Genetic tracing of the epithelial lineage during mammalian kidney repair

Developing new therapeutic approaches to treat acute kidney injury requires a detailed understanding of endogenous cellular repair. Genetic fate mapping defines cellular hierarchies in vivo and we used this technique to assess a possible contribution of non-epithelial stem cells to renal repair after ischemic injury. Mice with efficient labeling of renal epithelial cells, but not non-epithelial interstitial cells, were subjected to a single cycle or sequential cycles of kidney injury and repair. No dilution of the epithelial cell fate marker was observed despite robust epithelial cell proliferation. Thus, non-tubular cells do not have the ability to migrate across the basement membrane and differentiate into epithelial cells in this model. Instead, surviving tubular epithelial cells are responsible for repair of the damaged nephron. Future studies will need to distinguish between uniform dedifferentiation and proliferation of all epithelial cells after injury versus selective expansion of an intratubular epithelial stem cell

Genetic tracing of the epithelial lineage during mammalian kidney repair

Developing new therapeutic approaches to treat acute kidney injury requires a detailed understanding of endogenous cellular repair. Genetic fate mapping defines cellular hierarchies in vivo and we used this technique to assess a possible contribution of non-epithelial stem cells to renal repair after ischemic injury. Mice with efficient labeling of renal epithelial cells, but not non-epithelial interstitial cells, were subjected to a single cycle or sequential cycles of kidney injury and repair. No dilution of the epithelial cell fate marker was observed despite robust epithelial cell proliferation. Thus, non-tubular cells do not have the ability to migrate across the basement membrane and differentiate into epithelial cells in this model. Instead, surviving tubular epithelial cells are responsible for repair of the damaged nephron. Future studies will need to distinguish between uniform dedifferentiation and proliferation of all epithelial cells after injury versus selective expansion of an intratubular epithelial stem cell

Mouse kidney nuclear isolation and library preparation for single-cell combinatorial indexing RNA sequencing

Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq3) enables high-throughput single-nucleus transcriptomic profiling of multiple samples in one experiment. Here, we describe an optimized protocol of mouse kidney nuclei isolation and sci-RNA-seq3 library preparation. The use of a dounce tissue homogenizer enables nuclei extraction with high yield. Fixed nuclei are processed for sci-RNA-seq3, and self-loaded transposome Tn5 is used for tagmentation in library generation. The step-by-step protocol allows researchers to generate scalable single-cell transcriptomic data with common laboratory supplies at low cost. For complete details on the use and execution of this protocol, please refer to Li et al. (2022)

Lineage-tracing methods and the kidney

The kidney is a complex organ with over 30 different cell types, and understanding the lineage relationships between these cells is challenging. During nephrogenesis, a central question is how the coordinated morphogenesis, growth, and differentiation of distinct cell types leads to development of a functional organ. In mature kidney, understanding cell division and fate during injury, regeneration and aging are critical topics for understanding disease. Genetic lineage tracing offers a powerful tool to decipher cellular hierarchies in both development and disease because it allows the progeny of a single cell, or group of cells, to be tracked unambiguously. Recent advances in this field include the use of inducible recombinases, multicolor reporters, and mosaic analysis. In this review, we discuss lineage-tracing methods focusing on the mouse model system and consider the impact of these methods on our understanding of kidney biology and prospects for future application

Mapping genomic regulation of kidney disease and traits through high-resolution and interpretable eQTLs

Expression quantitative trait locus (eQTL) studies illuminate genomic variants that regulate specific genes and contribute to fine-mapped loci discovered via genome-wide association studies (GWAS). Efforts to maximize their accuracy are ongoing. Using 240 glomerular (GLOM) and 311 tubulointerstitial (TUBE) micro-dissected samples from human kidney biopsies, we discovered 5371 GLOM and 9787 TUBE genes with at least one variant significantly associated with expression (eGene) by incorporating kidney single-nucleus open chromatin data and transcription start site distance as an integrative prior for Bayesian statistical fine-mapping. The use of an integrative prior resulted in higher resolution eQTLs illustrated by (1) smaller numbers of variants in credible sets with greater confidence, (2) increased enrichment of partitioned heritability for GWAS of two kidney traits, (3) an increased number of variants colocalized with the GWAS loci, and (4) enrichment of computationally predicted functional regulatory variants. A subset of variants and genes were validated experimentally in vitro and using a Drosophila nephrocyte model. More broadly, this study demonstrates that tissue-specific eQTL maps informed by single-nucleus open chromatin data have enhanced utility for diverse downstream analyses

Multimodal single cell sequencing implicates chromatin accessibility and genetic background in diabetic kidney disease progression

The proximal tubule is a key regulator of kidney function and glucose metabolism. Diabetic kidney disease leads to proximal tubule injury and changes in chromatin accessibility that modify the activity of transcription factors involved in glucose metabolism and inflammation. Here we use single nucleus RNA and ATAC sequencing to show that diabetic kidney disease leads to reduced accessibility of glucocorticoid receptor binding sites and an injury-associated expression signature in the proximal tubule. We hypothesize that chromatin accessibility is regulated by genetic background and closely-intertwined with metabolic memory, which pre-programs the proximal tubule to respond differently to external stimuli. Glucocorticoid excess has long been known to increase risk for type 2 diabetes, which raises the possibility that glucocorticoid receptor inhibition may mitigate the adverse metabolic effects of diabetic kidney disease

A single-cell multiomic analysis of kidney organoid differentiation

Kidney organoids differentiated from pluripotent stem cells are powerful models of kidney development and disease but are characterized by cell immaturity and off-target cell fates. Comparing the cell-specific gene regulatory landscape during organoid differentiation with human adult kidney can serve to benchmark progress in differentiation at the epigenome and transcriptome level for individual organoid cell types. Using single-cell multiome and histone modification analysis, we report more broadly open chromatin in organoid cell types compared to the human adult kidney. We infer enhancer dynamics by cis-coaccessibility analysis and validate an enhancer driving transcription o