Transcriptomic, epigenomic, and spatial metabolomic cell profiling redefines regional human kidney anatomy.

A large-scale multimodal atlas that includes major kidney regions is lacking. Here, we employed simultaneous high-throughput single-cell ATAC/RNA sequencing (SHARE-seq) and spatially resolved metabolomics to profile 54 human samples from distinct kidney anatomical regions. We generated transcriptomes of 446,267 cells and chromatin accessibility profiles of 401,875 cells and developed a package to analyze 408,218 spatially resolved metabolomes. We find that the same cell type, including thin limb, thick ascending limb loop of Henle and principal cells, display distinct transcriptomic, chromatin accessibility, and metabolomic signatures, depending on anatomic location. Surveying metabolism-associated gene profiles revealed non-overlapping metabolic signatures between nephron segments and dysregulated lipid metabolism in diseased proximal tubule (PT) cells. Integrating multimodal omics with clinical data identified PLEKHA1 as a disease marker, and its in vitro knockdown increased gene expression in PT differentiation, suggesting possible pathogenic roles. This study highlights previously underrepresented cellular heterogeneity underlying the human kidney anatomy.

Tnik depletion induces inflammation and apoptosis ininjured renal proximal tubule epithelial cells.

In the aftermath of acute kidney injury (AKI), surviving proximal tubule epithelia repopulate injured tubules to promote repair. However, a portion of cells fail to repair (termed Failed Repair Proximal Tubule Cells, FR-PTCs) and exert ongoing proinflammatory and profibrotic effects. To better understand the molecular drivers of the FR-PTC state, we reanalyzed a mouse ischemia-reperfusion injury single nucleus RNA-sequencing (snRNA-seq) atlas to identify Traf2 and Nck Interacting Kinase, Tnik, to be exclusively expressed in FR-PTCs, but not in healthy or acutely injured proximal tubules post AKI (2 and 6 weeks) in mice. We confirmed expression of Tnik protein in injured mouse and human tissues by immunofluorescence. Then, to determine the functional role of Tnik in FR-PTCs, we depleted Tnik using siRNA in two human renal proximal tubule epithelial cell lines (primary and immortalized hRPTECs) and analyzed each by bulk RNA-sequencing. Pathway analysis revealed significant upregulation of inflammatory signaling pathways, while pathways associated with differentiated proximal tubule such as organic acid transport were significantly downregulated. TNIK knockdown drove reduced cell viability and increased apoptosis, including differentially expressed PARP family members, cleaved PARP-1 fragments and increased Annexin V binding to phosphatidylserine. Together, these results indicate that Tnik upregulation in FR-PTCs acts in a compensatory fashion to suppress inflammation and promote proximal tubule epithelial cell survival following injury. Modulating Tnik activity may represent a pro-repair therapeutic strategy after AKI.

Tubular CPT1A deletion minimally affects aging and chronic kidney injury.

Kidney tubules use fatty acid oxidation (FAO) to support their high energetic requirements. Carnitine palmitoyltransferase 1A (CPT1A) is the rate-limiting enzyme for FAO, and it is necessary to transport long-chain fatty acids into mitochondria. To define the role of tubular CPT1A in aging and injury, we generated mice with tubule-specific deletion of Cpt1a (Cpt1aCKO mice), and the mice were either aged for 2 years or injured by aristolochic acid or unilateral ureteral obstruction. Surprisingly, Cpt1aCKO mice had no significant differences in kidney function or fibrosis compared with wild-type mice after aging or chronic injury. Primary tubule cells from aged Cpt1aCKO mice had a modest decrease in palmitate oxidation but retained the ability to metabolize long-chain fatty acids. Very-long-chain fatty acids, exclusively oxidized by peroxisomes, were reduced in kidneys lacking tubular CPT1A, consistent with increased peroxisomal activity. Single-nuclear RNA-Seq showed significantly increased expression of peroxisomal FAO enzymes in proximal tubules of mice lacking tubular CPT1A. These data suggest that peroxisomal FAO may compensate in the absence of CPT1A, and future genetic studies are needed to confirm the role of peroxisomal ß-oxidation when mitochondrial FAO is impaired.

Predicting proximal tubule failed repair drivers through regularized regression analysis of single cell multiomic sequencing.

Renal proximal tubule epithelial cells have considerable intrinsic repair capacity following injury. However, a fraction of injured proximal tubule cells fails to undergo normal repair and assumes a proinflammatory and profibrotic phenotype that may promote fibrosis and chronic kidney disease. The healthy to failed repair change is marked by cell state-specific transcriptomic and epigenomic changes. Single nucleus joint RNA- and ATAC-seq sequencing offers an opportunity to study the gene regulatory networks underpinning these changes in order to identify key regulatory drivers. We develop a regularized regression approach to construct genome-wide parametric gene regulatory networks using multiomic datasets. We generate a single nucleus multiomic dataset from seven adult human kidney samples and apply our method to study drivers of a failed injury response associated with kidney disease. We demonstrate that our approach is a highly effective tool for predicting key cis- and trans-regulatory elements underpinning the healthy to failed repair transition and use it to identify NFAT5 as a driver of the maladaptive proximal tubule state.

Epigenetic reprogramming driving successful and failed repair in acute kidney injury.

Acute kidney injury (AKI) causes epithelial damage followed by subsequent repair. While successful repair restores kidney function, this process is often incomplete and can lead to chronic kidney disease (CKD) in a process called failed repair. To better understand the epigenetic reprogramming driving this AKI-to-CKD transition we generated a single nucleus multiomic atlas for the full mouse AKI time course, consisting of ~280,000 single nucleus transcriptomes and epigenomes. We reveal cell-specific dynamic alterations in gene regulatory landscapes reflecting especially activation of proinflammatory pathways. We further generated single nucleus multiomic data from four human AKI samples including validation by genome-wide identification of NF-kB binding sites. A regularized regression analysis identifies key regulators involved in both successful and failed repair cell fate, identifying the transcription factor CREB5 as a regulator of both successful and failed tubular repair that also drives proximal tubule cell proliferation after injury. Our interspecies multiomic approach provides a foundation to comprehensively understand cell states in AKI.

Targeting de novo lipogenesis to mitigate kidney disease.

Ten percent of the population worldwide suffers from chronic kidney disease (CKD), but the mechanisms driving CKD pathology are incompletely understood. While dysregulated lipid metabolism is one hallmark of CKD, the pathogenesis of cellular lipid accumulation remains unclear. In this issue of the JCI, Mukhi et al. Identify acyl-CoA synthetase short-chain family 2 (ACSS2) as a disease risk gene and demonstrate a role for ACSS2 in de novo lipogenesis (DNL). Notably, genetic or pharmacological inhibition of DNL protected against kidney disease progression in mice. These findings warrant evaluation of DNL inhibition with respect to efficacy and safety in people with CKD.

High resolution spatial profiling of kidney injury and repair using RNA hybridization-based in situ sequencing.

Emerging spatially resolved transcriptomics technologies allow for the measurement of gene expression in situ at cellular resolution. We apply direct RNA hybridization-based in situ sequencing (dRNA HybISS, Cartana part of 10xGenomics) to compare male and female healthy mouse kidneys and the male kidney injury and repair timecourse. A pre-selected panel of 200 genes is used to identify cell state dynamics patterns during injury and repair. We develop a new computational pipeline, CellScopes, for the rapid analysis, multi-omic integration and visualization of spatially resolved transcriptomic datasets. The resulting dataset allows us to resolve 13 kidney cell types within distinct kidney niches, dynamic alterations in cell state over the course of injury and repair and cell-cell interactions between leukocytes and kidney parenchyma. At late timepoints after injury, C3+ leukocytes are enriched near pro-inflammatory, failed-repair proximal tubule cells. Integration of snRNA-seq dataset from the same injury and repair samples also allows us to impute the spatial localization of genes not directly measured by dRNA HybISS.

Mosaic loss of Y chromosome is associated with aging and epithelial injury in chronic kidney disease.

BACKGROUND: Mosaic loss of Y chromosome (LOY) is the most common chromosomal alteration in aging men. Here, we use single-cell RNA and ATAC sequencing to show that LOY is present in the kidney and increases with age and chronic kidney disease. RESULTS: The likelihood of a cell having LOY varies depending on its location in the nephron. Cortical epithelial cell types have a greater proportion of LOY than medullary or glomerular cell types, which may reflect their proliferative history. Proximal tubule cells are the most abundant cell type in the cortex and are susceptible to hypoxic injury. A subset of these cells acquires a pro-inflammatory transcription and chromatin accessibility profile associated with expression of HAVCR1, VCAM1, and PROM1. These injured epithelial cells have the greatest proportion of LOY and their presence predicts future kidney function decline. Moreover, proximal tubule cells with LOY are more likely to harbor additional large chromosomal gains and express pro-survival pathways. Spatial transcriptomics localizes injured proximal tubule cells to a pro-fibrotic microenvironment where they adopt a secretory phenotype and likely communicate with infiltrating immune cells. CONCLUSIONS: We hypothesize that LOY is an indicator of increased DNA damage and potential marker of cellular senescence that can be applied to single-cell datasets in other tissues.

Leveraging multimodal chromatin profiling to identify a new potential driver of diabetic kidney disease.

The 3-dimensional nature of chromatin architecture plays crucial roles in regulating gene expression in development, homeostasis, and disease. Until recently, however, comprehensive chromatin profiling in human kidneys has been lacking. In this issue, Eun and Kim et al. employed a multimodal approach by integrating a single-nucleus assay for transposase-accessible chromatin sequencing, chromatin immunoprecipitation sequencing, and Hi-C (a method to comprehensively detect chromatin interactions) to investigate how the epigenetic landscape is altered in diabetic kidney disease.

Sequencing of Physically Interacting Cells in Human Kidney Allograft Rejection to Infer Contact-dependent Immune Cell Transcription.

BACKGROUND: Rejection requires cell-cell contact involving immune cells. Inferring the transcriptional programs of cell-cell interactions from single-cell RNA-sequencing (scRNA-seq) data is challenging as spatial information is lost. METHODS: We combined a CD45 pos enrichment strategy with Cellular Indexing of Transcriptomes and Epitopes by sequencing based quantification of leukocyte surface proteins to analyze cell-cell interactions in 11 human kidney transplant biopsies encompassing a spectrum of rejection diagnoses. scRNA-seq was performed using the 10X Genomics platform. We applied the sequencing physically interacting cells computational method to deconvolute the transcriptional profiles of heterotypic physically interacting cells. RESULTS: The 11 human allograft biopsies generated 31 203 high-quality single-cell libraries. Clustering was further refined by combining Cellular Indexing of Transcriptomes and Epitopes by sequencing data from 6 different leukocyte-specific surface proteins. Three of 6 doublet clusters were identified as physically interacting cell complexes; macrophages or dendritic cells bound to B cells or plasma cells; natural killer (NK) or T cells bound to macrophages or dendritic cells and NK or T cells bound to endothelial cells. Myeloid-lymphocyte physically interacting cell complexes expressed activated and proinflammatory genes. Lymphocytes physically interacting with endothelial cells were enriched for NK and CD4 T cells. NK cell-endothelial cell contact caused increased expression of endothelial proinflammatory genes CXCL9 and CXCL10 and NK cell proinflammatory genes CCL3 , CCL4 , and GNLY . CONCLUSIONS: The transcriptional profiles of physically interacting cells from human kidney transplant biopsies can be inferred from scRNA-seq data using the sequencing physically interacting cells method. This approach complements previous methods that estimate cell-cell physical contact from scRNA-seq data.

Elucidating the Proximal Tubule HNF4A Gene Regulatory Network in Human Kidney Organoids.

SIGNIFICANCE STATEMENT: HNF4 genes promote proximal tubule differentiation in mice, but their function in human nephrogenesis is not fully defined. This study uses human pluripotent stem cell (PSC)-derived kidney organoids as a model to investigate HNF4A and HNF4G functions. The loss of HNF4A , but not HNF4G , impaired reabsorption-related molecule expression and microvilli formation in human proximal tubules. Cleavage under targets and release using nuclease (CUT&RUN) sequencing and CRISPR-mediated transcriptional activation (CRISPRa) further confirm that HNF4A directly regulates its target genes. Human kidney organoids provide a good model for studying transcriptional regulation in human kidney development. BACKGROUND: The proximal tubule plays a major role in electrolyte homeostasis. Previous studies have shown that HNF4A regulates reabsorption-related genes and promotes proximal tubule differentiation during murine kidney development. However, the functions and gene regulatory mechanisms of HNF4 family genes in human nephrogenesis have not yet been investigated. METHODS: We generated HNF4A -knock out (KO), HNF4G -KO, and HNF4A/4G -double KO human pluripotent stem cell lines, differentiated each into kidney organoids, and used immunofluorescence analysis, electron microscopy, and RNA-seq to analyze them. We probed HNF4A-binding sites genome-wide by cleavage under targets and release using nuclease sequencing in both human adult kidneys and kidney organoid-derived proximal tubular cells. Clustered Regularly Interspaced Short Palindromic Repeats-mediated transcriptional activation validated HNF4A and HNF4G function in proximal tubules during kidney organoid differentiation. RESULTS: Organoids lacking HNF4A , but not HNF4G , showed reduced expression of transport-related, endocytosis-related, and brush border-related genes, as well as disorganized brush border structure in the apical lumen of the organoid proximal tubule. Cleavage under targets and release using nuclease revealed that HNF4A primarily bound promoters and enhancers of genes that were downregulated in HNF4A -KO, suggesting direct regulation. Induced expression of HNF4A or HNF4G by CRISPR-mediated transcriptional activation drove increased expression of selected target genes during kidney organoid differentiation. CONCLUSIONS: This study reveals regulatory mechanisms of HNF4A and HNF4G during human proximal tubule differentiation. The experimental strategy can be applied more broadly to investigate transcriptional regulation in human kidney development.

Genetically engineering endothelial niche in human kidney organoids enables multilineage maturation, vascularization and de novo cell types.

Vascularization plays a critical role in organ maturation and cell type development. Drug discovery, organ mimicry, and ultimately transplantation in a clinical setting thereby hinges on achieving robust vascularization of in vitro engineered organs. Here, focusing on human kidney organoids, we overcome this hurdle by combining an inducible ETS translocation variant 2 (ETV2) human induced pluripotent stem cell (iPSC) line, which directs endothelial fate, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive vascularization by endothelial cells with an identity most closely related to endogenous kidney endothelia. Vascularized organoids also show increased maturation of nephron structures including more mature podocytes with improved marker expression, foot process interdigitation, an associated fenestrated endothelium, and the presence of renin+ cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Furthermore, this approach is orthogonal to native tissue differentiation paths, hence readily adaptable to other organoid systems and thus has the potential for a broad impact on basic and translational organoid studies.

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 of HNF1B by CRISPR interference both in cultured proximal tubule cells and also during organoid differentiation. Our approach provides an experimental framework to judge the cell-specific maturation state of human kidney organoids and shows that kidney organoids can be used to validate individual gene regulatory networks that regulate differentiation.

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.

International Society of Nephrology first consensus guidance for preclinical animal studies in translational nephrology.

Preclinical tests in animal models are key steps in early drug development. Consequently, the International Society of Nephrology held a consensus meeting that connected experts in the global kidney community in order to provide guidance on optimal management of translational animal studies for the development of new drugs to treat kidney disease, entitled "TRANSFORM; TRAnslational Nephrology Science FOR new Medications." The meeting covered various themes, including the following: (i) selection of disease model; (ii) pharmacokinetics; (iii) interventions in late preclinical models; (iv) choice of animal; (v) statistical power; (vi) organoids and organ-on-a-chip models; and (vii) reporting of results. This guidance is the first to be provided on the optimal conduct of translational animal studies for the development of new drugs to treat kidney disease. These recommendations are designed to accelerate development of new drugs for efficacious treatment of kidney diseases, and to improve the prognosis and quality of life of patients with a variety of kidney diseases.

Epigenetic and transcriptomic characterization reveals progression markers and essential pathways in clear cell renal cell carcinoma.

Identifying tumor-cell-specific markers and elucidating their epigenetic regulation and spatial heterogeneity provides mechanistic insights into cancer etiology. Here, we perform snRNA-seq and snATAC-seq in 34 and 28 human clear cell renal cell carcinoma (ccRCC) specimens, respectively, with matched bulk proteogenomics data. By identifying 20 tumor-specific markers through a multi-omics tiered approach, we reveal an association between higher ceruloplasmin (CP) expression and reduced survival. CP knockdown, combined with spatial transcriptomics, suggests a role for CP in regulating hyalinized stroma and tumor-stroma interactions in ccRCC. Intratumoral heterogeneity analysis portrays tumor cell-intrinsic inflammation and epithelial-mesenchymal transition (EMT) as two distinguishing features of tumor subpopulations. Finally, BAP1 mutations are associated with widespread reduction of chromatin accessibility, while PBRM1 mutations generally increase accessibility, with the former affecting five times more accessible peaks than the latter. These integrated analyses reveal the cellular architecture of ccRCC, providing insights into key markers and pathways in ccRCC tumorigenesis.