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Molecular detection of maturation stages in the developing kidney

ABSTRACT: To identify maturation-dependent genes, we here performed single cell RNA sequencing (scRNA-seq) analysis using developing kidneys at different stages in the mouse, followed by highly sensitive in situ hybridization. We identified multiple genes expressed abundantly in newborn kidneys, but minimally at embryonic day 15.5. We then applied these maturation markers to the transplanted embryonic kidneys and found that the maturation process did not occur equally throughout nephron segments upon transplantation: glomeruli and proximal renal tubules became more mature than the other nephron segments. Thus, our scRNA-seq data at multiple stages of kidney development and identified maturation-dependent genes will serve as useful bases for assessing maturation of the developing kidney, and eventually of kidney organoids.

ORGANISM(S): Mus musculus

PROVIDER: GSE149134 | GEO | 2020/11/19

REPOSITORIES: GEO

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Project description:To gain molecular insight into kidney function, we performed a high-resolution quantitative analysis of gene expression in glomeruli and nine different nephron seg-ments dissected from mouse kidney using the long-SAGE method. We also developed dedicated bioinformatics tools and databases to annotate mRNA tags as transcripts. Over 800,000 mRNA SAGE tags were sequenced corresponding to >20,000 different mRNA tags present at least twice in at least one library. Hierarchical clustering analysis of tags demonstrated similarities between the three anatomical sub-segments of the proximal tubule, between the cortical and medullary segments of the thick ascending limb of Henle’s loop, and between the three segments constituting the aldosterone sensitive distal nephron segments, whereas the glomerulus and distal convoluted tubule clusterized independently. We also identified highly specific mRNA markers of each subgroup of nephron segments and of most nephron segments. Tag annotation also identified numbers of putative antisense mRNAs. This database constitutes a reference resource in which the quantitative expression of a given gene can be compared with that of other genes in the same nephron segment, or between different segments of the nephron. To illustrate possible applications of this database, we performed a deeper analysis of the glomerulus transcriptome which unexpectedly revealed expression of several ion and water carriers; within the glomerulus, they were found to be preferentially expressed in the parietal sheet. It also revealed the major role of the zinc finger transcription factor Wt1 in the specificity of gene expression in the glomerulus. Finally, functional annotation of glomerulus-specific transcripts showed the high proliferation activity of glomerular cells. Immunolabelling with anti-PCNA antibodies confirmed a high percentage of proliferating glomerular parietal cells. Approximately 1000 tubules from each different nephron segments were microdissected from 6-8 male CD1 mice. Over 800,000 mRNA SAGE tags were sequenced corresponding to >20,000 different mRNA tags present at least twice in at least one library.

Project description: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.

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Molecular detection of maturation stages in the developing kidney

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