Xinli Shao

A transcriptional network in polycystic kidney disease

Mutations in cystic kidney disease genes represent a major genetic cause of end‐stage renal disease. However, the molecular cascades controlling the expression of these genes are still poorly understood. Hepatocyte Nuclear Factor 1β (HNF1β) is a homeoprotein predominantly expressed in renal, pancreatic and hepatic epithelia. We report here that mice with renal‐specific inactivation of HNF1β develop polycystic kidney disease. We show that renal cyst formation is accompanied by a drastic defect in the transcriptional activation of Umod, Pkhd1 and Pkd2 genes, whose mutations are responsible for distinct cystic kidney syndromes. In vivo chromatin immunoprecipitation experiments demonstrated that HNF1β binds to several DNA elements in murine Umod, Pkhd1, Pkd2 and Tg737/Polaris genomic sequences. Our results uncover a direct transcriptional hierarchy between HNF1β and cystic disease genes. Interestingly, most of the identified HNF1β target gene products colocalize to the primary cilium, a crucial organelle that plays an important role in controlling the proliferation of tubular cells. This may explain the increased proliferation of cystic cells in MODY5 patients carrying autosomal dominant mutations in HNF1β.

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

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

Smad2 Protects against TGF-β/Smad3-Mediated Renal Fibrosis

Smad2 and Smad3 interact and mediate TGF-beta signaling. Although Smad3 promotes fibrosis, the role of Smad2 in fibrogenesis is largely unknown. In this study, conditional deletion of Smad2 from the kidney tubular epithelial cells markedly enhanced fibrosis in response to unilateral ureteral obstruction. In vitro, Smad2 knockdown in tubular epithelial cells increased expression of collagen I, collagen III, and TIMP-1 and decreased expression of the matrix-degrading enzyme MMP-2 in response to TGF-beta1 compared with similarly treated wild-type cells. We obtained similar results in Smad2-knockout fibroblasts. Mechanistically, Smad2 deletion promoted fibrosis through enhanced TGF-beta/Smad3 signaling, evidenced by greater Smad3 phosphorylation, nuclear translocation, promoter activity, and binding of Smad3 to a collagen promoter (COL1A2). Moreover, deletion of Smad2 increased autoinduction of TGF-beta1. Conversely, overexpression of Smad2 attenuated TGF-beta1-induced Smad3 phosphorylation and collagen I matrix expression in tubular epithelial cells. In conclusion, in contrast to Smad3, Smad2 protects against TGF-beta-mediated fibrosis by counteracting TGF-beta/Smad3 signaling.

Role of the hepatocyte nuclear factor-1β (HNF-1β) C-terminal domain in Pkhd1 (ARPKD) gene transcription and renal cystogenesis

Hepatocyte nuclear factor-1β (HNF-1β) is a homeodomain-containing transcription factor that regulates tissue-specific gene expression in the kidney and other epithelial organs. Mutations of HNF-1β produce congenital cystic abnormalities of the kidney, and previous studies showed that HNF-1β regulates the expression of the autosomal recessive polycystic kidney disease (ARPKD) gene, Pkhd1. Here we show that the C-terminal region of HNF-1β contains an activation domain that is functional when fused to a heterologous DNA-binding domain. An HNF-1β deletion mutant lacking the C-terminal domain interacts with wild-type HNF-1β, binds DNA, and functions as a dominant-negative inhibitor of a chromosomally integrated Pkhd1 promoter. The activation of the Pkhd1 promoter by wild-type HNF-1β is stimulated by sodium butyrate or coactivators CREB (cAMP-response element)-binding protein (CBP) and P/CAF. The interaction with CBP and P/CAF requires the C-terminal domain. Expression of an HNF-1β C-terminal deletion mutant in transgenic mice produces renal cysts, increased cell proliferation, and dilatation of the ureter similar to mice with kidney-specific inactivation of HNF-1β. Pkhd1 expression is inhibited in cystic collecting ducts but not in non-cystic proximal tubules, despite transgene expression in this nephron segment. We conclude that the C-terminal domain of HNF-1β is required for the activation of the Pkhd1 promoter. Deletion mutants lacking the C-terminal domain function as dominant-negative mutants, possibly by preventing the recruitment of histone acetylases to the promoter. Cyst formation correlates with inhibition of Pkhd1 expression, which argues that mutations of HNF-1β produce kidney cysts by down-regulating the ARPKD gene, Pkhd1. Expression of HNF-1α in proximal tubules may protect against cystogenesis.

The lupus-susceptibility gene kallikrein downmodulates antibody-mediated glomerulonephritis

Sle3 is a NZM2410/NZW-derived lupus-susceptibility interval on murine chromosome 7, which is associated with spontaneous lupus nephritis (SLN), and also anti-GBM-induced glomerulonephritis (GN). The tissue kallikrein gene cluster is located within the Sle3 interval and constitutes potential candidate genes for this locus. We have recently reported that renal kallikrein expression was upregulated by anti-GBM antibody challenge in a strain-specific manner and that it was significantly underexpressed in the anti-GBM-sensitive strains, including B6.Sle3. Further sequencing and functional studies reported earlier provided evidence that kallikreins could constitute disease genes in lupus. In this report, we have used an adenoviral vector to deliver the klk1 gene to B6.Sle3 congenics to directly test if kallikreins might have a protective effect against anti-GBM-induced nephritis. Our data show that klk1 gene delivery ameliorated anti-GBM-induced nephritis in B6.Sle3 congenics. Taken together with earlier studies, these findings indicate that kallikreins play an important protective role in autoantibody-initiated GN and could constitute potential candidate genes for anti-GBM-induced GN and SLN.

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

Autosomal recessive polycystic kidney disease, an inherited disorder characterized by the formation of cysts in renal collecting ducts and biliary dysgenesis, is caused by mutations of the polycystic kidney and hepatic disease 1 (PKHD1) gene. Expression of PKHD1 is tissue specific and developmentally regulated. Here, we show that a 2.0-kb genomic fragment containing the proximal promoter of mouse Pkhd1 directs tissue-specific expression of a lacZ reporter gene in transgenic mice. LacZ is expressed in renal collecting ducts beginning during embryonic development but is not expressed in extrarenal tissues. The Pkhd1 promoter contains a binding site for the transcription factor hepatocyte nuclear factor (HNF)-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.

Inducible expression of kallikrein in renal tubular cells protects mice against spontaneous lupus nephritis

OBJECTIVE To ascertain whether engineered expression of kallikreins within the kidneys, using an inducible Cre/loxP system, can ameliorate murine lupus nephritis. METHODS In mice with a lupus-prone genetic background, we engineered the expression of tamoxifen-inducible Cre recombinase under the control of a kidney-specific promoter whose activation initiates murine kallikrein-1 expression within the kidneys. These transgenic mice were injected with either tamoxifen or vehicle at age 2 months and then were monitored for 8 months for kallikrein expression and disease. RESULTS Elevated expression of kallikrein was detected in the kidney and urine of tamoxifen-injected mice but not in controls. At age 10 months, all vehicle-injected mice developed severe lupus nephritis, as evidenced by increased proteinuria (mean ± SD 13.43 ± 5.65 mg/24 hours), increased blood urea nitrogen (BUN) and serum creatinine levels (39.86 ± 13.45 mg/dl and 15.23 ± 6.89 mg/dl, respectively), and severe renal pathology. In contrast, the tamoxifen-injected mice showed significantly reduced proteinuria (6.6 ± 4.12 mg/24 hours), decreased BUN and serum creatinine levels (15.71 ± 8.17 mg/dl and 6.64 ± 3.39 mg/dl, respectively), and milder renal pathology. Tamoxifen-induced up-regulation of renal kallikrein expression increased nitric oxide production and dampened renal superoxide production and inflammatory cell infiltration, alluding to some of the pathways through which kallikreins may be operating within the kidneys. CONCLUSION Local expression of kallikreins within the kidney has the capacity to dampen lupus nephritis, possibly by modulating inflammation and oxidative stress.

LOSS OF PRIMARY CILIA PRECEDES KIDNEY CYST FORMATION IN KIF3A KNOCKOUT MICE.: 352

LOSS OF PRIMARY CILIA PRECEDES KIDNEY CYST FORMATION IN KIF3A KNOCKOUT MICE. V. Patel, P. Cobo, X. Shao, P. Igarashi, The University of Texas Southwestern Medical School, Dallas, TX. KIF3A is a motor protein that is essential for antegrade intraflagellar transport and the maintenance of primary cilia. We have previously shown that kidney-specific excision of Kif3a mediated by Ksp/Cre transgenic mice leads to renal cyst formation by postnatal day (P)5 (Lin et al, PNAS 2003;100:5286–91). The cyst epithelial cells lack primary cilia, but it is not clear if the loss of cilia precedes cyst formation. To better characterize the time course of the mutant phenotype, we produced Pkhd1/Cre transgenic mice that express Cre recombinase under the control of 4.7 kb of the mouse Pkhd1 (ARPKD) promoter. Pkhd1/Cre mice were crossed with R26R mice that express lacZ after Cre/loxP recombination. X-gal staining of bitransgenic Pkhd1/Cre;R26R progeny at P1 revealed that lacZ was specifically expressed in tubular epithelial cells in the renal collecting ducts, intrahepatic bile ducts, and epididymis. No lacZ expression was observed in embryos at E12.5. Pkhd1/Cre mice were crossed with mice carrying a floxed allele of Kif3a and the R26R reporter gene. Analysis of Pkhd1/Cre;Kif3a;R262R progeny at P7 showed that the kidneys were morphologically normal and that lacZ was expressed in noncystic collecting ducts. Antibody staining for acetylated tubulin showed that primary cilia were absent in the lacZ-positive collecting duct cells in the Kif3a mutant mice but were present in control littermates. Kidney cysts were observed in mutant mice at P14 and progressively increased in size and number at P21, P35, and P56. All cysts were derived from the collecting ducts and were lined by lacZ-positive cyst epithelial cells. The cyst epithelial cells lacked primary cilia and showed apical localization of ZO-1 and basolateral localization of aquaporin 3. Staining for PCNA showed that cyst epithelial cells had higher rates of cell proliferation compared with wild-type collecting duct cells. No morphologic abnormalities were detected in the livers of Kif3a mutant mice. In conclusion, the formation of kidney cysts in Kif3a mutant mice is directly due to inactivation of Kif3a, which produces a loss of renal cilia that precedes tubular dilatation. Pkhd1/Cre transgenic mice should be useful for tissue-specific targeting of other ciliary genes in the kidney, liver, and epididymis.