We have recently demonstrated that treatment of aged mice with a pan-ERR agonist reverses age-related increase in urinary albumin, decrease in podocyte density, impaired mitochondrial function, and inflammation. The contribution of individual isoforms of ERRs however has not been determined. Since the aging kidney showed a possible compensatory increased expression of ERRγ in the podocytes, in the face of decreased ERRα expression, in the present study we aimed to determine the role of ERRγ in aging podocyte. To this end, we cross bred ERRγ floxed mice with podocin-Cre mice to achieve a podocyte-specific ERRγ deletion. While these mice at 3 months of age showed no effect on albuminuria compared to the wild type, when the mice were aged to 21 months of age, there was a significant increase in albuminuria and decrease in podocyte density. Furthermore, we found that the podocyte deletion of ERRγ primarily targeted the expression of mitochondrial biogenesis regulator PGC-1α, and mitochondrial fatty acid oxidation enzymes CPT1a and MCAD in the kidney. Electron Microscopy (EM) revealed thickened glomerular basement membrane and diffuse podocyte foot process effacement, as well as severe mitochondrial damage including cristae abnormalities, fragmentation, and changes indicative of altered fusion and fission dynamics. Fluorescence Lifetime Imaging Microscopy (FLIM) to determine NADH and FAD lifetimes indicate a metabolic shift from mitochondrial oxidative phosphorylation towards glycolysis, and decrease in mitochondrial redox capacity. Considering a significantly decreased expression of ERRα in aging podocytes plus its traditional role in mitochondrial function, these studies using podocyte ERRγ deletion suggested an overlapping mechanism for ERRα/ERRγ to act as modulators of age-related mitochondrial dysfunction and age-related kidney disease.

Introduction:Heart failure with preserved ejection fraction (HFpEF) is strongly associated with cardiometabolic comorbidities, including obesity, diabetes, hypertension, chronic kidney disease and aging, yet the mechanistic contribution of cellular senescence to HFpEF pathogenesis remains poorly defined. Methods and Results:To model clinically relevant HFpEF, we subjected p16-3MR mice to a novel chronic "four-hit" cardiovascular-kidney-metabolic stress regimen (10 months of a high-fat diet, low-dose streptozotocin, L-NAME, and aging). These mice developed a robust HFpEF phenotype characterized by left ventricular hypertrophy, impaired diastolic function (reduced E'/A' and elevated E/E'), preserved ejection fraction, reduced -dP/dt, exercise intolerance, pulmonary congestion, and increased cardiac CD68⁺ macrophage infiltration. Cardiac proteomics identified 821 proteins significantly altered by four-hit stress. Selective genetic ablation of p16⁺ senescent cells using ganciclovir ameliorated HFpEF phenotypes, reduced cardiac p16 expression and inflammation, and normalized proteomic remodeling, without affecting body weight or glycemic status. Comparative network analysis of mouse and human HFpEF cardiac proteomes revealed highly concordant upstream regulatory networks, prominently involving cell-cycle control, DNA damage responses, and inflammatory signaling. Immunohistochemical analysis of human HFpEF cardiac biopsies confirmed increased p16, γH2AX, STING, IRF3, NF-κB p65, and CD68⁺ macrophages, mirroring the murine findings. The 4-Hit mice also developed chronic diabetic kidney disease with increased kidney inflammation, both of which were attenuated by Senolytic therapy. Mechanistically, the cGAS-STING (cyclic GMP-AMP synthase - stimulator of interferon genes) is activated in response to damaged DNA, which in turn activates the downstream immune responses, including NF-κB and interferons. Cross-species validation further demonstrated that combined metabolic stress impaired cardiac function and nephrocyte function in Drosophila. Cardiac and nephrocyte dysfunctions were independently rescued by cardiomyocyte-specific and nephrocyte-specific inhibition of the cGAS-STING pathway, respectively. In human iPSC-derived cardiomyocytes, irradiation and palmitate induced senescence, DNA damage sensing via ZBP1, and activation of the cGAS-STING-IRF3 signaling axis; ZBP1 knockdown or senolytic treatment suppressed this inflammatory axis. Conclusions:Across mouse, human, fly, and human iPSC models, our findings identify DNA damage-driven senescence and ZBP1-cGAS-STING signaling as conserved, causal mechanisms linking cardiovascular-kidney-metabolic comorbidities to HFpEF, highlighting senescence and innate immune pathways as promising therapeutic targets.

OBJECTIVES:Active proliferative LN is associated with elevated urinary neutrophil granule proteins such as PR3, implicating active cellular degranulation in LN. Yet, mature neutrophils are rarely observed in LN biopsies, raising the possibility of an unrecognized cellular source. We aimed to identify and characterize PR3-expressing myeloid cells in LN kidneys using a spatially resolved multi-omics approach. METHODS:Serial immunohistochemistry (10-plex) and spatial transcriptomics were employed to assess the distribution of PR3+ cells across renal tissue and characterize their cellular phenotype. RESULTS:PR3+ cells were abundant in LN kidney biopsies, but with marked inter-patient heterogeneity. These cells were predominantly localized to the tubulointerstitium and showed similar distribution in both proliferative and membranous LN. In contrast, intraglomerular PR3+ cells were enriched in proliferative LN and correlated with the NIH activity index. Phenotype analysis identified four subsets of PR3+ cells, consistent with a monocyte/neutrophil lineage. Most PR3+ cells lacked multilobate nuclei, distinguishing them from mature neutrophils, suggesting that these cells may be overlooked by conventional light microscopy. CONCLUSIONS:PR3+ myeloid cells are a prominent and heterogeneous population in LN. While intraglomerular PR3+ cells were specific to proliferative LN and correlated with histologic activity, the widespread presence of PR3+ cells in the tubulointerstitium suggests broader involvement in renal inflammation than previously recognized. These findings reveal a potentially overlooked immune cell population that may contribute to renal injury in LN.

Background: Traditional immunohistochemistry (IHC) with chromogen detection has limited multiplex capacity, detecting at most 4 protein markers per tissue section simultaneously, thereby restricting comprehensive spatial analysis of valuable human biopsies. We developed and validated a robust serial IHC (sIHC) staining method to detect multiple antigens on a single kidney biopsy slide, maximizing data yield for diagnosing and studying complex kidney diseases. Methods: Formalin-fixed, paraffin-embedded kidney biopsy sections were subjected to repeated IHC/imaging cycles with antibody removal using an optimized sodium dodecyl sulfate-glycerol buffer stripping protocol. Images were then co-registered, and analysis was performed using a variety of methodologies, including color deconvolution, cell segmentation, and spatial clustering. Results: This optimized sIHC method successfully detected up to 20 antigens on a single slide. Combining image analysis and artificial intelligence software, for example with HALO (Indica Labs), the assay assembles high-dimensional images and enables quantitative histology and single-cell spatial analysis. Using this advanced method, we were able to identify rare cell populations, such as double-negative T cells, that are challenging to detect conventionally. Conclusion: We have developed a validated, high-capacity sIHC protocol that uses standard IHC procedures with commercially available, clinically validated off-the-shelf antibodies. This method is a valuable, cost-effective tool for obtaining extensive, high-dimensional single-cell-resolved spatial data from limited pathology samples, such as a human kidney biopsy.

Mitochondrial dysfunction is a hallmark of aging and a key contributor to age-related diseases including cardiovascular disease. However, molecular pathways that safeguard mitochondrial homeostasis in the aging heart remain poorly understood. Here, we identify MTFR1L as a regulator of mitophagy that binds p-S65-Ub, a key signal amplifying the PINK1/Parkin axis. We find that MTFR1L is enriched in metabolically active tissues, particularly in the heart, where it regulates Parkin signaling. Genetic deletion of Mtfr1l in mice impairs stress-induced mitophagy and Parkin activation, leading to accumulation of damaged mitochondria, increased inflammation and senescence, and accelerated age-related cardiac dysfunction. Strikingly, cardiac expression of MTFR1L progressively decreases along with aging in mice, primates, and humans, coinciding with cardiomyocyte senescence and lipofuscin accumulation. Together, these findings uncover a role for MTFR1L in regulation of the p-S65-Ub/Parkin mitophagy axis and maintenance of mitochondrial homeostasis during cardiac aging and suggest that age-associated loss of MTFR1L may contribute to age-related cardiac dysfunction. Based on these findings, we propose a therapeutic paradigm for the prevention of heart aging by restoring MTFR1L function.

Background: Chronic kidney disease (CKD) is associated with renal metabolic disturbances, including impaired fatty acid oxidation (FAO). Nicotinamide adenine dinucleotide (NAD + ) is a small molecule that participates in hundreds of metabolism-related reactions. NAD + levels are decreased in CKD, and NAD + supplementation is protective. However, both the mechanism of how NAD + supplementation protects from CKD, as well as the cell types involved, are poorly understood. Drawing inspiration from investigations on NAD + supplementation in models of acute kidney injury, we hypothesized that NAD + supplementation protects from CKD by restoring renal metabolism in the proximal tubule. Methods: Control (Col4a3 +/- ) and Alport (Col4a3 -/- ) mice on the C57BL/6J background were treated with or without nicotinamide riboside (NR), an NAD + precursor. Both sexes were investigated at two timepoints: moderate disease (25-wk) and end-stage (35-wk). Kidney NAD + levels, plasma creatinine, and 24-hr urinary albumin excretion were quantified with commercial kits. Picrosirius red staining was performed to visualize renal fibrosis. Podocytes and immune cells were identified by immunohistochemistry for p57 kip2 and CD45, respectively. Kidney PGC-1α, CPT1α, and MCAD were quantified by immunoblotting. Bulk RNA-sequencing (RNA-seq) was performed on isolated kidney cortex. Single nuclei RNA-seq (snRNA-seq) and spatial transcriptomics were performed on transverse kidney sections. Results: NR-treatment prevented kidney disease in both sexes and at both timepoints. Plasma creatinine and urinary albumin excretion were increased in Alport mice, and NR-treatment ameliorated this in both sexes. On histology, Alport mice had increased renal fibrosis, increased renal inflammatory infiltrate, mesangial matrix expansion, glomerular hypertrophy, podocyte hypertrophy, and reduced volumetric podocyte density. NR-treatment prevented these pathological changes in both sexes. Bulk RNA-seq identified renal metabolic pathways, including FAO, were reduced in vehicle-treated Alport mice (vs. vehicle-treated control mice) and restored in NR-treated Alport mice (vs. vehicle-treated Alport mice). Importantly, metabolic pathways were increased in NR-treated control mice (vs. vehicle-treated control mice), suggesting that these changes are the direct effect of NR treatment. Transcription factor analyses identified PPARα as responsible for these changes. NR treatment increased protein expression of the PPARα coactivator, PGC-1α, in both control and Alport mice. Kidney protein expression of CPT1α and MCAD, key players in mitochondrial FAO, were reduced in Alport mice and restored by NR treatment. On snRNA-seq, NR treatment restored several vital cellular functions in the proximal tubule cells: translation (ribosome), metabolism, endocrine function, fatty acid degradation, and PPAR signaling. In the podocytes, NR treatment also restored PPAR signaling and fatty acid degradation, although fewer genes were differentially expressed, possibly due to the smaller sample size. Cpt1a and Acadm (MCAD) expression by snRNA-seq were increased in the proximal tubule S1 and S2 cells of Alport mice after NR treatment. On spatial transcriptomics, Cpt1a and Acadm (MCAD) expression were reduced in the pseudobulk, cortex, and outer medullary stripe in Alport mice, and NR treatment restored their expression. Conclusions: NAD + supplementation protects from CKD via the NAD + PGC-1α–PPARα–FAO axis, and the proximal tubule cells substantially contribute to this benefit. This work was supported by NIH award numbers F30DK129003 (BJ), TL1TR001431 (BJ), R01DK116567 (ML), R01DK127830 (ML), P50DK133943 (SJ), U01DK114923 (ME), and P30CA051008. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Cockayne Syndrome (CS) is a premature aging disorder caused by mutations in the CSA and CSB genes involved in DNA metabolism and other cellular processes. CS patients display many features including premature aging, neurodegeneration, and kidney abnormalities. Nicotinamide dinucleotide (NAD+) deprivation has been observed in CS patient-derived cells. NAD+ has essential roles in regulating cellular health, stress responses, and renal homeostasis. While kidney dysfunction is a common feature in CS patients, its molecular pathogenesis is not understood. Here, we report that severe kidney pathology is present in CS A and B mice. We find that the NAD+ biosynthetic pathways are impaired in kidneys from these mice. Using human renal tubular epithelial cells, we show that CSA/B downregulation causes persistent activation of the ATF3 transcription factor on the quinolinate phosphoribosyl transferase gene locus, a rate-limiting enzyme in de novo NAD+ biosynthesis in the kidney, causing impaired transcription and deficient NAD+ homeostasis.

Chronic kidney disease (CKD) is associated with renal metabolic disturbances, including impaired fatty acid oxidation (FAO). Nicotinamide adenine dinucleotide (NAD+) is a small molecule that participates in hundreds of metabolism-related reactions. NAD+ levels are decreased in CKD, and NAD+ supplementation is protective. However, both the mechanism of how NAD+ supplementation protects from CKD, as well as the cell types involved, are poorly understood. Using a mouse model of Alport syndrome, we show that nicotinamide riboside (NR), an NAD+ precursor, stimulated renal PPARα signaling and restored FAO in the proximal tubules, thereby protecting from CKD in both sexes. Bulk RNA-sequencing showed that renal metabolic pathways were impaired in Alport mice and activated by NR in both sexes. These transcriptional changes were confirmed by orthogonal imaging techniques and biochemical assays. Single-nuclei RNA sequencing and spatial transcriptomics, both the first of their kind to our knowledge from Alport mice, showed that NAD+ supplementation restored FAO in proximal tubule cells. Finally, we also report, for the first time to our knowledge, sex differences at the transcriptional level in this Alport model. In summary, the data herein identify a nephroprotective mechanism of NAD+ supplementation in CKD, and they demonstrate that this benefit localizes to the proximal tubule cells.

Chronic kidney disease (CKD) is associated with renal metabolic disturbances, including impaired fatty acid oxidation (FAO). Nicotinamide adenine dinucleotide (NAD + ) is a small molecule that participates in hundreds of metabolism-related reactions. NAD + levels are decreased in CKD, and NAD + supplementation is protective. However, both the mechanism of how NAD + supplementation protects from CKD, as well as the cell types involved, are poorly understood. Using a mouse model of Alport syndrome, we show that nicotinamide riboside (NR), an NAD + precursor, stimulates renal peroxisome proliferator-activated receptor alpha signaling and restores FAO in the proximal tubules, thereby protecting from CKD in both sexes. Bulk RNA-sequencing shows that renal metabolic pathways are impaired in Alport mice and activated by NR in both sexes. These transcriptional changes are confirmed by orthogonal imaging techniques and biochemical assays. Single nuclei RNA-sequencing and spatial transcriptomics, both the first of their kind from Alport mice, show that NAD + supplementation restores FAO in proximal tubule cells. Finally, we also report, for the first time, sex differences at the transcriptional level in this Alport model. In summary, we identify a nephroprotective mechanism of NAD + supplementation in CKD, and we demonstrate that the proximal tubule cells substantially contribute to this benefit.

Chronic kidney disease (CKD) progresses through the replacement of functional tissue compartments with fibrosis, a maladaptive repair process. Shifting kidney repair towards a physiologically intact architecture, rather than fibrosis, is key to blocking CKD progression. Much research into the mechanisms of fibrosis is performed in rodent models with less attention to the human genetic context. Recently, human induced pluripotent stem cell (iPSC)-derived organoids have shown promise in overcoming the limitation. In this study, we developed a fibrosis model that uses human iPSC-based three-dimensional renal organoids, in which exogenous Transforming growth factor- β1 (TGF-β1) induced the production of extracellular matrix. TGF-β1-treated organoids showed tubulocentric collagen 1α1 production by regulating downstream transcriptional regulators, Farnesoid X receptor, phosphorylated mothers against decapentaplegic homolog 3 (p-SMAD3), and transcriptional coactivator with PDZ-binding motif (TAZ). Increased nuclear TAZ expression was confirmed in the tubular epithelium in human kidney biopsies with tubular injury and early fibrosis. A dual bile acid receptors agonist (INT-767) increased Farnesoid X receptor and reduced p-SMAD3 and TAZ, attenuating TGF-β1-induced fibrosis in kidney organoids. Finally, we show that TAZ interacted with TEA-domain transcription factors and p-SMAD3 with TAZ and TEA-domain transcription factor 4 co-regulating collagen 1α1 gene transcription. In summary, we establish a novel, readily manipulable fibrogenesis model and posit a role for bile acid receptor agonism early in renal parenchymal fibrosis.

HIV disease remains prevalent in the USA and chronic kidney disease remains a major cause of morbidity in HIV-1-positive patients. Host double-stranded RNA (dsRNA)-activated protein kinase (PKR) is a sensor for viral dsRNA, including HIV-1. We show that PKR inhibition by compound C16 ameliorates the HIV-associated nephropathy (HIVAN) kidney phenotype in the Tg26 transgenic mouse model, with reversal of mitochondrial dysfunction. Combined analysis of single-nucleus RNA-seq and bulk RNA-seq data revealed that oxidative phosphorylation was one of the most downregulated pathways and identified signal transducer and activator of transcription (STAT3) as a potential mediating factor. We identified in Tg26 mice a novel proximal tubular cell cluster enriched in mitochondrial transcripts. Podocytes showed high levels of HIV-1 gene expression and dysregulation of cytoskeleton-related genes, and these cells dedifferentiated. In injured proximal tubules, cell-cell interaction analysis indicated activation of the pro-fibrogenic PKR-STAT3-platelet-derived growth factor (PDGF)-D pathway. These findings suggest that PKR inhibition and mitochondrial rescue are potential novel therapeutic approaches for HIVAN.

Chronic kidney disease progresses through the replacement of functional tissue compartments with fibrosis, a maladaptive repair process. Shifting kidney repair toward a physiologically intact architecture, rather than fibrosis, is key to blocking chronic kidney disease progression. Much research into the mechanisms of fibrosis is performed in rodent models with less attention to the human genetic context. Recently, human induced pluripotent stem cell (iPSC)-derived organoids have shown promise in overcoming the limitation. In this study, we developed a fibrosis model that uses human iPSC-based 3-dimensional renal organoids, in which exogenous transforming growth factor-I31 (TGF-I31) induced the production of extracellular matrix. TGF-I31-treated organoids showed tubulocentric collagen 1a1 production by regulating downstream transcriptional regulators, Farnesoid X receptor, phosphorylated mothers against decapentaplegic homolog 3 (pSMAD3), and transcriptional coactivator with PDZ-binding motif (TAZ). Increased nuclear TAZ expression was confirmed in the tubular epithelium in human kidney biopsies with tubular injury and early fibrosis. A dual bile acid receptor agonist (INT-767) increased Farnesoid X receptor and reduced p-SMAD3 and TAZ, attenuating TGF-I31-induced fibrosis in kidney organoids. Finally, we show that TAZ interacted with TEA-domain transcription factors and p-SMAD3 with TAZ and TEAdomain transcription factor 4 coregulating collagen 1a1 gene transcription. In summary, we establish a novel, readily manipulable fibrogenesis model and posit a role for bile acid receptor agonism early in renal parenchymal fibrosis. (c) 2024 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.

Diabetes mellitus is the leading cause of cardiovascular and renal disease in the United -States. Despite the beneficial interventions available for patients with diabetes, there remains a need for additional therapeutic targets and therapies in diabetic kidney disease (DKD). Inflammation and oxidative stress are increasingly recognized as important causes of renal diseases. Inflammation is closely associated with mitochondrial damage. The molecular connection between inflammation and mitochondrial metabolism remains to be elucidated. Recently, nicotinamide adenine nucleotide (NAD+) metabolism has been found to regulate immune function and inflammation. In the present studies, we tested the hypothesis that enhancing NAD metabolism could prevent inflammation in and progression of DKD. We found that treatment of db/db mice with type 2 diabetes with nicotinamide riboside (NR) prevented several manifestations of kidney dysfunction (i.e., albuminuria, increased urinary kidney injury marker-1 (KIM1) excretion, and pathologic changes). These effects were associated with decreased inflammation, at least in part via inhibiting the activation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling pathway. An antagonist of the serum stimulator of interferon genes (STING) and whole-body STING deletion in diabetic mice showed similar renoprotection. Further analysis found that NR increased SIRT3 activity and improved mitochondrial function, which led to decreased mitochondrial DNA damage, a trigger for mitochondrial DNA leakage which activates the cGAS-STING pathway. Overall, these data show that NR supplementation boosted NAD metabolism to augment mitochondrial function, reducing inflammation and thereby preventing the progression of diabetic kidney disease.

ABSTRACTBackgroundPodocyte depletion is an established indicator of glomerular injury and predicts clinical outcomes. The semi-quantitative nature of existing podocyte estimation methods or podometrics hinders incorporation of such analysis into experimental and clinical pathologic workflows. Computational image analysis offers a robust approach to automate podometrics through objective quantification of cell and tissue structure. Toward this goal, we developed PodoCount, a computational tool for quantitative analysis of podocytes, and validated the generalizability of the tool across a diverse dataset.MethodsPodocyte nuclei and glomerular boundaries were labeled in murine whole kidney sections,n= 135, from six disease models and human kidney biopsies,n= 45, from diabetic nephropathy (DN) patients. Digital whole slide images (WSIs) of tissues were then acquired. Classical image analysis was applied to obtain podocyte nuclear and glomerular morphometrics. Statistically significant morphometric features, which correlated with each murine disease, were identified. Engineered features were also assessed for their ability to predict outcomes in human DN.PodoCounthas been disbursed for other researchers as an open-source, cloud-based computational tool.ResultsPodoCountoffers highly accurate quantification of podocytes. Engineered podometric features were benchmarked against routine glomerular histopathology and were found to be significant predictors of disease diagnosis, proteinuria level, and clinical outcomes.ConclusionsPodoCountoffers high quantification performance in diverse murine disease models as well as in human DN. Resultant podometric features offers significant correlation with associated metadata as well as outcome. Our cloud-based end-user tool will provide a standardized approach for podometric analysis from gigapixel size WSIs in basic research and clinical practice.

Diabetic kidney disease (DKD) is the most common cause of kidney failure in the world, and novel predictive biomarkers and molecular mechanisms of disease are needed. Endothelial cell-specific molecule-1 (Esm-1) is a secreted proteoglycan that attenuates inflammation. We previously identified that a glomerular deficiency of Esm-1 associates with more pronounced albuminuria and glomerular inflammation in DKD-susceptible relative to DKD-resistant mice, but its contribution to DKD remains unexplored. In this study, we show that lower circulating Esm-1 predicts progressive stages of albuminuria in patients with diabetes. In DKD-susceptible mice, Esm-1 inversely correlates with albuminuria and glomerular leukocyte infiltration. Using hydrodynamic tail-vein injection, we show that over-expression of either mouse or human Esm-1 reduces diabetes-induced albuminuria relative to saline-injected controls independent of leukocyte infiltration. Using a complementary approach, we find that constitutive deletion of Esm-1 in DKD-resistant mice increases the degree of diabetes-induced albuminuria versus wild-type controls. Mechanistically, over-expression of Esm-1 attenuates diabetes-induced podocyte injury. By glomerular RNAseq, we identify that Esm-1 attenuates diabetes-induced up-regulation of interferon-stimulated genes, and Esm-1 inhibits expression of kidney disease-promoting and interferon-related genes, including Ackr2 and Cxcl11 . In conclusion, we demonstrate that Esm-1 protects against diabetes-induced albuminuria, and podocytopathy, possibly through select interferon signaling.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 as its primary infection mechanism. Interactions between S and endogenous proteins occur after infection but are not well understood. We profiled binding of S against >9000 human proteins and found an interaction between S and human estrogen receptor α (ERα). Using bioinformatics, supercomputing, and experimental assays, we identified a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit. In cultured cells, S DNA transfection increased ERα cytoplasmic accumulation, and S treatment induced ER-dependent biological effects. Non-invasive imaging in SARS-CoV-2–infected hamsters localized lung pathology with increased ERα lung levels. Postmortem lung experiments from infected hamsters and humans confirmed an increase in cytoplasmic ERα and its colocalization with S in alveolar macrophages. These findings describe the discovery of a S-ERα interaction, imply a role for S as an NRC, and advance knowledge of SARS-CoV-2 biology and coronavirus disease 2019 pathology.

Chronic kidney disease (CKD) progresses by replacement of functional tissue compartments with fibrosis, representing a maladaptive repair process. Shifting kidney repair towards a physiologically-intact architecture, rather than fibrosis, is key to blocking CKD progression. In this study, we developed a fibrosis model that uses human induced pluripotent stem cell (iPSC)-based three-dimensional renal organoids, in which exogenous TGF-β1 induces production of extracellular matrix. In these organoids, TGF- β1 increased transcription factor tafazzin (TAZ) expression. Further, in human kidney biopsies, nuclear TAZ expression was markedly increased in mild and moderate fibrosis. In cultured renal tubular cells expressing a fibrogenic program, TAZ formed a trimeric complex with phosphorylated mothers against decapentaplegic homolog 3 (p-SMAD3) and TEA domain protein (TEAD)-4. Overexpression of TEAD4 protein suppressed collagen-1α1 (COL1A1) promoter activity, and expression of TAZ attenuated this inhibition. INT-767, a dual bile acid receptor agonist binding farnesoid X receptor (FXR) and the Takeda G protein-coupled receptor 5 (TGR5), decreased the TGF-β1-induced increase in p-SMAD3 and TAZ, and preserved renal organoid architecture. These data demonstrate, in an iPSC-derived renal organoid fibrosis model, that INT767 prevents fibrosis programs early in the course of tubular injury through modulation of the TEAD4/TAZ pathway.

Background Skeletal muscle myofibers can be separated into functionally distinct cell types that differ in gene and protein expression. Current single cell expression data is generally based upon single nucleus RNA, rather than whole myofiber material. We examined if a whole-cell flow sorting approach could be applied to perform single cell RNA-seq (scRNA-seq) in a single muscle type. Methods We performed deep, whole cell, scRNA-seq on intact and fragmented skeletal myofibers from the mouse fast-twitch flexor digitorum brevis muscle utilizing a flow-gated method of large cell isolation. We performed deep sequencing of 763 intact and fragmented myofibers. Results Quality control metrics across the different gates indicated only 171 of these cells were optimal, with a median read count of 239,252 and an average of 12,098 transcripts per cell. scRNA-seq identified three clusters of myofibers (a slow/fast 2A cluster and two fast 2X clusters). Comparison to a public skeletal nuclear RNA-seq dataset demonstrated a diversity in transcript abundance by method. RISH validated multiple genes across fast and slow twitch skeletal muscle types. Conclusion This study introduces and validates a method to isolate intact skeletal muscle myofibers to generate deep expression patterns and expands the known repertoire of fiber-type-specific genes.

Skeletal muscle myocytes have evolved into slow and fast-twitch types. These types are functionally distinct as a result of differential gene and protein expression. However, an understanding of the complexity of gene and protein variation between myofibers is unknown. We performed deep, whole cell, single cell RNA-seq on intact and fragments of skeletal myocytes from the mouse flexor digitorum brevis muscle. We compared the genomic expression data of 171 of these cells with two human proteomic datasets. The first was a spatial proteomics survey of mosaic patterns of protein expression utilizing the Human Protein Atlas (HPA) and the HPASubC tool. The second was a mass-spectrometry (MS) derived proteomic dataset of single human muscle fibers. Immunohistochemistry and RNA-ISH were used to understand variable expression. scRNA-seq identified three distinct clusters of myocytes (a slow/fast 2A cluster and two fast 2X clusters). Utilizing 1,605 mosaic patterned proteins from visual proteomics, and 596 differentially expressed proteins by MS methods, we explore this fast 2X division. Only 36 genes/proteins had variable expression across all three studies, of which nine are newly described as variable between fast/slow twitch myofibers. An additional 414 genes/proteins were identified as variable by two methods. Immunohistochemistry and RNA-ISH generally validated variable expression across methods presumably due to species-related differences. In this first integrated proteogenomic analysis of mature skeletal muscle myocytes we confirm the main fiber types and greatly expand the known repertoire of twitch-type specific genes/proteins. We also demonstrate the importance of integrating genomic and proteomic datasets.