Loss of Zeb2 in mesenchyme-derived nephrons causes primary glomerulocystic disease
2016; Elsevier BV; Volume: 90; Issue: 6 Linguagem: Inglês
10.1016/j.kint.2016.06.037
ISSN1523-1755
AutoresHila Milo Rasouly, Sudhir Kumar, Stefanie Chan, Anna Pisarek-Horowitz, Richa Sharma, Qiongchao Xi, Yuriko Nishizaki, Yujiro Higashi, David J. Salant, Richard L. Maas, Lu W,
Tópico(s)Renal cell carcinoma treatment
ResumoPrimary glomerulocystic kidney disease is a special form of renal cystic disorder characterized by Bowman's space dilatation in the absence of tubular cysts. ZEB2 is a SMAD-interacting transcription factor involved in Mowat-Wilson syndrome, a congenital disorder with an increased risk for kidney anomalies. Here we show that deletion of Zeb2 in mesenchyme-derived nephrons with either Pax2-cre or Six2-cre causes primary glomerulocystic kidney disease without tubular cysts in mice. Glomerulotubular junction analysis revealed many atubular glomeruli in the kidneys of Zeb2 knockout mice, which explains the presence of glomerular cysts in the absence of tubular dilatation. Gene expression analysis showed decreased expression of early proximal tubular markers in the kidneys of Zeb2 knockout mice preceding glomerular cyst formation, suggesting that defects in proximal tubule development during early nephrogenesis contribute to the formation of congenital atubular glomeruli. At the molecular level, Zeb2 deletion caused aberrant expression of Pkd1, Hnf1β, and Glis3, three genes causing glomerular cysts. Thus, Zeb2 regulates the morphogenesis of mesenchyme-derived nephrons and is required for proximal tubule development and glomerulotubular junction formation. Our findings also suggest that ZEB2 might be a novel disease gene in patients with primary glomerular cystic disease. Primary glomerulocystic kidney disease is a special form of renal cystic disorder characterized by Bowman's space dilatation in the absence of tubular cysts. ZEB2 is a SMAD-interacting transcription factor involved in Mowat-Wilson syndrome, a congenital disorder with an increased risk for kidney anomalies. Here we show that deletion of Zeb2 in mesenchyme-derived nephrons with either Pax2-cre or Six2-cre causes primary glomerulocystic kidney disease without tubular cysts in mice. Glomerulotubular junction analysis revealed many atubular glomeruli in the kidneys of Zeb2 knockout mice, which explains the presence of glomerular cysts in the absence of tubular dilatation. Gene expression analysis showed decreased expression of early proximal tubular markers in the kidneys of Zeb2 knockout mice preceding glomerular cyst formation, suggesting that defects in proximal tubule development during early nephrogenesis contribute to the formation of congenital atubular glomeruli. At the molecular level, Zeb2 deletion caused aberrant expression of Pkd1, Hnf1β, and Glis3, three genes causing glomerular cysts. Thus, Zeb2 regulates the morphogenesis of mesenchyme-derived nephrons and is required for proximal tubule development and glomerulotubular junction formation. Our findings also suggest that ZEB2 might be a novel disease gene in patients with primary glomerular cystic disease. Cystic kidney disease is a group of genetically heterogeneous disorders that are characterized mainly by renal tubular dilatation.1Kurschat C.E. Muller R.U. Franke M. et al.An approach to cystic kidney diseases: the clinician's view.Nat Rev Nephrol. 2014; 10: 687-699Crossref PubMed Scopus (13) Google Scholar, 2Bonsib S.M. The classification of renal cystic diseases and other congenital malformations of the kidney and urinary tract.Arch Pathol Lab Med. 2010; 134: 554-568PubMed Google Scholar Glomerulocystic kidney disease (GCKD) is a special form of cystic kidney disease that confines cystic dilatation to the Bowman's space.3Bissler J.J. Siroky B.J. Yin H. Glomerulocystic kidney disease.Pediatr Nephrol. 2010; 25 (quiz 2056–2049): 2049-2056Crossref PubMed Scopus (41) Google Scholar, 4Lennerz J.K. Spence D.C. Iskandar S.S. et al.Glomerulocystic kidney: one hundred-year perspective.Arch Pathol Lab Med. 2010; 134: 583-605PubMed Google Scholar, 5Bernstein J. Glomerulocystic kidney disease–nosological considerations.Pediatr Nephrol. 1993; 7: 464-470Crossref PubMed Scopus (88) Google Scholar GCKD is defined as a 2- to 3-fold dilatation of Bowman's space in >5% of identifiable glomeruli in the plane of a kidney section, and the glomerular cysts in primary GCKD are mainly localized to the subcapsular region of the kidney.4Lennerz J.K. Spence D.C. Iskandar S.S. et al.Glomerulocystic kidney: one hundred-year perspective.Arch Pathol Lab Med. 2010; 134: 583-605PubMed Google Scholar, 5Bernstein J. Glomerulocystic kidney disease–nosological considerations.Pediatr Nephrol. 1993; 7: 464-470Crossref PubMed Scopus (88) Google Scholar, 6Gusmano R. Caridi G. Marini M. et al.Glomerulocystic kidney disease in a family.Nephrol Dial Transplant. 2002; 17: 813-818Crossref PubMed Scopus (24) Google Scholar Glomerular cysts in association with tubular cysts can be found in several syndromes, including tuberous sclerosis complex, orofaciodigital syndrome 1, and Meckel-Gruber syndrome.3Bissler J.J. Siroky B.J. Yin H. Glomerulocystic kidney disease.Pediatr Nephrol. 2010; 25 (quiz 2056–2049): 2049-2056Crossref PubMed Scopus (41) Google Scholar, 7Feather S.A. Winyard P.J. Dodd S. Woolf A.S. Oral-facial-digital syndrome type 1 is another dominant polycystic kidney disease: clinical, radiological and histopathological features of a new kindred.Nephrol Dial Transplant. 1997; 12: 1354-1361Crossref PubMed Scopus (65) Google Scholar, 8Galliani C.A. Gomez A.M. Panniello G. Bisceglia M. Selected case from the Arkadi M. 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Ellard S. et al.Mutations in the hepatocyte nuclear factor-1beta gene are associated with familial hypoplastic glomerulocystic kidney disease.Am J Hum Genet. 2001; 68: 219-224Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar and in patients with autosomal dominant polycystic kidney disease with PKD1 mutations, other ciliopathies such as nephronophthisis, multicystic dysplastic kidney, or urinary tract obstruction.3Bissler J.J. Siroky B.J. Yin H. Glomerulocystic kidney disease.Pediatr Nephrol. 2010; 25 (quiz 2056–2049): 2049-2056Crossref PubMed Scopus (41) Google Scholar However, the genetic basis of primary GCKD without tubular dilatation remains largely unknown. ZEB2 is a zinc finger E-box–binding homeobox transcription factor that promotes epithelial-mesenchymal transition via a transforming growth factor (TGF)-β/SMAD/ZEB/microRNA (miR)-200 signaling network.12Xiong M. Jiang L. Zhou Y. et al.The miR-200 family regulates TGF-beta1-induced renal tubular epithelial to mesenchymal transition through Smad pathway by targeting ZEB1 and ZEB2 expression.Am J Physiol Renal Physiol. 2012; 302: F369-F379Crossref PubMed Scopus (224) Google Scholar, 13Tang O. Chen X.M. Shen S. et al.MiRNA-200b represses transforming growth factor-beta1-induced EMT and fibronectin expression in kidney proximal tubular cells.Am J Physiol Renal Physiol. 2013; 304: F1266-F1273Crossref PubMed Scopus (71) Google Scholar, 14Gregory P.A. Bracken C.P. Smith E. et al.An autocrine TGF-beta/ZEB/miR-200 signaling network regulates establishment and maintenance of epithelial-mesenchymal transition.Mol Biol Cell. 2011; 22: 1686-1698Crossref PubMed Scopus (441) Google Scholar ZEB2 mutations in humans cause Mowat-Wilson syndrome (MWS; Online Mendelian Inheritance in Man 235730), a congenital disorder characterized by intellectual disability, craniofacial abnormalities, Hirschsprung disease, congenital heart defects, and an increased risk for congenital kidney anomalies.15Garavelli L. Mainardi P.C. Mowat-Wilson syndrome.Orphanet J Rare Dis. 2007; 2: 42Crossref PubMed Scopus (113) Google Scholar However, the pathological features of its kidney defects have not been examined and defined.15Garavelli L. Mainardi P.C. Mowat-Wilson syndrome.Orphanet J Rare Dis. 2007; 2: 42Crossref PubMed Scopus (113) Google Scholar, 16Yamada Y. Nomura N. Yamada K. et al.The spectrum of ZEB2 mutations causing the Mowat-Wilson syndrome in Japanese populations.Am J Med Genet A. 2014; 164A: 1899-1908Crossref PubMed Scopus (31) Google Scholar ZEB2 is also a known target for miR-200.17Gregory P.A. Bert A.G. Paterson E.L. et al.The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1.Nat Cell Biol. 2008; 10: 593-601Crossref PubMed Scopus (3156) Google Scholar Recently, it has been shown that upregulation of ZEB2 in Hnf1b knockout mice and downregulation of miR-200 in Dicer knockout mice are associated with glomerular and tubular cysts.18Hajarnis S.S. Patel V. Aboudehen K. et al.Transcription Factor HNF-1beta regulates microRNA-200 expression through a long noncoding RNA.J Biol Chem. 2015; 290: 24793-24805Crossref PubMed Scopus (39) Google Scholar, 19Patel V. Hajarnis S. Williams D. et al.MicroRNAs regulate renal tubule maturation through modulation of Pkd1.J Am Soc Nephrol. 2012; 23: 1941-1948Crossref PubMed Scopus (76) Google Scholar However, no direct link between ZEB2 downregulation and renal cystic disease has been established, and the pathological effect of ZEB2 downregulation in early nephrogenesis remains unknown. This study reports that deletion of Zeb2 in early mouse nephrogenesis with either Pax2-cre or Six2-cre resulted in primary glomerulocystic disease without tubular dilatation. We found that loss of Zeb2 caused renal proximal tubule hypotrophy and reduced glomerulotubular junction integrity and maturation, which contributed to the formation of congenital atubular glomeruli leading to glomerulocystic disease. By gene expression analysis, we found that Zeb2 nephron-specific knockout kidney had aberrant expression of Pkd1, Hnf1β, and Glis3, which are 3 genes associated with glomerular cysts. These results suggest that Zeb2 regulates the morphogenesis of metanephric mesenchyme derived early nephrons and is required for proximal tubule development and normal glomerulotubular junction formation. Because aberrant expressions of Zeb2 and miR-200 have been reported in young mice with a cystic kidney phenotype, and ZEB2 loss-of-function mutations are associated with an increased risk for renal anomalies in MWS patients,15Garavelli L. Mainardi P.C. Mowat-Wilson syndrome.Orphanet J Rare Dis. 2007; 2: 42Crossref PubMed Scopus (113) Google Scholar, 18Hajarnis S.S. Patel V. Aboudehen K. et al.Transcription Factor HNF-1beta regulates microRNA-200 expression through a long noncoding RNA.J Biol Chem. 2015; 290: 24793-24805Crossref PubMed Scopus (39) Google Scholar, 19Patel V. Hajarnis S. Williams D. et al.MicroRNAs regulate renal tubule maturation through modulation of Pkd1.J Am Soc Nephrol. 2012; 23: 1941-1948Crossref PubMed Scopus (76) Google Scholar we hypothesized that ZEB2 plays an important role in early kidney development. To test this hypothesis, we analyzed a Zeb2 floxed conditional knockout (cKO) mouse line20Higashi Y. Maruhashi M. Nelles L. et al.Generation of the floxed allele of the SIP1 (Smad-interacting protein 1) gene for Cre-mediated conditional knockout in the mouse.Genesis. 2002; 32: 82-84Crossref PubMed Scopus (85) Google Scholar because Zeb2 null mice die at E9.5 before kidney development begins.21Van de Putte T. Maruhashi M. Francis A. et al.Mice lacking ZFHX1B, the gene that codes for Smad-interacting protein-1, reveal a role for multiple neural crest cell defects in the etiology of Hirschsprung disease-mental retardation syndrome.Am J Hum Genet. 2003; 72: 465-470Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar We first crossed the Zeb2 floxed homozygotes (Zeb2flox/flox) with a Pax2-cre+ deleter strain22Ohyama T. Groves A.K. Generation of Pax2-Cre mice by modification of a Pax2 bacterial artificial chromosome.Genesis. 2004; 38: 195-199Crossref PubMed Scopus (202) Google Scholar that expresses the Cre recombinase in both the metanephric mesenchyme and ureteric bud (Supplementary Figure S1a). After genotyping 96 3-week old weanlings of Zeb2flox/+;Pax2-cre+ heterozygous matings, we did not find any Zeb2flox/flox;Pax2-cre+ homozygotes (Supplementary Figure S1b). We then analyzed newborn mice and E18.5 embryos from timed-pregnant females. Five dead newborn Zeb2flox/flox;Pax2-cre+ homozygotes and 21 of 55 (38%) E18.5 homozygous embryos were found (Supplementary Figure S1b), which suggest that Zeb2flox/flox;Pax2-cre+ homozygotes die at birth. The E18.5 Zeb2flox/flox;Pax2-cre+ homozygous embryos did not display discernible gross structural defects of the kidney or ureter. However, histologic examination of the kidneys from 8 E18.5 Zeb2flox/flox;Pax2-cre+ homozygous embryos revealed that 100% of the homozygotes had renal cysts, whereas none of the 7 littermate controls (0%) had a cystic phenotype (Table 1 and Figure 1). The renal cysts first appeared at E16.5 as no cysts could be detected at E15.5 (Table 1 and Figure 1). The renal cysts were apparently from a glomerular origin because 47% of the glomeruli were cystic in the E16.5 Zeb2 cKO embryos, and 30% of the cysts had a visible glomerular tuft (Tables 1 and 2). This phenotype is sufficient for a diagnosis of GCKD according to the established criteria.4Lennerz J.K. Spence D.C. Iskandar S.S. et al.Glomerulocystic kidney: one hundred-year perspective.Arch Pathol Lab Med. 2010; 134: 583-605PubMed Google ScholarTable 1Cystic phenotype observed in hematoxylin and eosin–stained kidney samples of Zeb2flox/flox;Pax2-cre+ and wild-type littermates between E16.5 and E18.5E16.5E17.5E18.5Zeb2+/+Zeb2flox/flox; Pax2-cre+Zeb2+/+Zeb2flox/flox; Pax2-cre+Zeb2+/+Zeb2flox/flox; Pax2-cre+Embryos with kidney cysts (>1 cyst)0/2 (0%)3/3 (100%)0/3 (0%)3/3 (100%)0/7 (0%)8/8 (100%)Glomerular cysts/number of glomeruli1/482%, (n = 4)26/5547%, (n = 5)0/400%, (n = 3)18/5930.5%, (n = 4)1/3630.2%, (n = 13)61/39115.6%, (n = 15)n = number of kidneys analyzed. Open table in a new tab Table 2Percentage of cysts with glomerular tufts on hematoxylin and eosin–stained kidney samples between E16.5 and E18.5Cysts with glomerular tufts/total cystsZeb2flox/flox;Pax2-cre+E16.526/85 (30%, n = 5)E17.518/46 (39%, n = 4)E18.561/226 (27%, n = 15)n = number of kidneys analyzed. Open table in a new tab n = number of kidneys analyzed. n = number of kidneys analyzed. To determine whether Zeb2 cKO embryonic kidneys also develop renal tubular cysts, we examined the kidney sections with both proximal tubule specific marker Lotus tetragonolobus lectin (LTL) and distal tubules and collecting duct specific marker Dolichos biflorus agglutinin (DBA). The cysts were negative for both LTL and DBA staining at E18.5 (Figure 1c and d), which suggest that the Zeb2flox/flox;Pax2-cre+ homozygous embryos develop primary glomerular cysts. To determine if glomerular cyst formation is due to a deletion of Zeb2 in the developing nephron, we examined ZEB2 expression during kidney development by analyzing a ZEB2-EGFP reporter mouse that was studied previously.23Nishizaki Y. Takagi T. Matsui F. Higashi Y. SIP1 expression patterns in brain investigated by generating a SIP1-EGFP reporter knock-in mouse.Genesis. 2014; 52: 56-67Crossref PubMed Scopus (15) Google Scholar In addition to the stromal cells, ZEB2 was detected in a subset of cells in which it could be deleted by Pax2-cre in the developing nephron (Supplementary Figure S2), including the S-shaped bodies (Supplementary Figure S2e) and the glomeruli (Supplementary Figure S2f). By co-staining, we confirmed that ZEB2 is co-expressed in a subset of cells with PAX2 and JAG1, which are 2 markers of developing nephrons (Supplementary Figure S3). Zeb2flox/flox;Pax2-cre+ homozygous mice died at birth (probably due to high expression of Pax2-cre in the nervous system, where ZEB2 also plays an important role),22Ohyama T. Groves A.K. Generation of Pax2-Cre mice by modification of a Pax2 bacterial artificial chromosome.Genesis. 2004; 38: 195-199Crossref PubMed Scopus (202) Google Scholar, 23Nishizaki Y. Takagi T. Matsui F. Higashi Y. SIP1 expression patterns in brain investigated by generating a SIP1-EGFP reporter knock-in mouse.Genesis. 2014; 52: 56-67Crossref PubMed Scopus (15) Google Scholar precluding a longitudinal analysis of GCKD progression after birth. A recent study shows that SIX2 regulates ZEB2 expression through miR-200.24Wang C.A. Drasin D. Pham C. et al.Homeoprotein Six2 promotes breast cancer metastasis via transcriptional and epigenetic control of E-cadherin expression.Cancer Res. 2014; 74: 7357-7370Crossref PubMed Scopus (37) Google Scholar SIX2 is also a nephron progenitor marker and is expressed in the metanephric mesenchyme that gives rise to all segments of the mature nephron, including the parietal and visceral epithelial cells in the glomeruli and the proximal and distal tubular epithelial cells.25Kobayashi A. Valerius M.T. Mugford J.W. et al.Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development.Cell Stem Cell. 2008; 3: 169-181Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar To further delineate the role of ZEB2 in nephron development and to study the pathologic effect of Zeb2 deletion in mesenchyme-derived nephrons, as well as glomerular cystic phenotype in the postnatal mature kidney, we crossed Zeb2flox/flox mice with Six2-cre mice. The Zeb2flox/flox;Six2-cre+ homozygous mice survived after birth and were weaned at a Mendelian distribution. Histologic examination of the kidneys from 9 Zeb2flox/flox;Six2-cre+ homozygous embryos revealed that, like the Zeb2flox/flox;Pax2-cre+ embryos, all homozygous embryos had glomerular cysts starting at E16.5 (Figure 2a). At postnatal day 12, the glomerular cysts in the Zeb2flox/flox;Six2-cre+ mice were mainly located in the subcapsular region of the kidney, which is consistent with the diagnosis of primary GCKD (Figure 2b).4Lennerz J.K. Spence D.C. Iskandar S.S. et al.Glomerulocystic kidney: one hundred-year perspective.Arch Pathol Lab Med. 2010; 134: 583-605PubMed Google Scholar The cysts were also negative for both LTL and DBA staining (Figure 2c and d), which confirmed their glomerular origin. In comparison, none of the 5 wild-type littermate controls had a cystic phenotype at postnatal day 12 (Figure 2b). To determine the longitudinal effect of GCKD on a mature kidney, we followed the Zeb2flox/flox;Six2-cre+ mice to adulthood. At 7 weeks of age, all Zeb2flox/flox;Six2-cre+ mice (n = 5; 100%) developed macroscopic renal cysts that were visualized on the surface of the kidney (Figure 3a). Histologic analysis revealed the presence of numerous large glomerular cysts in the area of the renal cortex and outer medulla (Figure 3b). Although no significant interstitial fibrosis was observed (Figure 4a), some remaining noncystic glomeruli in adult Zeb2flox/flox;Six2-cre+ mice displayed glomerulosclerosis lesions (Figure 4b). Similar lesions were also observed in few noncystic glomeruli in postnatal day 8 Zeb2 cKO kidneys (Supplementary Figure S4). By 8 weeks of age, all Zeb2flox/flox;Six2-cre+ mice (n = 5; 100%) developed albuminuria (Figure 4c and 4d) with significantly elevated serum blood urea nitrogen levels (Figure 4e). Albuminuria was not directly caused by loss of ZEB2 in podocytes as deletion of Zeb2 specifically in the podocyte using Nphs2-cre did not lead to proteinuria (data not shown). The expressions of megalin and cubilin were upregulated in Zeb2 adult cKO kidneys (Supplementary Figure S5), which might not decrease proximal tubule endocytosis of albumin.26Terryn S. Tanaka K. Lengele J.P. et al.Tubular proteinuria in patients with HNF1alpha mutations: HNF1alpha drives endocytosis in the proximal tubule.Kidney Int. 2016; 89: 1075-1089Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar These data suggest that loss of Zeb2 in mesenchyme-derived nephrons alone was sufficient to cause glomerulocystic disease in mature kidneys, which leads to secondary glomerulosclerosis, albuminuria, and renal failure in adult mice.Figure 4Mesenchyme-specific deletion of Zeb2 with Six2-cre causes glomerulosclerosis, albuminuria and renal failure in adult mice. (a) Masson Trichrome staining (MTS) shows minimal fibrosis (blue) in the kidney of a 7-week-old Zeb2flox/flox;Six2-cre+ mouse. Upper panels, original magnification ×100; lower panels, high magnification of boxed regions in the upper panels (n = 3 for each group). (b) Periodic-acid Schiff (PAS) staining shows glomerulosclerosis (arrow) in the noncystic glomeruli of a 7-week-old Zeb2flox/flox;Six2-cre+ mouse kidney. Upper panels, original magnification ×100; lower panels, high magnification of boxed regions in the upper panels (n = 3 for each group). (c) Representative sodium dodecylsulfate–polyacrylamide gel electrophoresis gel with Coomassie blue staining shows albuminuria (arrow) in 2 Zeb2flox/flox;Six2-cre+ 5-week-old mice but not in 2 littermate controls (n = 5 mice in each group); (d) Increased albumin-to-creatinine ratio (ACR) in 5-week-old Zeb2flox/flox;Six2-cre+ mice compared to Zeb2+/+ littermate controls (n = 4 mice in each group; *P < 0.05). (e) Significantly elevated blood urea nitrogen (BUN) in 5-week-old Zeb2flox/flox;Six2-cre+ mice compared with Zeb2+/+ littermate controls (n = 3 mice in each group; **P < 0.001). Data are represented as means ± SD. alb, albumin.View Large Image Figure ViewerDownload (PPT) One common cause of acquired glomerular cysts is the loss of glomerulotubular junction integrity leading to atubular glomeruli.27Chevalier R.L. Forbes M.S. Generation and evolution of atubular glomeruli in the progression of renal disorders.J Am Soc Nephrol. 2008; 19: 197-206Crossref PubMed Scopus (91) Google Scholar, 28Gibson I.W. Downie T.T. More I.A. Lindop G.B. Atubular glomeruli and glomerular cysts–a possible pathway for nephron loss in the human kidney?.J Pathol. 1996; 179: 421-426Crossref PubMed Scopus (39) Google Scholar Glomerulotubular integrity can be quantified by examining the connection of the proximal tubule to the Bowman's capsule with the proximal tubule marker LTL.29Forbes M.S. Thornhill B.A. Chevalier R.L. Proximal tubular injury and rapid formation of atubular glomeruli in mice with unilateral ureteral obstruction: a new look at an old model.Am J Physiol Renal Physiol. 2011; 301: F110-F117Crossref PubMed Scopus (88) Google Scholar To determine whether the Zeb2 knockout mice have decreased glomerulotubular integrity, we examined glomeruli from 5 postnatal day 12 Zeb2flox/flox;Six2-cre+ mice and 5 littermate controls by serial sections. We found that only 63 of 604 (10%) glomeruli had a visible LTL positive staining (i.e., glomerulotubular junction) in the Zeb2 cKO mice compared with 115 of 324 (36%) in the wild-type littermate controls (Figure 5), which was a statistically significant decrease of glomerulotubular integrity. This suggests that Zeb2 deletion causes formation of atubular glomeruli. Acquired atubular glomeruli can be caused by atrophy of the proximal tubules in polycystic kidney disease.30Galarreta C.I. Grantham J.J. Forbes M.S. et al.Tubular obstruction leads to progressive proximal tubular injury and atubular glomeruli in polycystic kidney disease.Am J Pathol. 2014; 184: 1957-1966Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar To determine if reduced glomerulotubular integrity in Zeb2 cKO is associated with renal proximal tubule atrophy (acquired defect) or hypotrophy (developmental defect), we analyzed the mRNA levels of markers for the proximal tubule (Hnf1a and Vil1), the podocyte (Nphs1 and Nphs2), and the collecting duct (Upk3a).31Massa F. Garbay S. Bouvier R. et al.Hepatocyte nuclear factor 1beta controls nephron tubular development.Development. 2013; 140: 886-896Crossref PubMed Scopus (95) Google Scholar We found that only the proximal tubule mRNA markers were significantly reduced in the Zeb2flox/flox;Six2-cre+ mice compared with the levels in the wild-type littermates at postnatal day 8 (Figure 6a). To determine if the reduction of proximal tubule mRNA at postnatal day 8 is caused by an early developmental defect preceding the formation of glomerular cysts in Zeb2 cKO mice, we analyzed mRNA markers in E14.5 kidneys. Similar to postnatal day 8, a significant decrease of the proximal tubular markers was detected in the E14.5 Zeb2flox/flox;Six2-cre+ kidneys compared with their wild-type littermates (Figure 6b). Consistent with this finding, the expression of the proximal tubule brush border protein villin 1 (VIL1),31Massa F. Garbay S. Bouvier R. et al.Hepatocyte nuclear factor 1beta controls nephron tubular development.Development. 2013; 140: 886-896Crossref PubMed Scopus (95) Google Scholar was also downregulated in both E15.5 Zeb2flox/flox;Pax2-cre+ and E16.5 Zeb2flox/flox;Six2-cre+ mutant kidneys (Figure 6c and d). Finally, the mean kidney size of the E16.5 Zeb2 cKO embryos was smaller than that of their wild-type littermate controls (Figure 6e). Taken together, these data suggest that loss of Zeb2 in the mesenchyme-derived nephrons causes early proximal tubule developmental defects, which results in tubular hypotrophy, reduced glomerulotubular junction integrity, and congenital atubular glomeruli formation. Glomerular cysts are reported in several mouse models of renal cystic kidney disease, including Wwtr1, Glis3, Ofd1, and Pkhd1 knockout mice, the Hnf1β and Dicer cKO mice, and Pkd1 over expression transgenic mice.19Patel V. Hajarnis S. Williams D. et al.MicroRNAs regulate renal tubule maturation through modulation of Pkd1.J Am Soc Nephrol. 2012; 23: 1941-1948Crossref PubMed Scopus (76) Google Scholar, 31Massa F. Garbay S. Bouvier R. et al.Hepatocyte nuclear factor 1beta controls nephron tubular development.Development. 2013; 140: 886-896Crossref PubMed Scopus (95) Google Scholar, 32Pritchard L. Sloane-Stanley J.A. 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Ko H.L. et al.Glomerulocystic kidney disease in mice with a targeted inactivation of Wwtr1.Proc Natl Acad Sci U S A. 2007; 104: 1631-1636Crossref PubMed Scopus (223) Google Scholar Interestingly, the Hnf1β cKO mice using Six2-cre develop glomerular cysts due to a drastic reduction in the levels of proximal tubular markers at E14.5, resembling the Zeb2 cKO phenotype.31Massa F. Garbay S. Bouvier R. et al.Hepatocyte nuclear factor 1beta controls nephron tubular development.Development. 2013; 140: 886-896Crossref PubMed Scopus (95) Google Scholar However, by immunostaining of JAG1, a marker for the renal vesicle and the S-shaped body,31Massa F. Garbay S. Bouvier R. et al.Hepatocyte nuclear factor 1beta controls nephron tubular development.Development. 2013; 140: 886-896Crossref PubMed Scopus (95) Google Scholar we did not observe differences between Zeb2 cKO and wild-type littermate controls (Supplementary Figure S6). To determine if loss of Zeb2 affects the expression of these 6 genes and the miR-200, we examined mRNA and miR levels in the kidney tissues of E14.5 and E18.5 Zeb2 cKO and wild-type controls. Although there was no significant difference in the expression levels of any of the 6 genes and miR-200 at E14.5 before glomerular cyst formation (Figure 7a), we detected a decreased expression of Glis3 and increased expression of Hnf1β and Pkd1 levels in E18.5 Zeb2 cKO compared with the wild-type littermate controls (Figure 7b). The expression level of Pkd1 mRNA was the most significantly upregulated in the Zeb2 cKO kidneys at postnatal day 8 (Figure 7c). Consistent with the mRNA levels, PKD1 coding protein polycystin-1 (PC1) was also found to be expressed at a higher level in the glomeruli of postnatal day 7 (P7) Zeb2flox/flox;Six2-cre+ mice but not at E16.5 and E17.5 when the glomerular cysts were initially observed (Figure 7d). Interestingly, the PC1 expression was upregulated in noncystic glomeruli but not in the glomeruli with a dilated Bowman's space (Figure 7e). These data suggest that loss of Zeb2 in the kidney leads to upregulation of polycystin-1 expression in noncystic glomeruli after the initial phase of glomerular cyst formation. Renal cystogenesis is often associated with increased cell proliferation.37Paul B.M. Vanden Heuvel G.B. Kidney: polycystic kidney disease.Wiley Interdiscip Rev Dev Biol. 2014; 3: 465-487Crossref PubMed Scopus (51) Google Scholar To determine if abnormal cell proliferation also plays a role in the formation of glomerular cysts in Zeb2 cKO mice, we quant
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