nuclei-isolation-from-adult-mouse-kidney-for-single-nucleus-rna

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin

Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC)

Grainyhead transcription factors control epithelial barriers, tissue morphogenesis, and differentiation, but their role in the kidney is poorly understood. Here, we report that nephric duct, ureteric bud, and collecting duct epithelia express high levels of grainyhead-like homolog 2 (Grhl2) and that nephric duct lumen expansion is defective in Grhl2-deficient mice. In collecting duct epithelial cells, Grhl2 inactivation impaired epithelial barrier formation and inhibited lumen expansion. Molecular analyses showed that GRHL2 acts as a transcriptional activator and strongly associates with histone H3 lysine 4 trimethylation. Integrating genome-wide GRHL2 binding as well as H3 lysine 4 trimethylation chromatin immunoprecipitation sequencing and gene expression data allowed us to derive a high-confidence GRHL2 target set. GRHL2 transactivated a group of genes including Ovol2, encoding the ovo-like 2 zinc finger transcription factor, as well as E-cadherin, claudin 4 (Cldn4), and the small GTPase Rab25. Ovol2 induction alone was sufficient to bypass the requirement of Grhl2 for E-cadherin, Cldn4, and Rab25 expression. Re-expression of either Ovol2 or a combination of Cldn4 and Rab25 was sufficient to rescue lumen expansion and barrier formation in Grhl2-deficient collecting duct cells. Hence, we identified a Grhl2/Ovol2 network controlling Cldn4 and Rab25 expression that facilitates lumen expansion and barrier formation in subtypes of renal epithelia. 

A Grainyhead-Like 2/Ovo-Like 2 Pathway Regulates Renal Epithelial Barrier Function and Lumen Expansion.

Proper renal function relies on the tightly regulated development of nephrons and collecting ducts. This process, known as tubulogenesis, involves dynamic cellular and molecular changes that instruct cells to form highly organized tubes of epithelial cells which compartmentalize the renal interstitium and tubular lumen via assembly of a selective barrier. The integrity and diversity of the various renal epithelia is achieved via formation of intercellular protein complexes along the apical-basal axis of the epithelial cells. In recent years, the evolutionarily conserved family of Grainyhead-like (GRHL) transcription factors which encompasses three mammalian family members (Grainyhead-like 1, 2, 3) has emerged as a group of critical regulators for organ development, epithelial differentiation, and barrier formation. Evidence from transgenic animal models supports the presence of Grainyhead-like-dependent transcriptional mechanisms that promote formation and maintenance of epithelial barriers in the kidney. In this review, we highlight different Grhl-dependent mechanisms that modulate epithelial differentiation in the kidney. Additionally, we discuss how disruptions in these mechanisms result in impaired renal function later in life.

Functional roles of Grainyhead-like transcription factors in renal development and disease.

Mix for Regeneration: Nephron Replacement by Transplanted Cells.

Single-cell transcriptomes from dissociated tissues provide insights into cell types and their gene expression and may harbor additional information on spatial position and the local microenvironment. The kidney's cells are embedded into a gradient of increasing tissue osmolality from the cortex to the medulla, which may alter their transcriptomes and provide cues for spatial reconstruction.

Article	Year
A Grainyhead-Like 2/Ovo-Like 2 Pathway Regulates Renal Epithelial Barrier Function and Lumen Expansion. Journal of the American Society of Nephrology : JASN\| PubMed ID: 25788534	2015
Functional roles of Grainyhead-like transcription factors in renal development and disease. Pediatric nephrology (Berlin, Germany)\| PubMed ID: 30554362	2020
Mix for Regeneration: Nephron Replacement by Transplanted Cells. Journal of the American Society of Nephrology : JASN\| PubMed ID: 33154176	2020
Kidney Single-cell Transcriptomes Predict Spatial Corticomedullary Gene Expression and Tissue Osmolality Gradients. Journal of the American Society of Nephrology : JASN\| PubMed ID: 33239393	2021
High rates of long-term renal recovery in survivors of coronavirus disease 2019-associated acute kidney injury requiring kidney replacement therapy. Kidney international\| PubMed ID: 33515574	2021
Long-term effects of COVID-19 on kidney function. Lancet (London, England)\| PubMed ID: 33992141	2021

Kai M. Schmidt-Ott

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