Jihwan Park

Single-cell transcriptomics of the mouse kidney reveals potential cellular targets of kidney disease

Touring the kidney, cell by cell Our kidneys play a critical role in keeping us healthy, a fact of which we are reminded several times each day. This organs cellular complexity has hindered progress in understanding the mechanisms underlying chronic kidney disease, which affects 10% of the worlds population. Using single-cell transcriptional profiling, Park et al. produced a comprehensive cell atlas of the healthy mouse kidney (see the Perspective by Humphreys). An unexpected cell type in the collecting duct appears to be a transitional state between two known cell types. The transition from one cell type to the other is regulated by the Notch signaling pathway and is associated with metabolic acidosis. The authors also find that genetically distinct kidney diseases with common clinical features share common cellular origins. Science, this issue p. 758; see also p. 709 A single-cell atlas of the mouse kidney reveals an unexpected cell type that likely contributes to kidney disease. Our understanding of kidney disease pathogenesis is limited by an incomplete molecular characterization of the cell types responsible for the organ’s multiple homeostatic functions. To help fill this knowledge gap, we characterized 57,979 cells from healthy mouse kidneys by using unbiased single-cell RNA sequencing. On the basis of gene expression patterns, we infer that inherited kidney diseases that arise from distinct genetic mutations but share the same phenotypic manifestation originate from the same differentiated cell type. We also found that the collecting duct in kidneys of adult mice generates a spectrum of cell types through a newly identified transitional cell. Computational cell trajectory analysis and in vivo lineage tracing revealed that intercalated cells and principal cells undergo transitions mediated by the Notch signaling pathway. In mouse and human kidney disease, these transitions were shifted toward a principal cell fate and were associated with metabolic acidosis.

PGC-1α Protects from Notch-Induced Kidney Fibrosis Development

Kidney fibrosis is the histologic manifestation of CKD. Sustained activation of developmental pathways, such as Notch, in tubule epithelial cells has been shown to have a key role in fibrosis development. The molecular mechanism of Notch-induced fibrosis, however, remains poorly understood. Here, we show that, that expression of peroxisomal proliferation g-coactivator (PGC-1α) and fatty acid oxidation-related genes are lower in mice expressing active Notch1 in tubular epithelial cells (Pax8-rtTA/ICN1) compared to littermate controls. Chromatin immunoprecipitation assays revealed that the Notch target gene Hes1 directly binds to the regulatory region of PGC-1α Compared with Pax8-rtTA/ICN1 transgenic animals, Pax8-rtTA/ICN1/Ppargc1a transgenic mice showed improvement of renal structural alterations (on histology) and molecular defect (expression of profibrotic genes). Overexpression of PGC-1α restored mitochondrial content and reversed the fatty acid oxidation defect induced by Notch overexpression in vitro in tubule cells. Furthermore, compared with Pax8-rtTA/ICN1 mice, Pax8-rtTA/ICN1/Ppargc1a mice exhibited improvement in renal fatty acid oxidation gene expression and apoptosis. Our results show that metabolic dysregulation has a key role in kidney fibrosis induced by sustained activation of the Notch developmental pathway and can be ameliorated by PGC-1α.

Notch pathway is activated via genetic and epigenetic alterations and is a therapeutic target in clear cell renal cancer

Clear cell renal cell carcinoma (CCRCC) is an incurable malignancy in advanced stages and needs newer therapeutic targets. Transcriptomic analysis of CCRCCs and matched microdissected renal tubular controls revealed overexpression of NOTCH ligands and receptors in tumor tissues. Examination of the TCGA RNA-seq data set also revealed widespread activation of NOTCH pathway in a large cohort of CCRCC samples. Samples with NOTCH pathway activation were also clinically distinct and were associated with better overall survival. Parallel DNA methylation and copy number analysis demonstrated that both genetic and epigenetic alterations led to NOTCH pathway activation in CCRCC. NOTCH ligand JAGGED1 was overexpressed and associated with loss of CpG methylation of H3K4me1-associated enhancer regions. JAGGED2 was also overexpressed and associated with gene amplification in distinct CCRCC samples. Transgenic expression of intracellular NOTCH1 in mice with tubule-specific deletion of VHL led to dysplastic hyperproliferation of tubular epithelial cells, confirming the procarcinogenic role of NOTCH in vivo. Alteration of cell cycle pathways was seen in murine renal tubular cells with NOTCH overexpression, and molecular similarity to human tumors was observed, demonstrating that human CCRCC recapitulates features and gene expression changes observed in mice with transgenic overexpression of the Notch intracellular domain. Treatment with the γ-secretase inhibitor LY3039478 led to inhibition of CCRCC cells in vitro and in vivo. In summary, these data reveal the mechanistic basis of NOTCH pathway activation in CCRCC and demonstrate this pathway to a potential therapeutic target.

Genetic-Variation-Driven Gene-Expression Changes Highlight Genes with Important Functions for Kidney Disease

Chronic kidney disease (CKD) is a complex gene-environmental disease affecting close to 10% of the US population. Genome-wide association studies (GWASs) have identified sequence variants, localized to non-coding genomic regions, associated with kidney function. Despite these robust observations, the mechanism by which variants lead to CKD remains a critical unanswered question. Expression quantitative trait loci (eQTL) analysis is a method to identify genetic variation associated with gene expression changes in specific tissue types. We hypothesized that an integrative analysis combining CKD GWAS and kidney eQTL results can identify candidate genes for CKD. We performed eQTL analysis by correlating genotype with RNA-seq-based gene expression levels in 96 human kidney samples. Applying stringent statistical criteria, we detected 1,886 genes whose expression differs with the sequence variants. Using direct overlap and Bayesian methods, we identified new potential target genes for CKD. With respect to one of the target genes, lysosomal beta A mannosidase (MANBA), we observed that genetic variants associated with MANBA expression in the kidney showed statistically significant colocalization with variants identified in CKD GWASs, indicating that MANBA is a potential target gene for CKD. The expression of MANBA was significantly lower in kidneys of subjects with risk alleles. Suppressing manba expression in zebrafish resulted in renal tubule defects and pericardial edema, phenotypes typically induced by kidney dysfunction. Our analysis shows that gene-expression changes driven by genetic variation in the kidney can highlight potential new target genes for CKD development.

Human Kidney Tubule-Specific Gene Expression Based Dissection of Chronic Kidney Disease Traits

Chronic kidney disease (CKD) has diverse phenotypic manifestations including structural (such as fibrosis) and functional (such as glomerular filtration rate and albuminuria) alterations. Gene expression profiling has recently gained popularity as an important new tool for precision medicine approaches. Here we used unbiased and directed approaches to understand how gene expression captures different CKD manifestations in patients with diabetic and hypertensive CKD. Transcriptome data from ninety-five microdissected human kidney samples with a range of demographics, functional and structural changes were used for the primary analysis. Data obtained from 41 samples were available for validation. Using the unbiased Weighted Gene Co-Expression Network Analysis (WGCNA) we identified 16 co-expressed gene modules. We found that modules that strongly correlated with eGFR primarily encoded genes with metabolic functions. Gene groups that mainly encoded T-cell receptor and collagen pathways, showed the strongest correlation with fibrosis level, suggesting that these two phenotypic manifestations might have different underlying mechanisms. Linear regression models were then used to identify genes whose expression showed significant correlation with either structural (fibrosis) or functional (eGFR) manifestation and mostly corroborated the WGCNA findings. We concluded that gene expression is a very sensitive sensor of fibrosis, as the expression of 1654 genes correlated with fibrosis even after adjusting to eGFR and other clinical parameters. The association between GFR and gene expression was mostly mediated by fibrosis. In conclusion, our transcriptome-based CKD trait dissection analysis suggests that the association between gene expression and renal function is mediated by structural changes and that there may be differences in pathways that lead to decline in kidney function and the development of fibrosis, respectively.

Comprehensive single cell RNAseq analysis of the kidney reveals novel cell types and unexpected cell plasticity

A key limitations to understand kidney function and disease development has been that specific cell types responsible for specific homeostatic kidney function or disease phenotypes have not been defined at the molecular level. To fill this gap, we characterized 57,979 cells from healthy mouse kidneys using unbiased single-cell RNA sequencing. We show that genetic mutations that present with similar phenotypes mostly affect genes that are expressed in a single unique differentiated cell type. On the other hand, we found unexpected cell plasticity of epithelial cells in the final segment of the kidney (collecting duct) that is responsible for final composition of the urine. Using computational cell trajectory analysis and in vivo linage tracing, we found that, intercalated cells (that secrete protons) and principal cells (that maintain salt, water and potassium balance) undergo a Notch mediated interconversion via a newly identified transitional cell type. In disease states this transition is shifted towards the principal cell fate. Loss of intercalated cells likely contributes to metabolic acidosis observed in kidney disease. In summary, single cell analysis advanced a mechanistic description of kidney diseases by identifying a defective homeostatic cell lineage. One Sentence Summary A comprehensive single cell atlas of the kidney reveals a transitional cell type and cell plasticity determined by Notch signaling which is defective in chronic kidney disease.

Increasing the level of peroxisome proliferator-activated receptor γ coactivator-1α in podocytes results in collapsing glomerulopathy

Inherited and acquired mitochondrial defects have been associated with podocyte dysfunction and chronic kidney disease (CKD). Peroxisome proliferator-activated receptor γ coactivator-1α (PGC1α) is one of the main transcriptional regulators of mitochondrial biogenesis and function. We hypothesized that increasing PGC1α expression in podocytes could protect from CKD. We found that PGC1α and mitochondrial transcript levels are lower in podocytes of patients and mouse models with diabetic kidney disease (DKD). To increase PGC1α expression, podocyte-specific inducible PGC1α-transgenic mice were generated by crossing nephrin-rtTA mice with tetO-Ppargc1a animals. Transgene induction resulted in albuminuria and glomerulosclerosis in a dose-dependent manner. Expression of PGC1α in podocytes increased mitochondrial biogenesis and maximal respiratory capacity. PGC1α also shifted podocytes towards fatty acid usage from their baseline glucose preference. RNA sequencing analysis indicated that PGC1α induced podocyte proliferation. Histological lesions of mice with podocyte-specific PGC1α expression resembled collapsing focal segmental glomerular sclerosis. In conclusion, decreased podocyte PGC1α expression and mitochondrial content is a consistent feature of DKD, but excessive PGC1α alters mitochondrial properties and induces podocyte proliferation and dedifferentiation, indicating that there is likely a narrow therapeutic window for PGC1α levels in podocytes.

Bulk Tissue Cell Type Deconvolution with Multi-Subject Single-Cell Expression Reference

We present MuSiC, a method that utilizes cell-type specific gene expression from single-cell RNA sequencing (RNA-seq) data to characterize cell type compositions from bulk RNA-seq data in complex tissues. When applied to pancreatic islet and whole kidney expression data in human, mouse, and rats, MuSiC outperformed existing methods, especially for tissues with closely related cell types. MuSiC enables characterization of cellular heterogeneity of complex tissues for identification of disease mechanisms.

DNMT1 in Six2 progenitor cells is essential for transposable element silencing and kidney development

Cytosine methylation (5mC) plays a key role in maintaining progenitor cell self-renewal and differentiation. Here, we analyzed the role of 5mC in kidney development by genome-wide methylation, expression profiling, and by systematic genetic targeting of DNA methyltransferases (Dnmt) and Ten-eleven translocation methylcytosine hydroxylases (Tet). In mice, nephrons differentiate from Six2+ progenitor cells, therefore we created animals with genetic deletion of Dnmt1, 3a, 3b, Tet1, and Tet2 in the Six2+ population (Six2Cre/Dnmt1flox/flox, Six2Cre/Dnmt3aflox/flox, Six2Cre/Dnmt3bflox/flox, Six2Cre/Tet2flox/flox and Tet1-/-). Animals with conditional deletion of Dnmt3a, 3b, Tet1 and Tet2 showed no significant structural or functional renal abnormalities. On the other hand, Six2Cre/Dnmt1flox/flox mice died within 24hrs of birth. Dnmt1 knock-out animals had small kidneys and significantly reduced nephron number. Genome-wide methylation analysis indicated marked loss of methylation mostly on transposable elements. RNA sequencing detected endogenous retroviral (ERV) gene transcripts and early embryonic genes. Increase in levels of interferon (and RIG-I signaling) and apoptosis (Trp53) in response to ERV activity likely contributed to the phenotype development. Once epithelial differentiation was established, loss of Dnmt1, 3a, 3b, Tet1 or Tet2 in glomerular epithelial cells did not lead to functional or structural differences at baseline or following toxic glomerular injury. Genome-wide cytosine methylation and gene expression profiling showed that Dnmt1-mediated DNA methylation is essential for kidney development by preventing regression of progenitor cells into a primitive undifferentiated state and demethylation of transposable elements. Significance Cytosine methylation of regulatory regions (promoters and enhancers) has been proposed to play a key role in establishing gene expression and thereby cellular phenotype. DNMT1 is the key enzyme responsible for maintaining methylation patterns during DNA replication. While the role of Dnmt1 has been described in multiple organs, here we identified a novel, critically important mechanism how Dnmt1 controls tissue progenitors. The greatest methylation difference in Dnmt1 knock-out mice was observed on transposable elements (TE), which resulted in increase of endogenous retroviruses and cell death. We believe that release of TE was a critically overlooked component of phenotype development in previous studies that our comprehensive genome wide methylation analysis allowed us to identify. Competing interests The Susztak lab receives research support from Biogen, Boehringer Ingelheim, Celgene, GSK, Merck, Regeneron and ONO Pharma for work not related to this manuscript.

Jagged1/Notch2 controls kidney fibrosis via Tfam-mediated metabolic reprogramming

While Notch signaling has been proposed to play a key role in fibrosis, the direct molecular pathways targeted by Notch signaling and the precise ligand and receptor pair that are responsible for kidney disease remain poorly defined. In this study, we found that JAG1 and NOTCH2 showed the strongest correlation with the degree of interstitial fibrosis in a genome-wide expression analysis of a large cohort of human kidney samples. Transcript analysis of mouse kidney disease models, including folic-acid (FA)–induced nephropathy, unilateral ureteral obstruction (UUO), or apolipoprotein L1 (APOL1)-associated kidney disease, indicated that Jag1 and Notch2 levels were higher in all analyzed kidney fibrosis models. Mice with tubule-specific deletion of Jag1 or Notch2 (Kspcre/Jag1flox/flox and Kspcre/Notch2flox/flox) had no kidney-specific alterations at baseline but showed protection from FA-induced kidney fibrosis. Tubule-specific genetic deletion of Notch1 and global knockout of Notch3 had no effect on fibrosis. In vitro chromatin immunoprecipitation experiments and genome-wide expression studies identified the mitochondrial transcription factor A (Tfam) as a direct Notch target. Re-expression of Tfam in tubule cells prevented Notch-induced metabolic and profibrotic reprogramming. Tubule–specific deletion of Tfam resulted in fibrosis. In summary, Jag1 and Notch2 play a key role in kidney fibrosis development by regulating Tfam expression and metabolic reprogramming.