3} Human HNF-1β contains 557 amino acids⁴ organized into an amino-terminal dimerization domain, a bipartite DNA-binding domain, and a carboxy-terminal transactivation domain ^{5; 6} (Figure 1). HNF-1β binds DNA as a homodimer or as a heterodimer with its paralogue, HNF-1α.⁷ HNF-1α is restricted to renal proximal tubules where it regulates genes involved in amino acid, phosphate, and organic acid transport, whereas HNF-1β is expressed more broadly in all nephron segments.⁷ The DNA-binding domains of HNF-1α and HNF-1β are highly conserved, and the proteins recognize a similar DNA sequence. However, the carboxy-terminal domains are divergent, which enables HNF-1β homodimers and HNF-1β/HNF-1α heterodimers to interact with different coactivators and corepressors.^5–7 Dimerization of HNF-1β and HNF-1α is mediated by their amino-terminal domains and promoted by interaction with dimerization cofactor of HNF1 (DCoH).^{8; 9}

Figure 1. Structure of HNF-1β and cyst-causing mutations.

Schematic representation of HNF-1β protein structure. The N-terminal dimerization domain (D), POU-specific (POU_s), POU-homeo (POU_H) domains, and C-terminal transactivation domain are shown. Locations of a subset of mutations that are referenced in this review and associated with kidney cysts are depicted above.^{6; 25; 26; 28; 29; 39; 52; 56; 57; 63; 70; 111}

The DNA-binding domain of HNF-1β is composed of a POU (Pit-1, Oct-1/2, Unc-86)-specific domain (POU_S) and POU-homeodomain (POU_H). The POU_S and POU_H domains adopt a helix-turn-helix tertiary structure that fits into the major groove of DNA^{10; 11} and mediates sequence-specific binding.^{5; 6} HNF-1β recognizes a pseudopalindromic consensus sequence, 5′-GGTTAATNATTAAC-3′, ^{7; 12} that is often present in the promoter region or enhancers of target genes. Once bound to DNA, HNF-1β modulates gene expression by inducing conformational changes in chromatin structure via its interaction with co-activators and corepressors. For example, interactions of the carboxy-terminal domain of HNF-1β with the histone acetylases CREB binding protein and P300/CBP-associated factor results in transcriptional activation⁶ Conversely, HNF-1β can function as a transcriptional repressor through interactions of the carboxy-terminal domain with histone deacetylases.⁶ An unusual property of HNF-1β is that it remains bound to DNA during cell division, which differs from most transcription factors that dissociate from mitotic chromosomes.¹³ This “bookmarking” function enables rapid restoration of HNF-1β target gene activation in daughter cells.¹⁴

1.2. Essential role in kidney development

HNF-1β is expressed in the embryonic kidney and is required at multiple steps of kidney development, including branching morphogenesis, nephrogenesis, nephron patterning, and tubulogenesis. Kidney development involves complex reciprocal interactions and signaling between two main cellular compartments: the ureteric bud (UB) and metanephric mesenchyme (MM). In mice at embryonic day 10.5 (E10.5), the UB forms as an outgrowth from the posterior Wolffian duct (WD). This process is induced by the adjacent MM, which contains nephron progenitor cells and stromal cells.^{15; 16} The UB invades the MM and undergoes branching morphogenesis giving rise to the renal collecting system. The tips of the branching UB induce the MM to condense and undergo mesenchymal-to-epithelial transition (MET) forming the nephrons proper. The first polarized epithelial MM-derived structures, the renal vesicles, subsequently differentiate into the comma-shaped- and S-shaped bodies, which then fuse with the collecting system forming maturing, segmented nephrons.^15–18 HNF-1β is expressed in the mouse WD epithelium at E9.5 and in the branching UB throughout later stages of kidney development.¹ HNF-1β is absent in the MM and first appears at the renal vesicle stage.^{19; 20} Expression is sustained in the mature nephron.^{1; 19}

To identify the role of HNF-1β in kidney development, Lokmane et al. generated HNF-1β-deficient mouse embryos using tetraploid embryo complementation.¹⁹ The initial branching of the UB is delayed in mutant embryos, and branching remains defective throughout subsequent organogenesis. As a consequence, HNF-1β mutant kidneys are hypoplastic.¹⁹ One major signaling pathway that regulates branching morphogenesis is Ret/Glial cell line-derived neurotrophic factor (GDNF) signaling. The MM expresses the ligand GDNF, which engages the Ret receptor and Gfrcd co-receptor expressed in the WD and UB.^{15; 16} In HNF-1β mutant kidneys, the expression of both Ret and GDNF is significantly reduced.¹⁹ To further elucidate the role of Ret/GDNF signaling, Desgrange et al. ablated HNF-1β specifically in the UB of embryonic mouse kidneys using Cre-loxP recombination.²¹ Mutant kidneys show severely impaired branching morphogenesis²¹ associated with downregulation of Gfrα, indicating that HNF-1β directly regulates Ret/GDNF signaling. In addition, the expression of Pax-2, a transcription factor that regulates branching morphogenesis by activating Wnt11 expression,²² is downregulated.²¹ Wnt11 plays an essential role in branching morphogenesis by maintaining GDNF expression via an autoregulatory loop with Ret/GDNF.²³ These findings demonstrate that HNF-1β is essential for kidney branching morphogenesis through regulation of Pax-2 and a Ret/GDNF/Wnt11 autoregulatory loop.

HNF-1β is also necessary for proper nephron induction and patterning. HNF-1β mutant kidneys show decreased expression of Wnt9b,¹⁹ the primary inducer of nephrogenesis²⁴ and a direct downstream target of HNF-1β.¹⁹ Wnt9b is secreted by the UB tips and acts as a paracrine signal initiating nephrogenesis in the MM. In addition, HNF-1β regulates MM-autonomous signaling pathways that drive nephrogenesis. Massa et al. ablated HNF-1β in the MM of the mouse kidney using Cre-lox recombination and found that renal vesicles still form, but the expression of nephron markers is delayed and S-shaped bodies exhibit structural defects.²⁰ The mid- and upper- limbs, which give rise to the proximal and distal convoluted tubules, are deformed and show increased apoptosis and decreased cell proliferation. At birth, the mutant kidneys contain greatly reduced proximal and distal convoluted tubules.²⁰ These results indicate that expression of HNF-1β in the MM is not required for initial nephrogenesis but is required for the differentiation of the proximal and distal tubules.

2. Human cystic diseases associated with mutations of HNF-1β

Heterozygous mutations of HNF1B are among the most common single-gene mutations found in developmental kidney disease.²⁵ In this section, we discuss the congenital kidney abnormalities that are associated with mutations of HNF1B focusing on cystic phenotypes.

2.1. HNF-1β mutations in human disease

More than 100 pathogenic mutations of HNF1B have been reported in humans.²⁶ The mutations range from intragenic mutations, including missense, frameshift, nonsense, and small insertions or deletions, to whole-gene deletions and large genomic rearrangements ^{25; 27; 28} (Figure 1). Mutations of HNF1B are usually present in the germline but can also occur de novo^{28; 29} Few genotype-phenotype correlations have been identified, in part because the severity of renal disease is highly variable even within families carrying the identical mutation.^{28; 30} This variable expressivity is reminiscent of ADPKD and is likely due to additional genetic, environmental, and epigenetic factors modulating the HNF1B mutant phenotype.^{2; 29} Developmental timing may also play a role as children with very early onset disease have more severe kidney abnormalities and accelerated loss of kidney function than those with later onset.³¹

One-third of the reported human HNF1B mutations consist of whole-gene deletions.²⁶ Such deletions are difficult to detect by conventional DNA sequencing, and specialized methods to measure copy number variation (CNV) must be employed. Most individuals with HNF1B deletions are heterozygous for the 17q12 deletion, which is a recurrent 1.4 Mb deletion that encompasses HNF1B and 14 neighboring genes. This locus is a hot spot for mutation since it is flanked by segmental repeats that can undergo homologous recombination during meiosis resulting in deletion of the intervening 1.4 Mb region.³² In addition to kidney abnormalities and diabetes caused by loss of HNF1B, individuals with 17q12 deletions often exhibit neurodevelopmental disorders, including autism spectrum disorder and attention-deficit/hyperactivity disorder, and facial dysmorphism.^{25; 32; 33} The neurological phenotypes are generally not seen in individuals with intragenic HNF1B deletions, suggesting that other genes within the deleted segment are responsible. One such candidate gene, LHX1, encodes a transcription factor that is involved in brain development and is also a downstream target of HNF1B³² The renal failure in 17q12 deletion individuals is less severe than those with an intragenic mutation,^{25; 30} possibly due to compensatory alterations in DNA methylation secondary to the 1,4-Mb deletion.²⁵

In contrast to humans, mice that are heterozygous for an Hnf1b-null allele do not develop kidney cysts or other abnormalities, at least over the period of observation.⁶ The reason for this discrepancy is unclear, although a similar phenomenon has been described for mutations of Pkd1 and Pkd2. Various mechanisms may explain how heterozygous germline mutations produce a mutant phenotype despite the presence of a wild-type allele, including loss of heterozygosity (“second hit”), haploinsufficiency, and dominant-negative mutations. Kidneys from humans who are heterozygous for HNF1B mutations show preserved expression of the wild-type allele, arguing against a second hit mechanism.³⁴ Some intragenic mutations are predicted to encode a mutant protein with an altered transcriptional activation domain but intact dimerization and DNA-binding domains. When co-expressed with wild-type HNF-1β in cell assays, such variants interfere with DNA-binding or transactivation and function as dominant-negative mutants.⁶ For other HNF1B mutations, haploinsufficiency may be responsible for the mutant phenotype.

2.2. Renal cysts and diabetes

Mutations of HNF1B in humans produce maturity-onset diabetes of the young, type 5 (MODY5). MODY is a monogenic disorder characterized by autosomal dominant inheritance of early-onset, non-autoimmune diabetes mellitus.³⁵ Most affected individuals are diagnosed before 25 years of age, lack islet autoantibodies, and are neither obese nor insulin-dependent. MODY is genetically heterogenous and can arise from mutations in at least 14 genes. Mutations of glucokinase (GCK) and HNF1A are responsible for most cases.³⁶ Mutations of HNF1B cause <5% of MODY and usually produce a more severe diabetic phenotype that often requires insulin replacement therapy. Nishigori et al. first reported the association of MODY5 with non-diabetic kidney disease presenting with renal dysfunction, hypertension, absent or low-grade proteinuria, and bilateral cystic kidneys.³⁷ Subsequent studies have confirmed this association and additionally found that the renal phenotype is more penetrant than diabetes, so the absence of diabetes does not exclude the diagnosis.

Bingham et al. coined the term renal cysts and diabetes (RCAD) to describes patients with renal cysts and maturity-onset diabetes of the young caused by mutations of HNF1B³⁸ Mutations can occur throughout the gene,²⁶ although they are most common in exons 1-5. Of these mutations, the ones that occur in exons 2-4 affect the DNA binding domain.³⁸ Cysts can be detected in utero.³⁸ Histological analysis reveal kidney cysts, renal dysplasia, and oligomeganephronia. There is also considerable variation in kidney size in RCAD patients, such that kidneys are enlarged in fetuses and newborns and hypoplastic in adults. This size difference is thought to be due to growth cessation of the kidney after childhood.³⁹ Most patients have impaired renal function, but the magnitude of renal failure varies among individuals with identical mutations.³⁸

In addition to diabetes and kidney cysts, manifestations of RCAD may include hyperuricemia, gout, genital-tract malformations,^{38; 40} and hypomagnesemia.⁴¹ HNF-1β is required for pancreas development, and diabetes is thought to be due to reduced insulin secretion as a result of pancreatic hypoplasia.³⁸ Hyperuricemia and hypomagnesemia arise from decreased expression of UMOD, organic anion transporters, and FXYD2, which regulate uric acid secretion and magnesium reabsorption in the nephron. HNF1B is expressed in the Müllerian duct, and HNF1B mutations are associated with female genital tract malformations such as bicornuate uterus, uterus didelphys, and vaginal aplasia. Hyperparathyroidism due to derepression of PTH and hypokalemia, which may be due to hypomagnesemia and down-regulation of KCNJ10 and SLC12A1, have also been described.^{42; 43}

2.3. Multicystic dysplastic kidneys

Consistent with the essential role of HNF-1β in kidney development in mice, mutations of human HNF1B are causally associated with renal developmental abnormalities, some of which present as cystic kidneys. Mutations of HNF1B account for 10-30% of cases of congenital anomalies of the kidney and urinary tract (CAKUT).⁴⁴ CAKUT includes renal agenesis, hypoplasia, dysplasia, hydronephrosis, horseshoe kidney, and multicystic dysplastic kidney (MCDK), all of which have been observed in humans with HNF1B mutations.^{45; 46} In several large pediatric series, mutations of HNF1B were among the most common genetic causes of renal hypodysplasia.^47–49 Kidney function can range from normal to ESRD depending on the severity of kidney abnormalities and whether they are unilateral or bilateral.

MCDK is a relatively common renal developmental abnormality in which normal kidney structure is replaced by clusters of non-communicating cysts.⁴⁶ MCDK can be inherited or sporadic, unilateral or bilateral, and may be associated with ipsilateral urinary tract abnormalities, such as ureteral atresia. Histology shows disorganized kidney architecture, loss of corticomedullary boundary, primitive tubules surrounded by fibromuscular collars, and nests of metaplastic cartilage.⁵⁰ MCDK has a frequency of 1 in 1,000 to 4,300 live births,^{46; 51} and one in 10 patients with unilateral MCDK have HNF1B mutations.⁵² A recent report describing a large autopsy series of fetuses carrying HNF1B mutations showed that most had unilateral or bilateral enlarged cystic kidneys with characteristic histological features of renal dysplasia.⁵³ Urinary tract abnormalities were seen in 25% of cases.

2.4. Glomerulocystic kidney disease

GCKD is a rare autosomally-inherited disorder characterized by cortical glomerular cysts that can lead to chronic renal failure. Kidney histology shows cystic dilation of Bowman’s capsule with atrophic glomerular tufts present in at least 5% of the cysts.⁵⁴ Concomitant tubular cysts may be present in the renal cortex or medulla but suggest alternative diagnoses such as ADPKD or tuberous sclerosis. GCKD is most common in neonates and infants with a family history of ADPKD;⁵⁵ however, linkage studies found that some affected individuals do not have PKD1 or PKD2 mutations.^{55; 56} Germline mutations in HNF1B were found in some families with the hypoplastic form of GCKD.⁵⁶ Individuals with HNF1B-related GCKD present with hypoplastic kidneys with glomerular cysts, malformations in the calyces and collecting systems, and early-onset diabetes.^{26; 56; 57}

The mechanism of glomerular cystogenesis is not known, but primary cilia, Wnt signaling, proximal tubule malformation, and urinary tract obstruction have been implicated.⁵⁴ HNF-1β is expressed in the parietal epithelium of Bowman’s capsule suggesting that the formation of glomerular cysts is cell autonomous. Glomerular cysts are also seen in Glis3 and TAZ/Wwtr1 knockout mice, and Jetten et al. have shown that cilia-localized Glis3 interacts with TAZ/Wwtr1 suggesting that they function in a common pathway.⁵⁸ Wwtr1, which is an effector in the Hippo signaling pathway, is a downstream target of HNF-1β (see below), raising the possibility that glomerular cysts in HNF1B mutants arise from down-regulation of Wwtr1. Recent studies have shown that deletion of the transcription factor ZEB2 in mice produces glomerular cysts,⁵⁹ and our studies have shown that HNF1b regulates ZEB2 expression. However, expression of ZEB2 is elevated in HNF-1β mutant kidneys rather than decreased, so the role of ZEB2 in HNF1B-related GCKD remains uncertain.

2.5. Autosomal dominant tubulointerstitial kidney disease

Autosomal dominant tubulointerstitial kidney disease (ADTKD) is a recently recognized monogenic disorder characterized by progressive renal insufficiency, normal or small kidney size, bland urinary sediment, and absence of nephrotoxin exposure or other known causes of CKD.⁴⁰ ADTKD includes the diseases previously known as medullary cystic kidney disease (MCKD) and familial juvenile hyperuricemic nephropathy (FJHN). Affected individuals present with elevated serum creatinine, absent or low-grade proteinuria, impaired urinary concentration, and frequently hyperuricemia. Renal biopsy is rarely performed, but kidney histology shows interstitial fibrosis, tubular atrophy, tubular microcysts or macrocysts, basement membrane abnormalities, and absence of immune complex deposition. ESRD can develop over a wide age range from 17 to 75 years with a mean age of 47 years. ADTKD can arise from mutations in UMOD encoding uromodulin (Tamm-Horsfall protein), REN encoding renin, MUC1 encoding mucin-1, SEC61A1 encoding the Sec61 translocon, DNAJ11 encoding the chaperone protein ERdj3, and HNF1B⁶⁰ Several of these genes are involved in proteostasis, and mutations result in accumulation of misfolded proteins, endoplasmic reticulum stress, induction of the unfolded protein response, and renal fibrosis.⁶¹

HNF1B-associated ADTKD (ADTKD-HNF1B), also known as HNF1B nephropathy, can be difficult to diagnose because the manifestations are non-specific and vary within affected families. Individuals with ADTKD-HNF1B may have multiple renal cysts and other abnormalities associated with CAKUT.^62,63 Unlike other forms of ADTKD, ADTKD-HNF1B can present with extrarenal abnormalities including diabetes, elevated liver enzymes, autism spectrum disorder, cognitive impairment, pancreatic hypoplasia, female genital tract malformations, and hypomagnesemia.^{63; 64} These extrarenal manifestations can assist with diagnosis of HNF1B-related ADTKD. Faguer et al. developed an HNF1B score system based on the major clinical phenotypes as a method of pre-screening individuals for genetic testing.⁶⁵ The score is calculated based on the occurrence of family history, renal, liver, pancreas and genital tract abnormalities.

2.6. Polycystic kidney disease

Although mutations of HNF1B do not cause typical PKD, there is a strong association between HNF-1β and both the dominant and recessive forms of PKD. As described in greater detail below, HNF-1β directly regulates the transcription of PKD2 and PKHD1, genes that cause ADPKD and ARPKD, respectively. Genetic studies using HNF-1β knockout mice and detailed molecular characterization of HNF-1β functions in kidney epithelial cells have shown that mutations of HNF-1β phenocopy many characteristics of PKD.⁶⁶ The cystic kidney disease observed in humans with HNF1B mutations resembles ADPKD, and HNF1B mutations and have been detected in some individuals who were initially diagnosed with ADPKD but were not linked to PKD1 or PKD2,^{67; 68} The presence of atypical features such as unilateral disease, very early-onset, or extrarenal manifestations may assist with the correct diagnosis. Some phenotypic overlap between ADTKD and ADPKD also exists. Mutations of UMOD, MUC1, SEC61A1, DNAJB11, and HNF1B, which cause ADTKD, can also produce kidney cysts that phenocopy early-onset ADPKD.^{67; 68} The reason why mutations of these genes can present with primarily cystic kidney disease or primarily tubulointerstitial fibrosis is not clear, but may relate to developmental timing.

Expression of HNF-1β is reduced in cystic kidneys from individuals with ADPKD. Song et al. performed RNA expression profiling of individual kidney cysts derived from humans with ADPKD due to mutations of PKD1 and found consistently reduced expression of HNF1B compared to normal kidney.⁶⁹ Corresponding reductions in expression of 9 of 15 HNF-1β-dependent target genes and derepression of SOCS3 were observed. These results suggest that HNF-1β is downstream to PKD1. Although the molecular mechanism is not known, the decreased expression of HNF-1β may contribute to the PKD1 cystic phenotype.

Consistent with the latter, HNF1B has been identified as a candidate modifier gene in human ADPKD. As described elsewhere in this series, ADPKD shows highly variable intrafamilial and interfamilial expressivity, which has been attributed to environmental factors and the effect of modifier genes. A small percentage of individuals with PKD1 or PKD2 mutations display an early onset of ADPKD that closely resembles autosomal recessive polycystic kidney disease (ARPKD) and can result in perinatal and neonatal death. Bergmann et al. genotyped families with early-onset ADPKD and found four individuals from three families who had inherited a PKD1 mutation from one parent and an HNF1B mutation from the other parent.⁷⁰ The occurrence of an HNF1B mutation together with a PKD1 mutation produced more severe, earlier-onset disease compared to either parent alone. Children or fetuses with both mutations presented with oligohydramnios, renal insufficiency, or early-onset ESRD, and renal ultrasound showed bilateral enlarged echogenic or cystic kidneys. These results suggest that mutations of HNF1B may contribute to the phenotypic variation seen in humans with ADPKD.

3. Identification of genes regulated by HNF-1β

3.1. Mouse models of HNF-1β-related kidney diseases

To gain insights into the molecular pathogenesis of HNF1B-related kidney diseases, our laboratory and others have generated mouse models. HNF-1β is essential for visceral endoderm formation, and constitutive ablation of HNF-1β in the mouse is embryonic lethal.^{6; 71} Therefore, kidney-specific transgenics and gene targeting were required. In the first such approach, we generated transgenic mice expressing a dominant-negative HNF-1β (DN-HNF-1β) mutant lacking the carboxy-terminal transactivation domain under the control of a kidney-specific cadherin-16 promoter.⁷² DN-HNF-1β transgenic mice develop glomerular and tubular cysts and ESRD, which recapitulates the phenotype of humans with HNF1B mutations. Analysis of candidate genes showed marked reduction of Pkhd1 mRNA transcripts in the cyst epithelium compared to wide-type tubules. Pkhd1 encodes fibrocystin or polyductin, a transmembrane protein localized in cilia, and is the most frequently mutated gene in autosomal recessive polycystic kidney disease (ARPKD).⁷³ The function of fibrocystin is not known, but its expression pattern closely overlaps with HNF-1β. Biochemical studies showed that transcription of Pkhd1 is directly regulated by HNF-1β in renal collecting duct cells.

Kidney-specific ablation of HNF-1β in renal tubules using Cre/loxP recombination also results in the formation of kidney cysts, confirming the results seen in DN-HNF-1β transgenic mice.⁷⁴ In addition to decreased expression of Pkhd1, the expression of cystic disease genes Pkd2, Umod, Ift88, and Nphp1 is also downregulated. Chromatin immunoprecipitation showed the presence of HNF-1β binding sites in the Pkhd1, Umod, Pkd2, and Ift88 genes, suggesting that the transcription of these genes is directly regulated by HNF-1β.

Interestingly, the effects of HNF-1β inactivation are dependent on cell proliferation.¹³ In proliferating cells, loss of HNF-1β inhibits transcription of downstream target genes, whereas expression of target genes is unaffected when HNF-1β is inactivated in quiescent cells. This property may be related to the bookmarking function described earlier and suggests that HNF-1β binding to mitotic chromosomes is required to maintain the expression of target genes in daughter cells. Consequently, inactivation of HNF-1β in mice at postnatal day 1 when tubules are rapidly proliferating results in decreased expression of Pkd2 and Pkhd1 and the formation of kidney cysts. In contrast, inactivation of HNF-1β in mice after 10 days of age, when tubules are more quiescent, does not affect Pkd2 or Pkhd1 expression and does not produce cysts for up to 6 months. When adult mutant mice are subjected to acute kidney injury, cell proliferation and cyst formation are stimulated. Similar findings have been made in Kif3a and Pkd1 knockout mice, which suggests that abnormal repair after kidney injury may be a common factor in cyst initiation.⁷⁵ Differences in kidney injury and cell proliferation may contribute to the phenotypic variability of HNF1B mutations.

3.2. Regulation of PKD-relevant genes

To identify additional gene networks regulated by HNF-1β, we performed genome-wide chromatin immunoprecipitation (ChIP-chip and ChIP-seq) in cultured renal epithelial cells.^{76; 77} By combining these approaches with RNA transcriptome profiling (microarrays and RNA-seq), we identified genes that are dysregulated in HNF-1β mutant cells, contain nearby HNF-1β binding sites, and are likely to represent direct transcriptional targets of HNF-1β.^{74; 77} These studies revealed that HNF-1β regulates the expression of many genes that are associated with cystic kidney disease in humans and rodents, including Pkhd1 (ARPKD); Pkd2 (ADPKD); Umod (ADTKD, MCKD); Cystin (cpk mouse); Nphp1, Nphp3, and Glis2 (nephronophthisis), Bicc1 (jcpk mouse); Kif12 (cpk modifier gene); and Wwtr1 (TAZ) (^{74; 76–80} and unpublished data). The above studies reveal that HNF-1β regulates a transcriptional network containing multiple cystic disease genes. Loss-of-function mutations of HNF1B decrease the expression of cystic disease genes, which leads to cyst formation.

The HNF-1β-regulated gene Kif12 was first identified as a candidate modifier gene in the congenital polycystic kidney (cpk) mouse model of polycystic kidney disease.⁸¹ We demonstrated that mutations of HNF-1β inhibit Kif12 transcription by altering histone modifications and coregulatory recruitment at its promoter region.⁸² As Kinesin-12 family members are involved in orienting cell division,⁸³ downregulation of Kif12 may underlie the abnormal planar cell polarity observed in HNF1B mutants (see below). In addition to transcriptional activation, HNF-1β can also function as a transcriptional repressor. In this case, mutations of HNF1B lead to increased target gene expression. One example is suppressor of cytokine signaling 3 (SOCS3), which is upregulated in HNF-1β mutant kidneys. SOCS3 inhibits HGF-induced tubulogenesis by reducing phosphorylation of Erk and STAT-3.⁷⁶ Interestingly, Socs3 mRNA is highly expressed in human polycystic kidneys in which HNF-1β expression is reduced,⁶⁹ and PKD1 mutant cells show activation of JAK2-STAT3 pathway with increased SOCS3 expression.⁸⁴ These findings suggest that upregulation of SOCS3 may be a common feature of cystic kidney diseases.

4. Regulation of PKD signaling pathways

Polycystic kidney disease is characterized by dysregulation of numerous intracellular and intercellular signaling pathways. Analysis of HNF-1β mutant cells and kidneys has revealed dysregulation of many of the same PKD pathways. In this section, we review the role of HNF-1β in signaling pathways that are disrupted in cystic kidney diseases, focusing on cAMP signaling, Wnt signaling, planar cell polarity, and renal fibrosis.

4.1. cAMP signaling

Activation of cAMP signaling is a hallmark of PKD and forms the basis for treatment with tolvaptan and other drugs that reduce intracellular cAMP levels.⁸⁵ Cyclic AMP levels are governed by the activities of G-protein coupled receptors, adenylyl cyclases, and cAMP phosphodiesterases.⁸⁶ Cyclic AMP levels are elevated in many animal models of PKD, including Hnf1b mutant mice;⁸⁷ however, the mechanism responsible for increased cAMP is poorly understood. We have shown that one protein complex in the primary cilium comprises adenylyl cyclase 5/6 (AC5/6), a scaffolding protein A-kinase anchoring protein 150 (AKAP150), and protein kinase A.⁸⁷ Genetic mutations of Pkd2 and Hnf1b in mice result in increased cAMP levels that are dependent on activation of AC5/6. Polycystin-2 interacts with the AC5/6 complex through its carboxy-terminus and thereby regulates cAMP levels, possibly by allowing Ca²⁺ entry and inhibiting the Ca²⁺-sensitive AC5/6 isoforms. Loss of polycystin-2 would relieve this inhibition resulting in increased cAMP levels. Consistent with this model, genetic ablation of AC5 reduces cAMP levels and cyst growth in Pkd2 mutant mice.⁸⁸ Since expression of polycystin-2 is controlled by HNF-1β, this mechanism could also explain the increased cAMP levels in HNF1B mutants.

In addition, the increased cAMP levels in HNF-1β mutant cells and kidneys may reflect transcriptional downregulation of a cAMP-specific member of the phosphodiesterase family, PDE4C.⁸⁷ Phosphodiesterases mediate the conversion of cAMP to AMP and thereby terminate cAMP signaling.⁸⁹ We found that PDE4C is located in the primary cilia of renal epithelial cells where it interacts with the AC5/6-AKAP150-PC2 complex.⁸⁷ ChIP-ChIP showed that HNF-1β binds to the proximal promoter of Pde4c and is required for its transcription.⁸⁷ Decreased expression of PDE4C in HNF1B mutant cells would result in reduced catabolism of cAMP and elevated cAMP levels. Collectively, these studies reveal novel mechanisms for dysregulation of cAMP signaling in cystic kidney diseases through down-regulation of Pkd2 and Pde4c. Inhibition of AC5 or stimulation of PDE4C may represent alternative approaches to decrease cAMP levels in cystic kidney diseases.

4.2. Canonical Wnt signaling

Wnts are secreted glycoproteins that play critical roles in many biological processes including embryonic development, cell fate determination, tissue homeostasis, and cell survival.⁹⁰ Deregulation of Wnt signaling is frequently seen in human diseases such as cancer.⁹¹ The Wnt signaling pathway can be classified into canonical Wnt/β-catenin dependent and non-canonical/β-catenin independent. Canonical Wnt signaling is activated by binding of Wnt ligands to Frizzled (Fzd) and LDL-related protein (LRP) cell surface receptors.⁹² Ligand binding results in the membrane sequestration of a multiprotein complex containing APC and GSK3β, which normally phosphorylates β-catenin and targets it for proteasomal degradation.⁹³ Sequestration of the destruction complex permits cytosolic accumulation of β-catenin, which translocates to the nucleus where it interacts with TCF/LEF transcription factors at Wnt Responsive Elements (WRE) to activate Wnt target gene expression.⁹⁴

Several studies suggest that canonical Wnt signaling plays an important role in cystic kidney disease. For example, genetic ablation of kinesin subunit II (Kif3a) in mice produces cystic kidneys with the upregulation of β-catenin in cyst epithelium.⁹⁵ Conditional inactivation of Ape, a scaffold protein contained in the cytosolic β-catenin destruction complex, produces kidney cysts lined by a hyperproliferative epithelium.⁹⁶ Upregulation of GSK3β expression is associated with cyst expansion in mice and humans with PKD.⁹⁷ The extracellular domain of polycystin-1 has been shown to bind to Wnt ligands and mediate Ca²⁺ influx through heterodimer complex formation with polycystin-2.⁹⁸ In addition, the cytosolic carboxy-terminal domain of PC1 binds to β-catenin and downregulates canonical Wnt signaling.⁹⁹ Conversely, mutation of Pkd2 increases β-catenin signaling in kidney tubules and results in cyst expansion, which can be prevented by Wnt inhibitors.¹⁰⁰

Transcriptomic analysis showed that canonical Wnt signaling is highly activated in HNF-1β knockout cells.^{79; 101} Ablation of HNF-1β in mIMCD3 renal epithelial cells produces hyperresponsiveness to exogenous Wnt ligands and increases expression of Wnt target genes, including Axin2, Ccdc80, and Rnf43. Levels of β-catenin and expression of Wnt target genes are also increased in HNF-1β mutant mouse kidneys. In agreement with the mouse model, humans with HNF1B mutations display bilateral enlarged multicystic kidneys with accumulation of β-catenin in the hyperplastic cyst-lining cells.³⁴ Genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) in wild-type and mutant cells showed that ablation of HNF-1β produces a 6-fold increase in the number of genomic sites that are occupied by β-catenin.¹⁰¹ Remarkably, 50% of the sites that are occupied by β-catenin in HNF-1β mutant cells colocalize with HNF-1β-occupied sites in wild-type cells. The striking association of β-catenin and HNF-1β on WREs suggests a direct and widespread transcriptional repression of canonical Wnt signaling by HNF-1β. We further demonstrated that HNF-1β directly competes with the binding of β-catenin/LEF complexes to WREs through a novel composite motif containing a TCF/LEF binding site and an overlapping HNF-1β half-site. This study reveals a novel mechanism whereby a transcription factor constrains canonical Wnt activation by competing with β-catenin/LEF chromatin binding. Hyperactivation of canonical Wnt signaling due to loss of HNF-1β may contribute to cyst initiation and expansion in cystic kidney disease.

4.3. Planar cell polarity

During normal kidney organogenesis, developing renal tubules elongate without increasing their diameter. Fischer et al. showed that the mitotic spindles in dividing tubular cells are oriented parallel to the tubule axis.¹⁰² Consequently, cell division produces daughter cells that are aligned longitudinally, which results in tubule elongation without tubular dilation. Karner et al. found evidence for convergent extension in the developing mouse kidney, such that cell intercalation results in tubular elongation and a decrease in the number of cells in tubular cross sections.¹⁰³ Both oriented cell division and convergent extension are manifestations of planar cell polarity (PCP). In HNF-1β mutant kidneys, the mitotic spindles in dividing tubular cells are no longer oriented parallel to the tubular axis.¹⁰² These data indicate that loss of HNF-1β disrupts PCP, which may contribute to tubular dilation and cyst formation. Disruption of PCP is also seen in a rat model of PKD caused by mutations of Pkhd1 (pck rat).¹⁰² In contrast, abnormalities in OCD were not observed in pre-cystic tubules in Pkd1 and Pkd2 mutant mice, indicating that early disruption of PCP is not a universal finding in PKD.¹⁰⁴

PCP is regulated by non-canonical (β-catenin-independent) Wnt signaling in which binding of Wnt ligands to Fzd receptors results in activation of RhoA kinase.⁷⁴ Studies by Karner et al. suggest that Wnt9b signaling through the non-canonical pathway may be involved in the establishment of PCP in the developing kidney, and mice homozygous for a hypomorphic Wnt9b allele form kidney cysts.¹⁰³ In developing Wnt9b mutant tubules, cell elongation occurs in random orientation and the number of cells in tubular cross sections does not decrease as in wild-type kidneys, suggesting a defect in convergent extension. Consistent with inhibition of the non-canonical Wnt pathway, the phosphorylation of downstream RhoA is decreased. Since HNF-1β directly regulates Wnt9b transcription (above), this mechanism may also underlie the PCP defects in HNF-1β mutant kidneys. In addition, ChIP-seq studies have shown that HNF-1β binds to genes encoding core PCP proteins, such as Pricklel.^{76; 79}

4.4. Renal fibrosis

Kidney fibrosis is a common final pathway in many forms of chronic kidney disease including cystic kidney diseases. Humans with HNF1B mutations can present with ADTKD, in which renal interstitial fibrosis is prominent. HNF-1β mutant mice produced using a collecting duct-specific Cre deletion develop more slowly progressive cystic kidney disease associated with significant renal fibrosis.² To unravel the mechanism whereby loss of HNF-1β in renal epithelial cells produces interstitial fibrosis, we generated HNF-1β-deficient mIMCD3 cells using CRISPR-based gene editing.¹⁰⁵ Characterization of the cells revealed that loss of HNF-1β induces epithelial-mesenchymal transition (EMT), as evidenced by spindle-shaped cell morphology, increased cell motility and invasiveness, and expression of mesenchymal markers.⁷⁹ EMT is also observed in vivo in HNF-1β mutant mice, but genetic lineage analysis showed that EMT does not directly contribute to the formation of renal interstitial fibroblasts. Instead, we identified Twst2 as a direct target of HNF-1β that regulates an EMT transcriptional network in renal epithelial cells. Upregulation of Twist2 increases the expression of EMT transcription factors such as SnaiH, Snail2, Zeb1 and Zeb2, which in turn induces aberrant TGF-β signaling and fibroblast activation within the kidney interstitium. Snai1 also represses Hnf1b itself, suggesting that a positive feed-forward loop may promote fibrosis.¹⁰⁵ The EMT mechanisms discovered in HNF-1β mutant cell and kidneys may underlie the pathophysiology of renal fibrosis in ADTKD and other cystic kidney diseases.

5. Regulation of noncoding RNAs

In addition to regulating the transcription of protein-coding genes, HNF-1β also regulates the expression of noncoding RNAs. As discussed in greater detail elsewhere in this series, much of the human genome is transcribed into RNA species that are not directly translated into proteins but instead have regulatory functions. Such noncoding RNAs include microRNAs (miRNA), long noncoding RNAs (IncRNA), enhancer RNAs (eRNA), circular RNAs (circRNA), and others. Microarray and RNA-seq experiments revealed that the expression of noncoding RNAs is dysregulated in HNF-1β mutant cells, and in some cases HNF-1β directly regulates the transcription of noncoding RNAs.

The first such noncoding RNA identified was a previously unannotated IncRNA encoding the miR-200 microRNA cluster.¹⁰⁶ Long noncoding RNAs are >200 nucleotides in length and do not contain a long open-reading frame. LncRNAs are epigenetic regulators that control recruitment of transcription factors and chromatin remodeling complexes, RNA editing and splicing, sequestration of miRNAs, and protein translation.¹⁰⁷ MicroRNA microarray analysis of renal epithelial cells expressing dominant-negative mutant HNF-1β revealed decreased expression of the miR-200b/200a/429 cluster. This microRNA cluster is of interest, since its expression is epithelial-specific, similar to HNF-1β, and down-regulation of miR-200 induces epithelial-mesenchymal transition, in part through disinhibition of the EMT transcription factors ZEB1 and ZEB2. Unexpectedly, we found that in renal epithelial cells, the miR-200b/a/429 cluster is encoded by a 28-kb IncRNA, the transcription of which is under the direct control of HNF-1β.¹⁰⁶ Ablation of HNF-1β results in decreased IncRNA and miR-200 expression, which leads to increased levels of downstream targets, including ZEB2 and PKD1¹⁰⁸ Re-expression of miR-200 reversed these abnormalities. These studies identified a novel mechanism for HNF-1β-dependent epigenetic regulation of PKD1 and revealed an additional pathway whereby loss of HNF-1β leads to EMT and renal fibrosis.

Some noncoding RNAs show increased expression following ablation of HNF-1β. Among these is the miR-17~92 cluster, overexpression of which is linked to PKD.¹⁰⁹ Expression of miR-17~92 is upregulated in HNF-1β mutant kidneys and other models of human and mouse PKD. In contrast to miR-200, the expression of miR17~92 does not appear to be directly regulated by HNF-1β. Rather, the transcription of miR-17~92 is regulated by c-Myc.¹¹⁰

6. Conclusions

HNF-1β is an essential transcription factor that regulates gene expression during kidney development. Human cystic kidney diseases such as RCAD, MCDK, GCKD, and ADTKD are associated with mutations in HNF1B. Mutations in HNF1B can be familial or de novo and produce a broad range of phenotypes. HNF-1β directly regulates the expression of PKHD1, PKD2, and other genes involved in cystic kidney diseases (Figure 3a). HNF-1β also regulates cAMP levels in ADPKD through PDE4C (Figure 3b). Both canonical and non-canonical Wnt signaling pathways play a role in cystogenesis and are regulated by HNF-1β (Figure 3c). HNF-1β inhibits EMT by inhibiting TWIST2 (Figure 3d). HNF-1β regulation of noncoding RNAs also inhibits EMT (Figure 3e). Studies on HNF-1β-regulated signaling pathways have provided general insights into the mechanism of cyst formation and identified potential targets for therapeutic intervention in human cystic kidney diseases.

Figure 3. Roles of HNF-1β in renal cystogenesis.

Schematic diagram showing the central role of HNF-1β in the transcriptional and post-transcriptional regulation of multiple pathways involved in renal cystogenesis, including a) Expression of cystic kidney disease genes; b) cAMP signaling; c) Wnt signaling; d) Renal fibrosis; and e) Noncoding RNAs. Mutations of HNF-1β produce kidney cysts through down-regulation of PKD2, PKHD1, and other cystic disease genes; down-regulation of PDE4C leading to increased cAMP; increased β-catenin/LEF binding leading to upregulation of canonical Wnt signaling and increased c-Myc and miR-17; and upregulation of Twist2 and down-regulation of miR-200 leading to EMT and renal fibrosis.

Figure 2. PKD-related signaling pathways.

Signaling pathways that are regulated by HNF-1β and perturbed in PKD. a) Regulation of cAMP levels. HNF-1β regulates the expression of polycystin-2 (PC2) and phosphodiesterase 4C (PDE4C). PC2 inhibits calcium-sensitive adenyl cyclase 5 (AC5), while PDE4C decreases cAMP levels by converting cAMP into AMP. b) Canonical and non-canonical Wnt signaling pathways. In the Wnt-on state of canonical Wnt signaling, β-catenin translocates to the nucleus and forms complexes with TCF/LEF that compete with HNF-1β for binding to Wnt target genes. Non-canonical Wnt signaling is regulated by Wnt9b in the kidney and plays a role in planar cell polarity, c) HNF-1β inhibits the expression of Twist2, thereby reducing downstream TGF-β signaling that drives renal interstitial fibrosis.

Highlights.

• HNF-1β is a POU-homeodomain transcription factor that regulates branching morphogenesis and mesenchymal-to-epithelial transition in the developing kidney

• Mutations of HNF-1β in humans produce cystic kidney diseases

• HNF-1β plays a central role in renal cystogenesis through the transcriptional and posttranscriptional regulation of cystic disease genes