ADAR regulates APOL1 via A-to-I RNA editing by inhibition of MDA5 activation in a paradoxical biological circuit.

APOL1 risk variants are associated with increased risk of kidney disease in patients of African ancestry, but not all individuals with the APOL1 high-risk genotype develop kidney disease. As APOL1 gene expression correlates closely with the degree of kidney cell injury in both cell and animal models, the mechanisms regulating APOL1 expression may be critical determinants of risk allele penetrance. The APOL1 messenger RNA includes Alu elements at the 3' untranslated region that can form a double-stranded RNA structure (Alu-dsRNA) susceptible to posttranscriptional adenosine deaminase acting on RNA (ADAR)-mediated adenosine-to-inosine (A-to-I) editing, potentially impacting gene expression. We studied the effects of ADAR expression and A-to-I editing on APOL1 levels in podocytes, human kidney tissue, and a transgenic APOL1 mouse model. In interferon-γ (IFN-γ)-stimulated human podocytes, ADAR down-regulates APOL1 by preventing melanoma differentiation-associated protein 5 (MDA5) recognition of dsRNA and the subsequent type I interferon (IFN-I) response. Knockdown experiments showed that recognition of APOL1 messenger RNA itself is an important contributor to the MDA5-driven IFN-I response. Mathematical modeling suggests that the IFN-ADAR-APOL1 network functions as an incoherent feed-forward loop, a biological circuit capable of generating fast, transient responses to stimuli. Glomeruli from human kidney biopsies exhibited widespread editing of APOL1 Alu-dsRNA, while the transgenic mouse model closely replicated the edited sites in humans. APOL1 expression in mice was inversely correlated with Adar1 expression under IFN-γ stimuli, supporting the idea that ADAR regulates APOL1 levels in vivo. ADAR-mediated A-to-I editing is an important regulator of APOL1 expression that could impact both penetrance and severity of APOL1-associated kidney disease.

Precision nephrology identified tumor necrosis factor activation variability in minimal change disease and focal segmental glomerulosclerosis.

The diagnosis of nephrotic syndrome relies on clinical presentation and descriptive patterns of injury on kidney biopsies, but not specific to underlying pathobiology. Consequently, there are variable rates of progression and response to therapy within diagnoses. Here, an unbiased transcriptomic-driven approach was used to identify molecular pathways which are shared by subgroups of patients with either minimal change disease (MCD) or focal segmental glomerulosclerosis (FSGS). Kidney tissue transcriptomic profile-based clustering identified three patient subgroups with shared molecular signatures across independent, North American, European, and African cohorts. One subgroup had significantly greater disease progression (Hazard Ratio 5.2) which persisted after adjusting for diagnosis and clinical measures (Hazard Ratio 3.8). Inclusion in this subgroup was retained even when clustering was limited to those with less than 25% interstitial fibrosis. The molecular profile of this subgroup was largely consistent with tumor necrosis factor (TNF) pathway activation. Two TNF pathway urine markers were identified, tissue inhibitor of metalloproteinases-1 (TIMP-1) and monocyte chemoattractant protein-1 (MCP-1), that could be used to predict an individual's TNF pathway activation score. Kidney organoids and single-nucleus RNA-sequencing of participant kidney biopsies, validated TNF-dependent increases in pathway activation score, transcript and protein levels of TIMP-1 and MCP-1, in resident kidney cells. Thus, molecular profiling identified a subgroup of patients with either MCD or FSGS who shared kidney TNF pathway activation and poor outcomes. A clinical trial testing targeted therapies in patients selected using urinary markers of TNF pathway activation is ongoing.

A glomerular transcriptomic landscape of apolipoprotein L1 in Black patients with focal segmental glomerulosclerosis.

Apolipoprotein L1 (APOL1)-associated focal segmental glomerulosclerosis (FSGS) is the dominant form of FSGS in Black individuals. There are no targeted therapies for this condition, in part because the molecular mechanisms underlying APOL1's pathogenic contribution to FSGS are incompletely understood. Studying the transcriptomic landscape of APOL1 FSGS in patient kidneys is an important way to discover genes and molecular behaviors that are unique or most relevant to the human disease. With the hypothesis that the pathology driven by the high-risk APOL1 genotype is reflected in alteration of gene expression across the glomerular transcriptome, we compared expression and co-expression profiles of 15,703 genes in 16 Black patients with FSGS at high-risk vs 14 Black patients with a low-risk APOL1 genotype. Expression data from APOL1-inducible HEK293 cells and normal human glomeruli were used to pursue genes and molecular pathways uncovered in these studies. We discovered increased expression of APOL1 and nine other significant differentially expressed genes in high-risk patients. This included stanniocalcin, which has a role in mitochondrial and calcium-related processes along with differential correlations between high- and low-risk APOL1 and metabolism pathway genes. There were similar correlations with extracellular matrix- and immune-related genes, but significant loss of co-expression of mitochondrial genes in high-risk FSGS, and an NF-κB-down regulating gene, NKIRAS1, as the most significant hub gene with strong differential correlations with NDUF family (mitochondrial respiratory genes) and immune-related (JAK-STAT) genes. Thus, differences in mitochondrial gene regulation appear to underlie many differences observed between high- and low-risk Black patients with FSGS.

Molecular programs associated with glomerular hyperfiltration in early diabetic kidney disease.

Hyperfiltration is a state of high glomerular filtration rate (GFR) observed in early diabetes that damages glomeruli, resulting in an iterative process of increasing filtration load on fewer and fewer remaining functional glomeruli. To delineate underlying cellular mechanisms of damage associated with hyperfiltration, transcriptional profiles of kidney biopsies from Pima Indians with type 2 diabetes with or without early-stage diabetic kidney disease were grouped into two hyperfiltration categories based on annual iothalamate GFR measurements. Twenty-six participants with a peak GFR measurement within two years of biopsy were categorized as the hyperfiltration group, and 26 in whom biopsy preceded peak GFR by over two years were considered pre-hyperfiltration. The hyperfiltration group had higher hemoglobin A1c, higher urine albumin-to-creatinine ratio, increased glomerular basement membrane width and lower podocyte density compared to the pre-hyperfiltration group. A glomerular 1240-gene transcriptional signature identified in the hyperfiltration group was enriched for endothelial stress response signaling genes, including endothelin-1, tec-kinase and transforming growth factor-ß1 pathways, with the majority of the transcripts mapped to endothelial and inflammatory cell clusters in kidney single cell transcriptional data. Thus, our analysis reveals molecular pathomechanisms associated with hyperfiltration in early diabetic kidney disease involving putative ligand-receptor pairs with downstream intracellular targets linked to cellular crosstalk between endothelial and mesangial cells.

Gene expression profiles of diabetic kidney disease and neuropathy in eNOS knockout mice: Predictors of pathology and RAS blockade effects.

Diabetic kidney disease (DKD) and diabetic peripheral neuropathy (DPN) are two common diabetic complications. However, their pathogenesis remains elusive and current therapies are only modestly effective. We evaluated genome-wide expression to identify pathways involved in DKD and DPN progression in db/db eNOS-/- mice receiving renin-angiotensin-aldosterone system (RAS)-blocking drugs to mimic the current standard of care for DKD patients. Diabetes and eNOS deletion worsened DKD, which improved with RAS treatment. Diabetes also induced DPN, which was not affected by eNOS deletion or RAS blockade. Given the multiple factors affecting DKD and the graded differences in disease severity across mouse groups, an automatic data analysis method, SOM, or self-organizing map was used to elucidate glomerular transcriptional changes associated with DKD, whereas pairwise bioinformatic analysis was used for DPN. These analyses revealed that enhanced gene expression in several pro-inflammatory networks and reduced expression of development genes correlated with worsening DKD. Although RAS treatment ameliorated the nephropathy phenotype, it did not alter the more abnormal gene expression changes in kidney. Moreover, RAS exacerbated expression of genes related to inflammation and oxidant generation in peripheral nerves. The graded increase in inflammatory gene expression and decrease in development gene expression with DKD progression underline the potentially important role of these pathways in DKD pathogenesis. Since RAS blockers worsened this gene expression pattern in both DKD and DPN, it may partly explain the inadequate therapeutic efficacy of such blockers.

Renin-angiotensin system inhibition reverses the altered triacylglycerol metabolic network in diabetic kidney disease.

OBJECTIVE: Dyslipidemia is a significant risk factor for progression of diabetic kidney disease (DKD). Determining the changes in individual lipids and lipid networks across a spectrum of DKD severity may identify lipids that are pathogenic to DKD progression. METHODS: We performed untargeted lipidomic analysis of kidney cortex tissue from diabetic db/db and db/db eNOS-/- mice along with non-diabetic littermate controls. A subset of mice were treated with the renin-angiotensin system (RAS) inhibitors, lisinopril and losartan, which improves the DKD phenotype in the db/db eNOS-/- mouse model. RESULTS: Of the three independent variables in this study, diabetes had the largest impact on overall lipid levels in the kidney cortex, while eNOS expression and RAS inhibition had smaller impacts on kidney lipid levels. Kidney lipid network architecture, particularly of networks involving glycerolipids such as triacylglycerols, was substantially disrupted by worsening kidney disease in the db/db eNOS-/- mice compared to the db/db mice, a feature that was reversed with RAS inhibition. This was associated with decreased expression of the stearoyl-CoA desaturases, Scd1 and Scd2, with RAS inhibition. CONCLUSIONS: In addition to the known salutary effect of RAS inhibition on DKD progression, our results suggest a previously unrecognized role for RAS inhibition on the kidney triacylglycerol lipid metabolic network.

Transcriptome analysis of primary podocytes reveals novel calcium regulated regulatory networks.

Podocytes are pivotal in establishing the selective permeability of the glomerular filtration barrier. Recently, we showed that an increase of the intracellular calcium ion concentration [Ca2+ ] causes a rapid and transient actin reset (CaAR) measurable through live imaging microscopy using lifeact-mCherry as an actin dye in different cell types including the podocyte. This and other studies show the critical role [Ca2+ ] and the actin cytoskeleton play in podocyte homeostasis. To further investigate the role of [Ca2+ ] and the actin cytoskeleton in podocytes, we used a double fluorescent reporter mouse model to establish a primary podocyte culture system. We treated these podocytes temporarily with a Calcium Ionophore and facultatively with Latrunculin A, an inhibitor of actin polymerization. Unbiased genome wide transcriptional analysis identified a transcriptional response in podocytes to elevated [Ca2+ ] levels, affecting mRNA levels of PDGF-BB, RICTOR, and MIR17HG as mediators of Ca2+ -signaling. Comparison of the ex vivo transcriptional response from the primary podocyte culture with glomerular transcripts across a wide spectrum of CKD disease confirmed co-regulation of transcript sets, establishing the disease relevance of the model system. Our findings demonstrate novel [Ca2+ ] regulated gene networks in podocytes deepening our understanding of podocyte biology and disease.

Comparative RNA-Seq transcriptome analyses reveal distinct metabolic pathways in diabetic nerve and kidney disease.

Treating insulin resistance with pioglitazone normalizes renal function and improves small nerve fibre function and architecture; however, it does not affect large myelinated nerve fibre function in mouse models of type 2 diabetes (T2DM), indicating that pioglitazone affects the body in a tissue-specific manner. To identify distinct molecular pathways regulating diabetic peripheral neuropathy (DPN) and nephropathy (DN), as well those affected by pioglitazone, we assessed DPN and DN gene transcript expression in control and diabetic mice with or without pioglitazone treatment. Differential expression analysis and self-organizing maps were then used in parallel to analyse transcriptome data. Differential expression analysis showed that gene expression promoting cell death and the inflammatory response was reversed in the kidney glomeruli but unchanged or exacerbated in sciatic nerve by pioglitazone. Self-organizing map analysis revealed that mitochondrial dysfunction was normalized in kidney and nerve by treatment; however, conserved pathways were opposite in their directionality of regulation. Collectively, our data suggest inflammation may drive large fibre dysfunction, while mitochondrial dysfunction may drive small fibre dysfunction in T2DM. Moreover, targeting both of these pathways is likely to improve DN. This study supports growing evidence that systemic metabolic changes in T2DM are associated with distinct tissue-specific metabolic reprogramming in kidney and nerve and that these changes play a critical role in DN and small fibre DPN pathogenesis. These data also highlight the potential dangers of a 'one size fits all' approach to T2DM therapeutics, as the same drug may simultaneously alleviate one complication while exacerbating another.

Integrative Genomics Identifies Novel Associations with APOL1 Risk Genotypes in Black NEPTUNE Subjects.

APOL1 variants have been associated with renal phenotypes in blacks. To refine clinical outcomes and discover mechanisms of APOL1-associated kidney injury, we analyzed clinical and genomic datasets derived from 90 black subjects in the Nephrotic Syndrome Study Network (NEPTUNE), stratified by APOL1 risk genotype. Ninety subjects with proteinuria ≥0.5 g/d were enrolled at first biopsy for primary nephrotic syndrome and followed. Clinical outcomes were determined, and renal histomorphometry and sequencing of Mendelian nephrotic syndrome genes were performed. APOL1 variants were genotyped, and glomerular and tubulointerstitial transcriptomes from protocol renal biopsy cores were analyzed for differential and correlative gene expression. Analyses were performed under the recessive model (high-risk genotype defined by two risk alleles). APOL1 high-risk genotype was significantly associated with a 17 ml/min per 1.73 m(2) lower eGFR and a 69% reduction in the probability of complete remission at any time, independent of histologic diagnosis. Neither APOL1 risk group was enriched for Mendelian mutations. On renal biopsy, high-risk genotype was associated with increased fractional interstitial area, interstitial fibrosis, and tubular atrophy. Risk genotype was not associated with intrarenal APOL1 mRNA expression levels. Differential expression analysis demonstrated an increased steady-state level of five genes associated with the high-risk genotype (CXCL9, CXCL11, and UBD in glomerulus; SNOR14B and MUC13 in tubulointerstitium). APOL1 tubulointerstitial coexpression analysis showed coexpression of APOL1 mRNA levels with a group of intrarenal transcripts that together were associated with increased interstitial fibrosis and tubular atrophy. These data indicate the high-risk APOL1 genotype confers renal risk across histopathologic diagnoses.

Defining cell-type specificity at the transcriptional level in human disease.

Cell-lineage-specific transcripts are essential for differentiated tissue function, implicated in hereditary organ failure, and mediate acquired chronic diseases. However, experimental identification of cell-lineage-specific genes in a genome-scale manner is infeasible for most solid human tissues. We developed the first genome-scale method to identify genes with cell-lineage-specific expression, even in lineages not separable by experimental microdissection. Our machine-learning-based approach leverages high-throughput data from tissue homogenates in a novel iterative statistical framework. We applied this method to chronic kidney disease and identified transcripts specific to podocytes, key cells in the glomerular filter responsible for hereditary and most acquired glomerular kidney disease. In a systematic evaluation of our predictions by immunohistochemistry, our in silico approach was significantly more accurate (65% accuracy in human) than predictions based on direct measurement of in vivo fluorescence-tagged murine podocytes (23%). Our method identified genes implicated as causal in hereditary glomerular disease and involved in molecular pathways of acquired and chronic renal diseases. Furthermore, based on expression analysis of human kidney disease biopsies, we demonstrated that expression of the podocyte genes identified by our approach is significantly related to the degree of renal impairment in patients. Our approach is broadly applicable to define lineage specificity in both cell physiology and human disease contexts. We provide a user-friendly website that enables researchers to apply this method to any cell-lineage or tissue of interest. Identified cell-lineage-specific transcripts are expected to play essential tissue-specific roles in organogenesis and disease and can provide starting points for the development of organ-specific diagnostics and therapies.

Integrative biology identifies shared transcriptional networks in CKD.

A previous meta-analysis of genome-wide association data by the Cohorts for Heart and Aging Research in Genomic Epidemiology and CKDGen consortia identified 16 loci associated with eGFR. To define how each of these single-nucleotide polymorphisms (SNPs) could affect renal function, we integrated GFR-associated loci with regulatory pathways, producing a molecular map of CKD. In kidney biopsy specimens from 157 European subjects representing nine different CKDs, renal transcript levels for 18 genes in proximity to the SNPs significantly correlated with GFR. These 18 genes were mapped into their biologic context by testing coregulated transcripts for enriched pathways. A network of 97 pathways linked by shared genes was constructed and characterized. Of these pathways, 56 pathways were reported previously to be associated with CKD; 41 pathways without prior association with CKD were ranked on the basis of the number of candidate genes connected to the respective pathways. All pathways aggregated into a network of two main clusters comprising inflammation- and metabolism-related pathways, with the NRF2-mediated oxidative stress response pathway serving as the hub between the two clusters. In all, 78 pathways and 95% of the connections among those pathways were verified in an independent North American biopsy cohort. Disease-specific analyses showed that most pathways are shared between sets of three diseases, with closest interconnection between lupus nephritis, IgA nephritis, and diabetic nephropathy. Taken together, the network integrates candidate genes from genome-wide association studies into their functional context, revealing interactions and defining established and novel biologic mechanisms of renal impairment in renal diseases.

Gene Expression Sets and Renal Profiling from the Renal AL Amyloid Involvement and NEOD00 (RAIN) Trial.

Introduction: There is an unmet need to understand the mechanisms by which amyloid deposition drives alterations in the kidney. We leveraged renal biopsies from amyloid light-chain (AL) amyloidosis participants of the Renal AL Amyloid Involvement and NEOD00 (RAIN) trial (NCT03168906) to perform transcriptional profiling and to employ a novel histologic scoring tool. Our objective was to utilize a transcriptome-driven approach to identify AL molecular signatures that may be prognostic. Methods: Clinical data were correlated to histologic and molecular findings. A composite scarring injury and amyloid score (AS) were assigned to each biopsy. Glomerular and tubulointerstitial (TI) compartments were microdissected and sequenced separately. Expression data were compared to healthy living donors and focal segmental glomerulosclerosis (FSGS) profiles. Differentially expressed genes were determined. Results: Cluster analysis revealed 2 distinct patient clusters (G1 and G2) based on gene expression. The AS was higher in the TI compartment (6.5 vs. 4.5; P = 0.0290) of G2. Glomeruli showed activation of fibrotic pathways and increased canonical signaling of LPS/IL-1. TNF activation was noted in TI. Enriched ingenuity canonical pathways included "coagulation system," "GADD45 signaling," and "Wnt/Ca+ pathway," among others. For AL versus living donors, ingenuity pathway analysis identified enrichment in PI3K/Akt signaling. Gene regulators of cellular proliferation were enriched in the amyloid group. Conclusion: Despite the small sample size, we identified 2 distinct groups of patients with AL based on molecular signatures. Detailed studies of a larger cohort encompassing omics technologies at a single cell resolution will further help to identify the response of individual kidney cell types to amyloid deposits, potentially leading to the development of novel therapeutic targets.

An integrated organoid omics map extends modeling potential of kidney disease.

Kidney organoids are a promising model to study kidney disease, but their use is constrained by limited knowledge of their functional protein expression profile. Here, we define the organoid proteome and transcriptome trajectories over culture duration and upon exposure to TNFα, a cytokine stressor. Older organoids increase deposition of extracellular matrix but decrease expression of glomerular proteins. Single cell transcriptome integration reveals that most proteome changes localize to podocytes, tubular and stromal cells. TNFα treatment of organoids results in 322 differentially expressed proteins, including cytokines and complement components. Transcript expression of these 322 proteins is significantly higher in individuals with poorer clinical outcomes in proteinuric kidney disease. Key TNFα-associated protein (C3 and VCAM1) expression is increased in both human tubular and organoid kidney cell populations, highlighting the potential for organoids to advance biomarker development. By integrating kidney organoid omic layers, incorporating a disease-relevant cytokine stressor and comparing with human data, we provide crucial evidence for the functional relevance of the kidney organoid model to human kidney disease.

Defining the molecular correlate of arteriolar hyalinosis in kidney disease progression by integration of single cell transcriptomic analysis and pathology scoring.

Arteriolar hyalinosis in kidneys is an independent predictor of cardiovascular disease, the main cause of mortality in chronic kidney disease (CKD). The underlying molecular mechanisms of protein accumulation in the subendothelial space are not well understood. Using single cell transcriptomic data and whole slide images from kidney biopsies of patients with CKD and acute kidney injury in the Kidney Precision Medicine Project, the molecular signals associated with arteriolar hyalinosis were evaluated. Co-expression network analysis of the endothelial genes yielded three gene set modules as significantly associated with arteriolar hyalinosis. Pathway analysis of these modules showed enrichment of transforming growth factor beta / bone morphogenetic protein (TGFß / BMP) and vascular endothelial growth factor (VEGF) signaling pathways in the endothelial cell signatures. Ligand-receptor analysis identified multiple integrins and cell adhesion receptors as over-expressed in arteriolar hyalinosis, suggesting a potential role of integrin-mediated TGFß signaling. Further analysis of arteriolar hyalinosis associated endothelial module genes identified focal segmental glomerular sclerosis as an enriched term. On validation in gene expression profiles from the Nephrotic Syndrome Study Network cohort, one of the three modules was significantly associated with the composite endpoint (> 40% reduction in estimated glomerular filtration rate (eGFR) or kidney failure) independent of age, sex, race, and baseline eGFR, suggesting poor prognosis with elevated expression of genes in this module. Thus, integration of structural and single cell molecular features yielded biologically relevant gene sets, signaling pathways and ligand-receptor interactions, underlying arteriolar hyalinosis and putative targets for therapeutic intervention.

Periostin is induced in glomerular injury and expressed de novo in interstitial renal fibrosis.

Matricellular proteins participate in the pathogenesis of chronic kidney diseases. We analyzed glomerular gene expression profiles from patients with proteinuric diseases to identify matricellular proteins contributing to the progression of human nephropathies. Several genes encoding matricellular proteins, such as SPARC, THBS1, and CTGF, were induced in progressive nephropathies, but not in nonprogressive minimal-change disease. Periostin showed the highest induction, and its transcript levels correlated negatively with glomerular filtration rate in both glomerular and tubulointerstitial specimen. In well-preserved renal tissue, periostin localized to the glomerular tuft, the vascular pole, and along Bowman's capsule; no signal was detected in the tubulointerstitial compartment. Biopsies from patients with glomerulopathies and renal dysfunction showed enhanced periostin expression in the mesangium, tubular interstitium, and sites of fibrosis. Periostin staining correlated negatively with renal function. α-smooth muscle actin-positive mesangial and interstitial cells localized close to periostin-positive sites, as indicated by co-immunofluorescence. In vitro stimulation of mesangial cells by external addition of TGF-ß1 resulted in robust induction of periostin. Addition of periostin to mesangial cells induced cell proliferation and decreased the number of cells expressing activated caspase-3, a marker of apoptosis. These human data indicate for the first time a role of periostin in glomerular and interstitial injury in acquired nephropathies.

Multi-Scalar Data Integration Links Glomerular Angiopoietin-Tie Signaling Pathway Activation With Progression of Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease (ESKD). Prognostic biomarkers reflective of underlying molecular mechanisms are critically needed for effective management of DKD. A three-marker panel was derived from a proteomics analysis of plasma samples by an unbiased machine learning approach from participants (N = 58) in the Clinical Phenotyping and Resource Biobank study. In combination with standard clinical parameters, this panel improved prediction of the composite outcome of ESKD or a 40% decline in glomerular filtration rate. The panel was validated in an independent group (N = 68), who also had kidney transcriptomic profiles. One marker, plasma angiopoietin 2 (ANGPT2), was significantly associated with outcomes in cohorts from the Cardiovascular Health Study (N = 3,183) and the Chinese Cohort Study of Chronic Kidney Disease (N = 210). Glomerular transcriptional angiopoietin/Tie (ANG-TIE) pathway scores, derived from the expression of 154 ANG-TIE signaling mediators, correlated positively with plasma ANGPT2 levels and kidney outcomes. Higher receptor expression in glomeruli and higher ANG-TIE pathway scores in endothelial cells corroborated potential functional effects in the kidney from elevated plasma ANGPT2 levels. Our work suggests that ANGPT2 is a promising prognostic endothelial biomarker with likely functional impact on glomerular pathogenesis in DKD.

A molecular profile of focal segmental glomerulosclerosis from formalin-fixed, paraffin-embedded tissue.

Focal segmental glomerulosclerosis (FSGS) is a common form of idiopathic nephrotic syndrome defined by the characteristic lesions of focal glomerular sclerosis and foot process effacement; however, its etiology and pathogenesis are unknown. We used mRNA isolated from laser-captured glomeruli from archived formalin-fixed, paraffin-embedded renal biopsies, until recently considered an unsuitable source of mRNA for microarray analysis, to investigate the glomerular gene expression profiles of patients with primary classic FSGS, collapsing FSGS (COLL), minimal change disease (MCD), and normal controls (Normal). Amplified mRNA was hybridized to an Affymetrix Human X3P array. Unsupervised (unbiased) hierarchical clustering revealed two distinct clusters delineating FSGS and COLL from Normal and MCD. Class comparison analysis of FSGS + COLL combined versus Normal + MCD revealed 316 significantly differentially regulated genes (134 up-regulated, 182 down-regulated). Among the differentially regulated genes were those known to be part of the slit diaphragm junctional complex and those previously described in the dysregulated podocyte phenotype. Analysis based on Gene Ontology categories revealed overrepresented biological processes of development, differentiation and morphogenesis, cell motility and migration, cytoskeleton organization, and signal transduction. Transcription factors associated with developmental processes were heavily overrepresented, indicating the importance of reactivation of developmental programs in the pathogenesis of FSGS. Our findings reveal novel insights into the molecular pathogenesis of glomerular injury and structural degeneration in FSGS.

Pro-cachectic factors link experimental and human chronic kidney disease to skeletal muscle wasting programs.

Skeletal muscle wasting is commonly associated with chronic kidney disease (CKD), resulting in increased morbidity and mortality. However, the link between kidney and muscle function remains poorly understood. Here, we took a complementary interorgan approach to investigate skeletal muscle wasting in CKD. We identified increased production and elevated blood levels of soluble pro-cachectic factors, including activin A, directly linking experimental and human CKD to skeletal muscle wasting programs. Single-cell sequencing data identified the expression of activin A in specific kidney cell populations of fibroblasts and cells of the juxtaglomerular apparatus. We propose that persistent and increased kidney production of pro-cachectic factors, combined with a lack of kidney clearance, facilitates a vicious kidney/muscle signaling cycle, leading to exacerbated blood accumulation and, thereby, skeletal muscle wasting. Systemic pharmacological blockade of activin A using soluble activin receptor type IIB ligand trap as well as muscle-specific adeno-associated virus-mediated downregulation of its receptor ACVR2A/B prevented muscle wasting in different mouse models of experimental CKD, suggesting that activin A is a key factor in CKD-induced cachexia. In summary, we uncovered a crosstalk between kidney and muscle and propose modulation of activin signaling as a potential therapeutic strategy for skeletal muscle wasting in CKD.