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. 2021 May 1;14(5):dmm047498.
doi: 10.1242/dmm.047498. Epub 2021 May 4.

Hnf1b haploinsufficiency differentially affects developmental target genes in a new renal cysts and diabetes mouse model

Leticia L Niborski  1 , Mélanie Paces-Fessy  1 , Pierbruno Ricci  1 , Adeline Bourgeois  1 , Pedro Magalhães  2   3 , Maria Kuzma-Kuzniarska  1 , Celine Lesaulnier  1 , Martin Reczko  4 , Edwige Declercq  1 , Petra Zürbig  2 , Alain Doucet  5 , Muriel Umbhauer  1 , Silvia Cereghini  1
Affiliations

Hnf1b haploinsufficiency differentially affects developmental target genes in a new renal cysts and diabetes mouse model

Leticia L Niborski et al. Dis Model Mech. .

Abstract

Heterozygous mutations in HNF1B cause the complex syndrome renal cysts and diabetes (RCAD), characterized by developmental abnormalities of the kidneys, genital tracts and pancreas, and a variety of renal, pancreas and liver dysfunctions. The pathogenesis underlying this syndrome remains unclear as mice with heterozygous null mutations have no phenotype, while constitutive/conditional Hnf1b ablation leads to more severe phenotypes. We generated a novel mouse model carrying an identified human mutation at the intron-2 splice donor site. Unlike heterozygous mice previously characterized, mice heterozygous for the splicing mutation exhibited decreased HNF1B protein levels and bilateral renal cysts from embryonic day 15, originated from glomeruli, early proximal tubules (PTs) and intermediate nephron segments, concurrently with delayed PT differentiation, hydronephrosis and rare genital tract anomalies. Consistently, mRNA sequencing showed that most downregulated genes in embryonic kidneys were primarily expressed in early PTs and the loop of Henle and involved in ion/drug transport, organic acid and lipid metabolic processes, while the expression of previously identified targets upon Hnf1b ablation, including cystic disease genes, was weakly or not affected. Postnatal analyses revealed renal abnormalities, ranging from glomerular cysts to hydronephrosis and, rarely, multicystic dysplasia. Urinary proteomics uncovered a particular profile predictive of progressive decline in kidney function and fibrosis, and displayed common features with a recently reported urine proteome in an RCAD pediatric cohort. Altogether, our results show that reduced HNF1B levels lead to developmental disease phenotypes associated with the deregulation of a subset of HNF1B targets. They further suggest that this model represents a unique clinical/pathological viable model of the RCAD disease.

Keywords: Gene dosage; Glomerular and proximal tubule cysts; HNF1B transcription factor; Mouse models; RCAD syndrome; Transcriptomics.

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Conflict of interest statement

Competing interests The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Expression levels of normal Hnf1b transcripts and protein from WT and Hnf1Sp2/+ heterozygous mutants. (A) qRT-PCR of normal Hnf1b transcripts in WT and Hnf1bSp2/+ kidneys at the indicated stages. WT versus Hnf1bSp2/+ sample numbers were as follows: E14.5, n=5 vs n=3; E15.5, n=9 vs n=5; E16.5, n=4 vs n=9; E17.5, n=4 vs n=6; P0, n=4 vs n=8; adult 3 months, n=7 vs n=7; adult 5 months, n=8 vs n=6. Significant decreases in Hnf1b transcript relative to WT were at E14.5 (58.5%), E15.5 (57%), E16.5 (50%) and P0 (64%). NS, not significant. (B) Western blot quantification of HNF1B protein levels in Hnf1bSp2/+ relative to WT. Significant decreases to 70%, 62%, 72% and 69% relative to WT were observed at E15.5, E17.5, P0 and 5 months, respectively. (C) Western blot quantification of 3-month-old WT and heterozygous Hnf1bLacZ/+ show a 98% increase in HNF1B levels in the mutants. Error bars represent s.e.m. Unpaired Student's t-test, *P<0.05, **P<0.01, ***P<0.001. (D) HNF1B immunostaining of E15.5 and E17.5 embryo sections show, in Hnf1bSp2/+ kidneys, a global decrease in the number of HNF1B+ structures, together with decreased nuclear staining in some regions (inset in E17.5). Note adjacent pancreatic ducts exhibiting decreased HNF1B expression (arrows), glomerular cysts (asterisks), tubular dilatations (cy) and HNF1B nuclear staining in both non-dilated and dilated renal tubules. Scale bar: 200 μm.
Fig. 2.
Fig. 2.
Histology of Hnf1bSp2/+ embryos from different genetic backgrounds highlights the early onset of renal cysts and hydronephrosis. (A-F) Representative Hematoxylin and Eosin (H&E)-stained sections of embryo kidneys show glomerular cysts and tubular dilatations in the medulla at E15.5 and E17.5 (arrows in E′, higher magnification of E). Arrow in F′ (higher magnification of F) shows cystic glomeruli. Embryos were from a mixed background (F1). (G-N) Inbreeding onto C57BL/6N and 129/sv backgrounds showed different phenotype severities. Note in the C57BL/6N background, pelvic dilatations at E17.5 (H), hydronephrosis and duplicated kidney at P0 (H,J) (see also Fig. S4), in addition to cystic glomeruli and medullar tubules dilatations (H,J), also observed in the 129/sv background (L,N). Images are representative of n=6 for each genotype. Scale bars: 200 μm.
Fig. 3.
Fig. 3.
Hnf1bSp2/+ embryo kidneys exhibit normal ureteric bud branching but glomerular cysts and delayed proximal tubule (PT) differentiation. (A-H′) Immunohistochemical analysis of WT and Hnf1bSp2/+ embryo kidneys with Pax2 (A,A′), HNF1B (C,C′), the PT markers HNF4A (E,E′,G,G′), HNF1A (B,B′) and LTA (F,F′), WT1 (D,D′, inset shows glomerular cyst magnification with partially disorganized podocyte expression, and the collecting duct lectin DBA (H,H′) at the indicated stages. Note in Hnf1bSp2/+ sections decreased HNF4A+ PT structures (E′,G′) and PT dilatations, particularly at E15.5 (E′), which correlate with HNF1B expression in a serial section (C′) of the same embryo, decreased HNF1A PT expression together with increased acinar pancreatic expression (B′), and the absence of lectins LTA (F′) and DBA (H′). Sections are co-stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars: 200 μm.
Fig. 4.
Fig. 4.
Glomerular cyst development, progressive tuft atrophy and abnormal PT implantation in Hnf1bSp2/+ embryos. (A-I) Histological comparison with human HNF1B mutant fetuses. Immunohistochemical analysis of E16.5 WT (A-C) and Hnf1bSp2/+ embryos (D-I). PTs are highlighted by the co-expression of HNF4A and HNF1B; developing glomeruli and condensed mesenchyme are shown by WT1 staining. Sections are co-stained with DAPI. Note that HNF4A staining in Hnf1bSp2/+ (E,H) was intentionally increased to better visualize positive PTs. Note in Hnf1bSp2/+ dilated PTs that were HNF4A+ (E,H), glomerular cysts with disorganized podocyte layer stained by WT1 (white arrowheads); non-cystic glomeruli show normal podocyte layer (green arrowheads). Yellow arrowheads (H,I) show abnormal lateral insertion of the glomerulotubular junction into Bowman's capsule and disorganized HNF4A expression (H). (J-P) H&E-staining of E17.5 Hnf1bSp2/+ embryos (J-L) and human mutant fetuses described in Haumaitre et al. (2006) (M-P). Black arrowheads indicate disorganized layer cells in mutants and human fetuses (K,O). Note also collapsed or disrupted tufts inside highly widened Bowman's capsules (L,P). Scale bars: 50 μm.
Fig. 5.
Fig. 5.
Cystic and dilated PT and thick loop of Henle cells exhibit abnormal apico-basal polarity and reduced number of cilia. (A-I) Confocal microscopy of co-stained sections with Vil1-HNF4A and Vil1-HNF1B, revealed that Vil1 expression (A-F), localized at the PT brush border, is interrupted in several regions of Hnf1bSp2/+ cystic structures, indicative of brush-border loss (magnifications in C and F). Confocal images of acetylated tubulin staining show some regions with cells devoid of primary cilia in E15.5 Hnf1bSp2/+ cystic PTs, while non-dilated tubules have apparent normal cilia distribution compared with WT (compare H,I with G). (J-M) Confocal images of Muc1-Lam1 and Lam1-ZO1 co-immunohistochemistry show that the collecting duct apical marker Muc1 in E15.5 Hnf1bSp2/+ (K) is expressed similar to WT (J), while the basement membrane Lam1 exhibits a partially disorganized pattern (K,M) compared to WT kidneys (J,L). The tight junction protein ZO1 (L,M) is normally expressed. (N-Q) Co-stained P0 kidney sections with NKCC2 (N,O) and merged NKCC2-CK (P,Q) show decreased staining of NKCC2 in Hnf1bSp2/+cystic medullar loops of Henle (O,Q) compared with WT (N,P). Apical CK expression in collecting ducts (P,Q) is not affected. Confocal images were captured with a ×20 objective (A-M). Optical pictures were captured with a ×20 (N-Q) objective. Scale bars: 50 μm.
Fig. 6.
Fig. 6.
Differential expression analysis of Hnf1bSp2/+ mutant versus WT kidneys at different embryonic stages. (A) Venn diagram showing the overlap of genes differentially expressed from E14.5 to P0. (B) Gene ontology (GO) analysis of downregulated genes showing the top enriched terms (Table S4). (C) Venn diagrams show the overlap of differentially expressed genes of E17.5 Hnf1bSp2/+ and P0 Hnf4a knockout (KO) kidneys (Marable et al., 2020). Also indicated are HNF1 and HNF4 common target genes (red) and HNF1 targets (blue).
Fig. 7.
Fig. 7.
Reduced expression of a subset of HNF1B targets in Hnf1bSp2/+ kidneys. (A-D′) Representative immunostainings of E17.5 WT (A,B,C,D) and Hnf1bSp2/+ kidneys (A′,B′,C′,D′) show strongly reduced expression in Hnf1bSp2/+ kidneys of TMEM27, CUBN and SPP1 (A′,B′,C′), whereas LRP2 expression (D′) is moderately reduced. (E) qRT-PCR analysis of selected HNF1B targets at different stages show, consistent with mRNA-seq data, significant and strong downregulation of Hnf4a, Cubn, Lrp2, Tmem27, Kcnj1, Umod, Slc22a6 and Slc3a1 during embryo stages up to P0. Also shown is qRT-PCR of adult mice: vertical line separates 3-month-old from two 6-month-old samples. Cubn and Tmem27 remained downregulated in adults, whereas Aqp2, downregulated at P0, becomes significantly upregulated in adults (6 months). Note downregulation of the target gene Nr1h4 in adults (6 months). The numbers (N) of WT and Hnf1bSp2/+ samples used at each stage are indicated. Unpaired Student's t-test, *P<0.05, **P<0.01 and ***P<0.001. Scale bars: 200 μm.
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