Chengxiang Qiu

Transgenic expression of human APOL1 risk variants in podocytes induces kidney disease in mice

African Americans have a heightened risk of developing chronic and end-stage kidney disease, an association that is largely attributed to two common genetic variants, termed G1 and G2, in the APOL1 gene. Direct evidence demonstrating that these APOL1 risk alleles are pathogenic is still lacking because the APOL1 gene is present in only some primates and humans; thus it has been challenging to demonstrate experimental proof of causality of these risk alleles for renal disease. Here we generated mice with podocyte-specific inducible expression of the APOL1 reference allele (termed G0) or each of the risk-conferring alleles (G1 or G2). We show that mice with podocyte-specific expression of either APOL1 risk allele, but not of the G0 allele, develop functional (albuminuria and azotemia), structural (foot-process effacement and glomerulosclerosis) and molecular (gene-expression) changes that closely resemble human kidney disease. Disease development was cell-type specific and likely reversible, and the severity correlated with the level of expression of the risk allele. We further found that expression of the risk-variant APOL1 alleles interferes with endosomal trafficking and blocks autophagic flux, which ultimately leads to inflammatory-mediated podocyte death and glomerular scarring. In summary, this is the first demonstration that the expression of APOL1 risk alleles is causal for altered podocyte function and glomerular disease in vivo.

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.

Epigenome-wide association studies identify DNA methylation associated with kidney function

Chronic kidney disease (CKD) is defined by reduced estimated glomerular filtration rate (eGFR). Previous genetic studies have implicated regulatory mechanisms contributing to CKD. Here we present epigenome-wide association studies of eGFR and CKD using whole-blood DNA methylation of 2264 ARIC Study and 2595 Framingham Heart Study participants to identify epigenetic signatures of kidney function. Of 19 CpG sites significantly associated (P < 1e-07) with eGFR/CKD and replicated, five also associate with renal fibrosis in biopsies from CKD patients and show concordant DNA methylation changes in kidney cortex. Lead CpGs at PTPN6/PHB2, ANKRD11, and TNRC18 map to active enhancers in kidney cortex. At PTPN6/PHB2 cg19942083, methylation in kidney cortex associates with lower renal PTPN6 expression, higher eGFR, and less renal fibrosis. The regions containing the 243 eGFR-associated (P < 1e-05) CpGs are significantly enriched for transcription factor binding sites of EBF1, EP300, and CEBPB (P < 5e-6). Our findings highlight kidney function associated epigenetic variation.Genome-wide association studies of kidney function show enrichment of associated genetic variants in regulatory regions. Here, the authors perform epigenome-wide association studies of kidney function and disease, identifying 19 CpG sites significantly associated with these.

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.

Genomic integration of ERRγ-HNF1β regulates renal bioenergetics and prevents chronic kidney disease

Significance Renal epithelial cells (RECs) contain abundant mitochondria that are essential to support renal reabsorption of electrolytes, glucose, and amino acids. However, it remains poorly understood how mitochondrial metabolism is coordinated with kidney reabsorptive functions. Here we show that deletion of estrogen-related receptor gamma (ERRγ) in RECs results in severe renal mitochondrial and reabsorptive dysfunction with fluid-filled cysts. ERRγ directly regulates mitochondrial metabolism and cooperates in regulating renal reabsorption genes with hepatic nuclear factor 1 beta (HNF1β), mutations of which cause strikingly similar renal dysfunction and cysts in animals and humans. These findings reveal a role for ERRγ in simultaneously coordinating a transcriptional program of renal energy-generating mitochondrial and energy-consuming reabsorptive functions relevant to kidney disease. Mitochondrial dysfunction is increasingly recognized as a critical determinant of both hereditary and acquired kidney diseases. However, it remains poorly understood how mitochondrial metabolism is regulated to support normal kidney function and how its dysregulation contributes to kidney disease. Here, we show that the nuclear receptor estrogen-related receptor gamma (ERRγ) and hepatocyte nuclear factor 1 beta (HNF1β) link renal mitochondrial and reabsorptive functions through coordinated epigenomic programs. ERRγ directly regulates mitochondrial metabolism but cooperatively controls renal reabsorption via convergent binding with HNF1β. Deletion of ERRγ in renal epithelial cells (RECs), in which it is highly and specifically expressed, results in severe renal energetic and reabsorptive dysfunction and progressive renal failure that recapitulates phenotypes of animals and patients with HNF1β loss-of-function gene mutations. Moreover, ERRγ expression positively correlates with renal function and is decreased in patients with chronic kidney disease (CKD). REC-ERRγ KO mice share highly overlapping renal transcriptional signatures with human patients with CKD. Together these findings reveal a role for ERRγ in directing independent and HNF1β-integrated programs for energy production and use essential for normal renal function and the prevention of kidney disease.

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.

Cytosine methylation predicts renal function decline in American Indians

Diabetic nephropathy accounts for most of the excess mortality in individuals with diabetes, but the molecular mechanisms by which nephropathy develops are largely unknown. Here we tested cytosine methylation levels at 397,063 genomic CpG sites for association with decline in the estimated glomerular filtration rate (eGFR) over a six year period in 181 diabetic Pima Indians. Methylation levels at 77 sites showed significant association with eGFR decline after correction for multiple comparisons. A model including methylation level at two probes (cg25799291 and cg22253401) improved prediction of eGFR decline in addition to baseline eGFR and the albumin to creatinine ratio with the percent of variance explained significantly improving from 23.1% to 42.2%. Cg22253401 was also significantly associated with eGFR decline in a case-control study derived from the Chronic Renal Insufficiency Cohort. Probes at which methylation significantly associated with eGFR decline were localized to gene regulatory regions and enriched for genes with metabolic functions and apoptosis. Three of the 77 probes that were associated with eGFR decline in blood samples showed directionally consistent and significant association with fibrosis in microdissected human kidney tissue, after correction for multiple comparisons. Thus, cytosine methylation levels may provide biomarkers of disease progression in diabetic nephropathy and epigenetic variations contribute to the development of diabetic 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.

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.