GSE159566
mRNA sequencing of embryonic kidney at different stages from wild-type and heterozygous mutants of a novel mouse model carrying a human splicing point-mutation in the Hnf1b-gene
Heterozygous mutations in HNF1B cause the complex syndrome Renal Cysts and Diabetes (RCAD), characterized by developmental abnormalities of kidney, genital tracts and pancreas and a variety of renal, pancreas and liver dysfunctions. The pathogenesis underlying this syndrome remained unclear since mice with Hnf1b heterozygous null mutations have no phenotype, while constitutive/conditional Hnf1b-ablation led to more severe phenotypes. We generated a novel mouse model carrying an identified human mutation at the intron-2 splice donor-site. Reflecting the disease and unlike previous heterozygous, heterozygous for the splicing mutation exhibited decreased HNF1B protein levels and bilateral renal cysts from embryonic stage E15, originated from glomeruli, early proximal tubules (PT) and intermediate nephron segments, concurrently with a delayed PT differentiation, hydronephrosis and rare genital tract anomalies. To further elucidate the cellular and molecular components sensitive to HNF1B levels, we performed RNA-sequencing on WT and heterozygous mutant kidneys at E14.5 (considered morphologically as pre-disease kidneys) and at disease stages (E15.5, E17.5 and P1).   Consistent with the histological and immunhistochemical findings, these analysis showed that most down-regulated genes in embryonic kidneys were primarily expressed in early PTs and Henle's Loop and involved in ion/drug transport, organic acid and lipid metabolic processes, while previous targets identified upon Hnf1b-ablation, including the cystic disease genes, were weakly or no 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, injury and fibrosis, and displayed common features with a recently reported urine proteome in a RCAD pediatric cohort. Our results show that HNF1B reduced levels lead to developmental disease phenotypes associated with the deregulation of a subset of its targets and further suggests that this new model represents a unique clinical/pathological viable model of the RCAD disease. Overall design: Stages analyzed: E14.5 (mutant kidneys were morphologically normal and considered as pre-disease stage) and at disease stages E15.5, E17.5 and postnatal day 1 (P1), presented various renal abnormalities (bilateral renal cysts from E15, originated from glomeruli, early proximal tubules (PT) and intermediate nephron segments, concurrently with a delayed PT differentiation, hydronephrosis. At E15.5 deep sequencing was from pooled samples of 3 WT and 3 Hnf1b Sp2/+ (6 kidneys each sample). At E17.5 kidneys were from embryos of 2 litters descendants of the same male and born de same day, allowing to pool n=3 (6 kidneys) for one of the two WT sample and the two heterozygous mutants   Total 14 samples (N : number of embryos or newborns (2 kidneys for each one) = E14.5: (4 samples) SCR10-E145-4wt( n=1), SCR13-E14.5wt (n=1), SCR12-E145 HET(n=1), SCR15-E145-HET (n=1); E15.5: (2 samples): SCR 2 WT(n=3), SCR1 Hnf1bSp2/+ ( n=3) ; E17.5 : (4 samples) GZS 17 WT (n=1), GZS 35WT(n=3), GSZ 34 Hnf1bSp2/+ (n=3), GSZ 36 Hnf1bSp2/+ (n=3) ; P1 : SCR20_P1_WT(n=1), SCR21_P1_WT (n=1), SCR22 P1 Hnf1bSp2/+ (n=1), SCR23_P1 Hnf1bSp2/+ (n=1).