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SnRNA-seq of human DKD A UMAP of snRNA-seq dataset. Six control and five DKD samples were aggregated, preprocessed, and filtered. A total of 39,176 cells are depicted. PT-proximal tubule, PT_VCAM1-VCAM1(+) proximal tubule, PEC-parietal epithelial cells, ATL-ascending thin limb, TAL1-CLDN16(-) thick ascending limb, TAL2-CLDN16(+) thick ascending limb, DCT1-early distal convoluted tubule, DCT2-late distal convoluted tubule, PC-principal cells, ICA-type A intercalated cells, ICB-type B intercalated cells, PODO-podocytes, ENDO-endothelial cells, MES-mesangial cells and vascular smooth muscle cells, FIB-fibroblasts, LEUK-leukocytes. B Proximal tubule DEG pathway enrichment. Significant cell-specific DEG from proximal tubule were used to perform gene ontology enrichment with Panther. Fold-enrichment for all significant GO biological processes is shown and the top 25 are highlighted (Source data are provided as a Source Data file). C DEG in DKD that are cell-specific or shared between cell types. DEG that were either shared between multiple cell types or unique to a specific cell type are displayed. DEG shared between multiple cell types are limited to groups that share ten or more DEG (Source data are provided in Supplementary Dataset 6). D Proximal tubule shows increased expression of gluconeogenic genes by snRNA-seq. Control proximal tubule was compared to DKD proximal tubule in the snRNA-seq dataset to identify differentially expressed genes with the FindMarkers function and visualized as violin plots and dot plots. DKD proximal tubule showed increased expression of PCK1, ALDOB, FBP1, and G6PC (see Supplementary Dataset 6 for adjusted p-values). E Proximal tubule-specific DAR and ATAC peaks in PCK1. snATAC-seq coverage plots for DKD and control PCT are displayed in relation to the PCK1 gene body. Orange bars indicate multiple DAR that show decreased accessibility in diabetic PCT (Supplementary Dataset 3). Green arcs depict the nodes of a cis-coaccessibility network (CCAN) surrounding the PCK1 gene body.
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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 k...
Citations
... Closing the CCNF promoter by CRISPRi caused a 73% decrease in CCNF mRNA expression ( Figure 5B-C). We next aligned our FOXM1 CUT&RUN dataset with datasets from ATAC-seq and CUT&RUN for H3K4me3 and H3K27ac in hRPTECs to identify active chromatin (57,58). We identified two regions -one proximal and another region distal to the CCNF promoter -with FOXM1 binding peaks that aligned with active chromatin peaks, suggestive of CREs ( Figure 5A, pink bars). ...
... Furthermore, mutant mice had increase expression of VCAM1 in proximal tubules, and VCAM1 has been shown to label a population of tubules with maladaptive repair (9,57). ...
... Omnibus database under accession number GSE234444. ATAC sequencing data in primary hRPTECs was previously published (57) and available under accession number For A-B, n=3 mice per group. *P < 0.05; **P < 0.01; ***P < 0.001, by two-tailed Student's t test. ...
... Heritability of serum creatinine level reveals a quantitatively larger role for the proximal tubule than other tubule cell types and prior studies have highlighted the involvement of the proximal tubule in kidney function heritability 12,13,74,75 . Because the proximal tubule actively secretes creatinine and therefore has a unique filtration-independent role in creatinine clearance, focus on serum creatinine as a sole measure of GFR likely overestimates the role of the proximal tubule in kidney function. ...
Kidney disease is highly heritable; however, the causal genetic variants, the cell types in which these variants function, and the molecular mechanisms underlying kidney disease remain largely unknown. To identify genetic loci affecting kidney function, we performed a GWAS using multiple kidney function biomarkers and identified 462 loci. To begin to investigate how these loci affect kidney function, we generated single-cell chromatin accessibility (scATAC-seq) maps of the human kidney and identified candidate cis -regulatory elements (cCREs) for kidney podocytes, tubule epithelial cells, and kidney endothelial, stromal, and immune cells. Kidney tubule epithelial cCREs explained 58% of kidney function SNP-heritability and kidney podocyte cCREs explained an additional 6.5% of SNP-heritability. In contrast, little kidney function heritability was explained by kidney endothelial, stromal, or immune cell-specific cCREs. Through functionally informed fine-mapping, we identified putative causal kidney function variants and their corresponding cCREs. Using kidney scATAC-seq data, we created a deep learning model (which we named ChromKid) to predict kidney cell type-specific chromatin accessibility from sequence. ChromKid and allele specific kidney scATAC-seq revealed that many fine-mapped kidney function variants locally change chromatin accessibility in tubule epithelial cells. Enhancer assays confirmed that fine-mapped kidney function variants alter tubule epithelial regulatory element function. To map the genes which these regulatory elements control, we used CRISPR interference (CRISPRi) to target these regulatory elements in tubule epithelial cells and assessed changes in gene expression. CRISPRi of enhancers harboring kidney function variants regulated NDRG1 and RBPMS expression. Thus, inherited differences in tubule epithelial NDRG1 and RBPMS expression may predispose to kidney disease in humans. We conclude that genetic variants affecting tubule epithelial regulatory element function account for most SNP-heritability of human kidney function. This work provides an experimental approach to identify the variants, regulatory elements, and genes involved in polygenic disease.
... Single-cell transcriptional and chromatin accessibility profiling (http:// humphreyslab.com/SingleCell/) [26] showed decreased expression and transcriptional activity of EHHADH in injured proximal tubules (Fig. S1F). Therefore, downregulation of EHHADH uniquely occurred in injured proximal tubules of patients with DN, and the endogenous expression of EHHADH was associated with the progression of DN. ...
... The result was in line with our findings in Ehhadh KO mice. We speculate that loss of EHHADH expression increases susceptibility to the development of DN, and analysis from the public database showed that EHHADH expression was specifically downregulated in damaged tubules of patients with DN [26]. As expected, although the weight and blood glucose of Ehhadh KO mice were slightly lower than those of WT mice under diabetic conditions, they still displayed more severe tubulointerstitial injury, indicating the importance of EHHADH in tubulointerstitial injury under hyperglycemia, which also is in agreement with previous findings that PEX11a deficiency aggravated renal interstitial lesions in mice [39]. ...
Peroxisomal l -bifunctional enzyme (EHHADH) plays a role in the classic peroxisomal fatty acid β-oxidation pathway; however, the relationship between EHHADH expression and diabetic kidney disease has not been well understood. Here, we found that endogenous EHHADH levels were strongly correlated with the progression and severity of diabetic nephropathy in T2D patients. EHHADH knockout mice exhibited worsened renal tubular injury in diabetic mice. Furthermore, EHHADH is a modulator of pexophagy. In renal tubular epithelial cells (RTECs) in vitro, the knockdown of EHHADH induced a dramatic loss of peroxisomes. The loss of peroxisomes in EHHADH-deficient RTECs was restored by either an autophagic inhibitor 3-methyladenine or bafilomycin A1 both in vitro and in vivo. NBR1 was required for pexophagy in EHHADH-knockdown cells, where the level of reactive oxygen species (ROS) was increased, while inhibition of ROS blocked pexophagy. In summary, our findings revealed EHHADH deficiency accelerated renal injury in DKD as a modulator of pexophagy.
... We and others have described Vcam1-expressing failed-repair proximal tubular cells (FR-PTC) after kidney injury in mice (10)(11)(12)26) and human (8,9,13,27). This subpopulation is characterized by pro-inflammatory, pro-fibrotic gene expression signature, implicated in progression of kidney diseases (13,27). ...
... We and others have described Vcam1-expressing failed-repair proximal tubular cells (FR-PTC) after kidney injury in mice (10)(11)(12)26) and human (8,9,13,27). This subpopulation is characterized by pro-inflammatory, pro-fibrotic gene expression signature, implicated in progression of kidney diseases (13,27). Of note, the frequency of FR-PTC in advanced ADPKD was markedly increased, replacing normal PTC(13), suggestive of its significant role in ADPKD progression (13). ...
Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and causes significant morbidity, ultimately leading to end-stage kidney disease. PKD pathogenesis is characterized by complex and dynamic alterations in multiple cell types during disease progression, hampering a deeper understanding of disease mechanism and the development of therapeutic approaches. Here, we generate a single nucleus multimodal atlas of an orthologous mouse PKD model at early, mid and late timepoints, consisting of 125,434 single-nucleus transcriptomic and epigenetic multiomes. We catalogue differentially expressed genes and activated epigenetic regions in each cell type during PKD progression, characterizing cell-type-specific responses to Pkd1 deletion. We describe heterogeneous, atypical collecting duct cells as well as proximal tubular cells that constitute cyst epithelia in PKD. The transcriptional regulation of the cyst lining cell marker GPRC5A is conserved between mouse and human PKD cystic epithelia, suggesting shared gene regulatory pathways. Our single nucleus multiomic analysis of mouse PKD provides a foundation to understand the earliest changes molecular deregulation in a mouse model of PKD at a single-cell resolution.
... Regrettably, studies on the role of metabolic memory in DKD are rare, especially studies on mesangial cells and the crosstalk between different cell types in the kidney. A recent study on the multimodal integration of single nucleus RNA (snRNA-seq) and an assay for transposase-accessible chromatin sequencing (snATAC-seq) in DKD may provide more information on the epigenetic regulation of chromatin accessibility, which could contribute to the long-term expression of DKD and metabolic memoryrelated genes (Wilson et al. 2022). However, further studies are still needed. ...
Diabetes mellitus, a chronic metabolic disease, often leads to numerous chronic complications, significantly contributing to global morbidity and mortality rates. High glucose levels trigger epigenetic modifications linked to pathophysiological processes like inflammation, immunity, oxidative stress, mitochondrial dysfunction, senescence and various kinds of cell death. Despite glycemic control, transient hyperglycemia can persistently harm organs, tissues, and cells, a latent effect termed "metabolic memory" that contributes to chronic diabetic complications. Understanding metabolic memory's mechanisms could offer a new approach to mitigating these complications. However, key molecules and networks underlying metabolic memory remain incompletely understood. This review traces the history of metabolic memory research, highlights its key features, discusses recent molecules involved in its mechanisms, and summarizes confirmed and potential therapeutic compounds. Additionally, we outline in vitro and in vivo models of metabolic memory. We hope this work will inform future research on metabolic memory's regulatory mechanisms and facilitate the development of effective therapeutic compounds to prevent diabetic complications.
... Notably, glucagon action in the kidney, which has the highest expression of GCGR after the liver, may play a key role in gluconeogenesis (159,160). Additionally, GCGR expression is lower in patients with T2D and CKD, and its expression is positively correlated with eGFR (161)(162)(163). Genetic studies link human GCGR variants to altered electrolyte handling, which complements findings in mice that kidney-specific GCGR knockdown leads to dysregulation in electrolyte and volume homeostasis (164)(165)(166). ...
... Within the Loop of Henle, glucagon's ability to enhance reabsorption is likely, at least partially, mediated by cAMP. Studies indicated that glucagon-stimulated cAMP formation in the kidney occurs predominantly in thick ascending limb and collecting duct (258), a pattern that aligns with more recent RNA sequencing datasets in mice (200,259) and humans (29,161,162). However, the distinct role of glucagon versus cAMP in the Loop of Henle and the collecting duct Figure 3. Schematic of direct and indirect effects of glucagon receptor (GCGR) activation by glucagon and analogs on the kidney. ...
Chronic kidney disease (CKD) is a debilitating condition associated with significant morbidity and mortality. In recent years, the kidney effects of incretin-based therapies, particularly glucagon-like peptide-1 receptor agonists (GLP-1RAs), have garnered substantial interest in the management of type 2 diabetes (T2D) and obesity. This review delves into the intricate interactions between the kidney, GLP-1RAs, and glucagon, shedding light on their mechanisms of action and potential kidney benefits. Both GLP-1 and glucagon, known for their opposing roles in regulating glucose homeostasis, improve systemic risk factors affecting the kidney, including adiposity, inflammation, oxidative stress, and endothelial function. Additionally, these hormones and their pharmaceutical mimetics may have direct impact on the kidney. Clinical studies have provided evidence that incretins, including those incorporating glucagon receptor agonism, are likely to exhibit improved kidney outcomes. Although further research is necessary, receptor polypharmacology holds promise for preserving kidney function through eliciting vasodilatory effects, influence on volume and electrolyte handling, and improvement of systemic risk factors.
... Previous studies suggested that HNF4A actions in the kidney are beneficial (39), because HNF4A also regulates gene expression through androgen-independent mechanisms. Mutations in HNF4A cause monogenic maturity-onset diabetes of the young type 1 (40) and renal Fanconi syndrome, characterized by altered PTEC reabsorption (41). Although androgen-independent HNF4A actions seem essential for PTECs, androgen-dependent actions of HNF4A may result in mitochondrial hyperactivity and predispose cells to oxidative stress. ...
... This is particularly important in diabetes, which results in altered amounts and activity of HNF4A (42,43) and is characterized by heightened oxidative stress. Furthermore, HNF4A regulates genes altered in PTECs from patients with diabetes (44), and HNF4A binding sites are enriched in open chromatin regions of proximal tubules in DKD (41). Because both DHT and HNF4A control metabolic processes that are altered by diabetes, they may play a synergistic role in DKD. ...
Diabetic kidney disease (DKD) is the main cause of chronic kidney disease (CKD) and progresses faster in males than in females. We identify sex-based differences in kidney metabolism and in the blood metabolome of male and female individuals with diabetes. Primary human proximal tubular epithelial cells (PTECs) from healthy males displayed increased mitochondrial respiration, oxidative stress, apoptosis, and greater injury when exposed to high glucose compared with PTECs from healthy females. Male human PTECs showed increased glucose and glutamine fluxes to the TCA cycle, whereas female human PTECs showed increased pyruvate content. The male human PTEC phenotype was enhanced by dihydrotestosterone and mediated by the transcription factor HNF4A and histone demethylase KDM6A. In mice where sex chromosomes either matched or did not match gonadal sex, male gonadal sex contributed to the kidney metabolism differences between males and females. A blood metabolomics analysis in a cohort of adolescents with or without diabetes showed increased TCA cycle metabolites in males. In a second cohort of adults with diabetes, females without DKD had higher serum pyruvate concentrations than did males with or without DKD. Serum pyruvate concentrations positively correlated with the estimated glomerular filtration rate, a measure of kidney function, and negatively correlated with all-cause mortality in this cohort. In a third cohort of adults with CKD, male sex and diabetes were associated with increased plasma TCA cycle metabolites, which correlated with all-cause mortality. These findings suggest that differences in male and female kidney metabolism may contribute to sex-dependent outcomes in DKD.
... Correlation analysis suggested that kidney medulla showed higher transcriptomic similarity with the papilla compared with the cortex, and high similarity between renal artery and ureter samples ( Figure S1C). We identified upregulation of SLC22A8 (encoding a PT transporter) in kidney cortex, RNF24 (specific to the Loop of Henle and distal nephron 29 ) in medulla and papilla and MYH11 (marker of smooth muscle cells [SMCs]) in the renal artery and ureter samples, confirming the sample quality ( Figures 1E and S1D). ...
... Surveying recently published single-cell studies further validated upregulation of PPFIBP1 and PLEKHA1 in PT cells in mouse models of kidney injury, 7 and in human autosomal dominant polycystic kidney disease 72 and diabetic kidney disease (Mendeley data). 29 In a similar approach, we were able to identify disease-related candidate genes that are specifically expressed in other cell types such as TAL and DCT ( Figure S7H; Mendeley data), further demonstrating the utilization of this single-cell human kidney atlas for translational studies. ...
... 30 For single-cell cluster annotation, a list of marker genes (summarized in Table S6) were curated from existing cell atlas datasets. 6,11,18,20,29,31,35,72,97 Each marker gene was first qualitatively visualized on the UMAP space and then its differential expression pattern was quantitatively evaluated, in which an adjusted p value lower than 0.01 in the Mann-Whitney-U test with the Benjamini-Hochberg correction was considered as significant. For cell type subclustering, cells annotated as the subtype in the above analysis were extracted and re-analyzed following the similar procedure (see Code Availability). ...
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.
... Recently several single-cell multi-omic analyses have greatly advanced our understanding of kidney cell states both in healthy and diseased kidneys [1][2][3][4][5][6] . A limitation of these single-cell multi-omic analyses is that they require cell or nuclear dissociation as a first step, resulting in the loss of spatial information which precludes a full analysis of how cells interact with each other in their tissue microenvironment 7 . ...
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.
... PT_VCAM1 resembles the failed repair proximal tubule state that arises following AKI. The PT_VCAM1 population appears to increase with age and in diabetic kidney disease 3,10,11 . These findings raise the possibility that subacute injuries incurred over a patient's life could lead to a gradual accumulation of FR-PT cells, resulting in a progressive increase in proinflammatory and profibrotic stimuli that drives CKD progression. ...
... Single cell sequencing has been successfully applied to characterize the transcriptional changes associated with specific cell types and cell states in a variety of human kidney diseases [2][3][4]11,[19][20][21][22] . Several current challenges limit our ability to leverage these datasets for the identification of regulatory factors coordinating gene expression changes. ...
... Partial nephrectomy kidney samples are similar to live donor samples and both contain FR-PT cells Failed repair proximal tubule cells (FR-PTC) have been identified in healthy human kidney samples derived from tumor-adjacent tissue after partial nephrectomy 10,11 . We hypothesize that FR-PTC in apparently healthy kidneys is attributable to subacute injury or stress sustained over a patient's lifetime, leading to accumulation of FR-PTC cells and a fibrosis-promoting phenotype. ...
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.
