Biology subjects

Rosenberg, A. Z.

Publications and source records attributed to Rosenberg, A. Z..

At least 19 recordsLinked to original sources

bioRxiv2026

Serial Immunohistochemistry for High-Dimensional Single-Cell Spatial Analysis of Human Kidney Biopsies

BackgroundTraditional 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. MethodsFormalin-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. ResultsThis 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. ConclusionWe 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.

pathology↗

bioRxiv2026

ERRγ deletion in podocytes accelerates aging related kidney disease

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 ERR{gamma} in the podocytes, in the face of decreased ERR expression, in the present study we aimed to determine the role of ERR{gamma} in aging podocyte. To this end, we cross bred ERR{gamma} floxed mice with podocin-Cre mice to achieve a podocyte-specific ERR{gamma} 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 ERR{gamma} primarily targeted the expression of mitochondrial biogenesis regulator PGC-1, 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 ERR in aging podocytes plus its traditional role in mitochondrial function, these studies using podocyte ERR{gamma} deletion suggested an overlapping mechanism for ERR/ERR{gamma} to act as modulators of age-related mitochondrial dysfunction and age-related kidney disease.

pharmacology and toxicology↗

bioRxiv2026

Senescence and DNA Damage-Induced Inflammation Drive Heart Failure with Preserved Ejection Fraction in Cardiovascular Kidney Metabolic Syndrome

IntroductionHeart 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 ResultsTo 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, {gamma}H2AX, STING, IRF3, NF-{kappa}B 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-{kappa}B 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. ConclusionsAcross 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.

pathology↗

bioRxiv2025

Spatial analysis reveals the cellular microenvironments and mechanisms of inflammation and kidney injury in acute interstitial nephritis

Acute interstitial nephritis (AIN) causes 15-20% of all acute kidney injury cases but lacks effective therapies beyond corticosteroids. Using high-resolution imaging mass cytometry and single-cell spatial transcriptomics to analyze human kidney biopsies with AIN, non-immunologic acute tubular injury (ATI), and reference tissue, the CXCL9-CXCR3 axis was identified as the defining immunologic signature of AIN, with 44-fold higher predicted CXCL9-CXCR3 interactions than ATI, creating homotypic inflammatory T cell amplification networks concentrated in lymphoid aggregates. C3AR1+ immune cells were enriched in peritubular neighborhoods of complement 3-expressing injured tubule cells, predominantly VCAM1+ injured proximal tubules, linking tubular injury to immune activation in AIN. Nicotinamide phosphoribosyltransferase (NAMPT) was the strongest predictor of VCAM1+ tubular microenvironments, with expression by both injured tubules and surrounding immune cells coordinating metabolic-inflammatory niches. These findings reveal distinct molecular circuits underlying AIN pathogenesis and identify potential therapeutic targets for improving clinical management and preventing progression to chronic kidney disease.

immunology↗

bioRxiv2024

Apolipoprotein-L1 G1 variant contributes to hydrocephalus but not to atherosclerosis in apolipoprotein-E knock-out mice

IntroductionIn USA, six million individuals with Sub-Saharan ancestry carry two APOL1 high-risk variants, which increase the risk for kidney diseases. Whether APOL1 high-risk variants are independent risk factors for cardiovascular diseases is unclear and requires further investigation. MethodsWe characterized a mouse model to investigate the role of APOL1 in dyslipidemia and cardiovascular diseases. Transgenic mice carrying APOL1 (G0 and G1 variants) on bacterial artificial chromosomes (BAC/APOL1 mice) were crossed with the ApoE knock-out (ApoE-KO) atherosclerosis mouse model. The compound transgenic mice were evaluated for the impact of APOL1 on systemic phenotypes. ResultsApoE-KO mice carrying APOL1-G0 and APOL1-G1 did not show differences in the extent of atherosclerotic lesions or aortic calcification, as evaluated by Sudan IV staining and radiographic examination, respectively. However, [~]20% of ApoE-KO; BAC/APOL1-G1 mice developed hydrocephalus and required euthanasia. The hydrocephalus was communicating and likely was due to excess cerebrospinal fluid produced by the choroid plexus, where epithelial cells expressed APOL1. Single-nuclear RNA-seq of choroid plexus identified solute transporter upregulation and mTORC2 pathway activation in APOL1-G1-expressing epithelial cells. Further, in the All of Us cohort, we found higher hydrocephalus prevalence among individuals with the APOL1-G1 variant in both recessive and dominant models, supporting the mouse findings. ConclusionWhile APOL1-G1 expression in ApoE-KO mice did not worsen cardiovascular disease phenotypes, we uncovered hydrocephalus as a novel APOL1 risk allele-mediated phenotype. These findings extend the spectrum of APOL1-associated pathologies.

molecular biology↗

bioRxiv2024

SFPQ-TFE3 gene fusion reciprocally regulates mTORC1 activity and induces lineage plasticity in a novel mouse model of renal tumorigenesis

The MiT/TFE family gene fusion proteins, such as SFPQ-TFE3, drive both epithelial (eg, translocation renal cell carcinoma, tRCC) and mesenchymal (eg, perivascular epithelioid cell tumor, PEComa) neoplasms with aggressive behavior. However, no prior mouse models for SFPQ-TFE3-related tumors exist and the mechanisms of lineage plasticity induced by this fusion remain unclear. Here, we demonstrate that constitutive murine renal expression of human SFPQ-TFE3 using Ksp Cadherin-Cre as a driver disrupts kidney development leading to early neonatal renal failure and death. In contrast, post-natal induction of SFPQ-TFE3 in renal tubular epithelial cells using Pax8 ERT-Cre induces infiltrative epithelioid tumors, which morphologically and transcriptionally resemble human PEComas. As seen in MiT/TFE fusion-driven human tumors, SFPQ-TFE3 expression is accompanied by the strong induction of mTORC1 signaling, which is partially amino acid-sensitive and dependent on increased SFPQ-TFE3-mediated RRAGC/D transcription. Remarkably, SFPQ-TFE3 expression is sufficient to induce lineage plasticity in renal tubular epithelial cells, with rapid down-regulation of the critical PAX2/PAX8 nephric lineage factors and tubular epithelial markers, and concomitant up-regulation of PEComa differentiation markers in transgenic mice, human cell line models and human tRCC. Pharmacologic or genetic inhibition of mTOR signaling downregulates expression of the SFPQ-TFE3 fusion protein and rescues nephric lineage marker expression and transcriptional activity in vitro. These data provide evidence of a potential epithelial cell-of-origin for TFE3-driven PEComas and highlight a reciprocal role for SFPQ-TFE3 and mTOR in driving lineage plasticity in the kidney, expanding our understanding of the pathogenesis of MiT/TFE-driven tumors.

cancer biology↗

bioRxiv2024

FUSION: A web-based application for in-depth exploration of multi-omics data with brightfield histology

Spatial -OMICS technologies facilitate the interrogation of molecular profiles in the context of the underlying histopathology and tissue microenvironment. Paired analysis of histopathology and molecular data can provide pathologists with otherwise unobtainable insights into biological mechanisms. To connect the disparate molecular and histopathologic features into a single workspace, we developed FUSION (Functional Unit State IdentificatiON in WSIs [Whole Slide Images]), a web-based tool that provides users with a broad array of visualization and analytical tools including deep learning-based algorithms for in-depth interrogation of spatial -OMICS datasets and their associated high-resolution histology images. FUSION enables end-to-end analysis of functional tissue units (FTUs), automatically aggregating underlying molecular data to provide a histopathology-based medium for analyzing healthy and altered cell states and driving new discoveries using "pathomic" features. We demonstrate FUSION using 10x Visium spatial transcriptomics (ST) data from both formalin-fixed paraffin embedded (FFPE) and frozen prepared datasets consisting of healthy and diseased tissue. Through several use-cases, we demonstrate how users can identify spatial linkages between quantitative pathomics, qualitative image characteristics, and spatial --omics

pathology↗

bioRxiv2024

Ontology-based modeling, integration, and analysis of heterogeneous clinical, pathological, and molecular kidney data for precision medicine

Many resources are now generating, processing, storing, or providing kidney-related molecular, pathological, and clinical data. Reference ontologies offer an opportunity to support knowledge and data organization and integration. The Kidney Precision Medicine Project (KPMP) team contributed to the representation and addition of 329 kidney phenotype terms to the Human Phenotype Ontology (HPO) and identified many subcategories of acute kidney injury (AKI) or chronic kidney disease (CKD). The Kidney Tissue Atlas Ontology (KTAO) imports and integrates kidney-related terms from existing ontologies (e.g., HPO, CL, and Uberon) and represents 259 kidney-related biomarkers. We also developed a precision medicine metadata ontology (PMMO) to integrate 50 variables from KPMP and CellxGene resources and applied PMMO for integrative analysis. The gene expression profiles of kidney gene biomarkers were specifically analyzed in healthy controls or AKI/CKD disease states. This work demonstrates how ontology-based approaches support multi-domain data and knowledge organization and integration to advance precision medicine.

bioinformatics↗

bioRxiv2024

Preeclamptic phenotype in transgenic mice with fetuses carrying APOL1 risk variants

African-American women have a maternal mortality rate approximately three times higher than European-American women. This is partially due to hypertensive disorders of pregnancy, including preeclampsia. Fetal APOL1 high-risk genotype increases preeclampsia risk, although mechanisms remain elusive. We characterized two mouse models to investigate whether fetal-origin APOL1 induces preeclampsia and which cell types contribute. We in vitro fertilized mice with sperm from two transgenic mouse lines: APOL1 transgenic mice carrying human genomic locus constructs from bacterial artificial chromosomes (BAC) containing the APOL1 gene, mimicking expression and function of human APOL1 (BAC/APOL1 mice) and albumin promoter APOL1 transgenic mice expressing APOL1 in liver and plasma (Alb/APOL1 mice). Dams carrying either BAC/APOL1-G1 or Alb/APOL1-G1 fetuses had elevated systolic blood pressure, while dams carrying BAC/APOL1-G0 or Alb/APOL1-G0 fetuses did not. BAC/APOL1-G1 and Alb/APOL1-G1 fetuses weighed less than littermates, indicating intrauterine growth restriction. Single-nucleus RNA-seq of APOL1-G1 placentas showed increased expression of osteopontin/Spp1, most prominently in vascular endothelial cells with robust APOL1 expression. Cell-cell interaction analysis indicated pro-inflammatory signaling between placental cells and maternal monocytes. These models show that fetal origin APOL1-G1 causes preeclampsia, inducing pro-inflammatory response in placenta and maternal monocytes. The APOL1-G1 variant poses a multi-generational problem, causing effects in mothers and offspring.

molecular biology↗

bioRxiv2024

Nicotinamide riboside activates renal metabolism and protects the kidney in a model of Alport syndrome

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.

molecular biology↗

bioRxiv2023

APOL1 kidney risk variants in glomerular diseases modeled in transgenic mice

APOL1 high-risk variants partially explain the high kidney disease prevalence among African ancestry individuals. Many mechanisms have been reported in cell culture models, but few have been demonstrated in mouse models. Here we characterize two models: (1) HIV- associated nephropathy (HIVAN) Tg26 mice crossed with bacterial artificial chromosome (BAC)/APOL1 transgenic mice and (2) interferon-{psi} administered to BAC/APOL1 mice. Both models showed exacerbated glomerular disease in APOL1-G1 compared to APOL1-G0 mice. HIVAN model glomerular bulk RNA-seq identified synergistic podocyte-damaging pathways activated by the APOL1-G1 allele and by HIV transgenes. Single-nuclear RNA-seq revealed podocyte-specific patterns of differentially-expressed genes as a function of APOL1 alleles. Eukaryotic Initiation factor-2 pathway was the most activated pathway in the interferon-{psi} model and the most deactivated pathway in the HIVAN model. HIVAN mouse model podocyte single-nuclear RNA-seq data showed similarity to human focal segmental glomerulosclerosis (FSGS) glomerular bulk RNA-seq data. Furthermore, single-nuclear RNA-seq data from interferon-{psi} mouse model podocytes (in vivo) showed similarity to human FSGS single-cell RNA- seq data from urine podocytes (ex vivo) and from human podocyte cell lines (in vitro) using bulk RNA-seq. These data highlight differences in the transcriptional effects of the APOL1-G1 risk variant in a model specific manner. Shared differentially expressed genes in podocytes in both mouse models suggest possible novel glomerular damage markers in APOL1 variant-induced diseases. Transcription factor Zbtb16 was downregulated in podocytes and endothelial cells in both models, possibly contributing to glucocorticoid-resistance. In summary, these findings in two mouse models suggest both shared and distinct therapeutic opportunities for APOL1 glomerulopathies. Significance statementCoding variants in APOL1, encoding apolipoprotein L1, contribute to kidney disease in individuals with African ancestry. The mechanisms for glomerular injury remain incompletely understood. We studied two transgenic mouse models, HIV-associated nephropathy and interferon-{psi} administration. Using glomerular and single-nuclear RNA sequencing, we identified genes differentially expressed among mice with kidney risk alleles (G1) and the common variant (G0). Both models exhibited up-regulation of genes that indicated podocyte damage with risk alleles compared to the common variant. One gene down-regulated in both models was Zbtb16, encoding a transcription factor, that may contribute to glucocorticoid-resistance. Overall, the findings suggest both shared and distinct alterations in the two disease models.

pathology↗

bioRxiv2023

Farnesoid X receptor agonism prevents neutrophil extracellular traps via reduced sphingosine-1-phosphate in chronic kidney disease

BackgroundActivation of the farnesoid X receptor (FXR) reduces renal inflammation, but the underlying mechanisms remain elusive. Neutrophil extracellular traps (NETs) are webs of DNA formed when neutrophils undergo specialized programmed cell death (NETosis). Sphingosine-1-phosphate (S1P) is a signaling lipid that stimulates NETosis via its receptor on neutrophils. Here, we identify FXR as a negative regulator of kidney NETosis via repressing S1P signaling in male but not female mice. MethodsWe determined the effects of the FXR agonist obeticholic acid (OCA) in mouse models of adenosine phosphoribosyltransferase deficiency and Alport syndrome. We assessed renal NETosis by immunofluorescence in these models and in biopsies from patients with Alport syndrome (6 male, 9 female). We also inhibited de novo sphingosine production in Alport mice to show a causal relationship between S1P signaling and renal NETosis. ResultsRenal FXR activity is greatly reduced in both models, and OCA prevents kidney fibrosis, inflammation, and lipotoxicity. OCA reduces renal neutrophilic inflammation and NETosis in male adenine and Alport mice, but not in female adenine mice. Extensive NETosis was also identified in human Alport kidney biopsies. Kidney sphingosine kinase 1 (Sphk1) expression is increased in mice with kidney disease and reduced by OCA in male but not female mice. Also, Sphk1 expression correlates with NETosis in male but not female mice. Short-term inhibition of sphingosine synthesis reduces neutrophilic inflammation and NETosis. ConclusionFXR agonism represses kidney Sphk1 expression in male but not female mice. This inhibits renal S1P signaling, thereby reducing neutrophilic inflammation and NETosis in a sex-dependent manner.

pharmacology and toxicology↗

bioRxiv2023

HIV viral protein R induces loss of DCT1-type renal tubules

Hyponatremia and salt wasting is a common occurance in patients with HIV/AIDS, however, the understanding of its contributing factors is limited. HIV viral protein R (Vpr) contributes to HIV-associated nephropathy. To investigate the effects of Vpr on the expression level of the Slc12a3 gene, encoding the Na-Cl cotransporter, which is responsible for sodium reabsorption in distal nephron segments, we performed single-nucleus RNA sequencing of kidney cortices from three wild-type (WT) and three Vpr-transgenic (Vpr Tg) mice. The results showed that the percentage of distal convoluted tubule (DCT) cells was significantly lower in Vpr Tg mice compared with WT mice (P < 0.05), and that in Vpr Tg mice, Slc12a3 expression was not different in DCT cell cluster. The Pvalb+ DCT1 subcluster had fewer cells in Vpr Tg mice compared with WT (P < 0.01). Immunohistochemistry demonstrated fewer Slc12a3+ Pvalb+ DCT1 segments in Vpr Tg mice. Differential gene expression analysis comparing Vpr Tg and WT in the DCT cluster showed Ier3, an inhibitor of apoptosis, to be the most downregulated gene. These observations demonstrate that the salt-wasting effect of Vpr in Vpr Tg mice is mediated by loss of Slc12a3+ Pvalb+ DCT1 segments via apoptosis dysregulation.

pathology↗

bioRxiv2022

PKR activation-induced mitochondrial dysfunction in HIV-transgenic mice with nephropathy

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 profibrogenic 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. Translational StatementThis work identified mitochondrial dysfunction in transgenic mice manifesting HIV-associated nephropathy mice kidney, using combination of single-nuclear and bulk RNA-seq analysis. Kidney damage was ameliorated by the PKR inhibitor C16, and mitochondrial rescue was shown by transcriptomic profiling and functional assay. These findings suggest that PKR inhibition and mitochondrial rescue are potential therapeutic approaches for HIV-associated nephropathy.

pathology↗

bioRxiv2022

Expression Microdissection for use in qPCR based analysis of miRNA in a single cell type

Cell-specific microRNA (miRNA) expression estimates are important in characterizing the localization of miRNA signaling within tissues. Much of this data is obtained from cultured cells, a process known to significantly alter miRNA expression levels. Thus, our knowledge of in vivo cell miRNA expression estimates is poor. We previously demonstrated expression microdissection-miRNA-sequencing (xMD-miRNA-seq) as a means to acquire in vivo estimates, directly from formalin fixed tissues, albeit with limited yield. Here we optimized each step of the xMD process including tissue retrieval, tissue transfer, film preparation, and RNA isolation to increase RNA yields and ultimately show strong enrichment for in vivo miRNA expression by qPCR array. These method improvements, including the development of a non-crosslinked ethylene vinyl acetate (EVA) membrane, resulted in a 23-45 fold increase in miRNA yield, depending on cell type. By qPCR, miR-200a was increased 14-fold in xMD-derived small intestine epithelial cells, with a concurrent 336-fold reduction in miR-143, relative to the matched non-dissected duodenal tissue. xMD is now an optimized method to obtain robust in vivo miRNA expression estimates from cells.

genomics↗

bioRxiv2022

The SARS-CoV-2 spike protein binds and modulates estrogen receptors

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 (ACE2) at the cell surface, which constitutes the primary mechanism driving SARS-CoV-2 infection. Molecular interactions between the transduced S and endogenous proteins likely occur post-infection, but such interactions are not well understood. We used an unbiased primary screen to profile the binding of full-length S against >9,000 human proteins and found significant S-host protein interactions, including one between S and human estrogen receptor alpha (ER). After confirming this interaction in a secondary assay, we used bioinformatics, supercomputing, and experimental assays to identify a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit and an S-ER binding mode. In cultured cells, S DNA transfection increased ER cytoplasmic accumulation, and S treatment induced ER-dependent biological effects and ACE2 expression. Noninvasive multimodal PET/CT imaging in SARS-CoV-2-infected hamsters using [18F]fluoroestradiol (FES) localized lung pathology with increased ER lung levels. Postmortem experiments in lung tissues from SARS-CoV-2-infected hamsters and humans confirmed an increase in cytoplasmic ER expression and its colocalization with S protein in alveolar macrophages. These findings describe the discovery and characterization of a novel S-ER interaction, imply a role for S as an NRC, and are poised to advance knowledge of SARS-CoV-2 biology, COVID-19 pathology, and mechanisms of sex differences in the pathology of infectious disease.

immunology↗

bioRxiv2022

Considerations for Deconvolution: A Case Study with GTEx Coronary Artery Tissues

Differential expression analyses are ubiquitous in the realm of statistical genomics, used to estimate functional differences between genomes of groups of subjects. However, differences in tissue composition between groups may contribute to changes in gene expression, potentially obscuring the detection of functionally significant genes of interest. Deconvolution techniques allow researchers to estimate the abundance of each cell type assumed to be in a tissue. While deconvolution is a useful tool to estimate composition, several crucial considerations must be made when setting up and employing such a workflow in an analysis. We perform a deconvolution on GTEx coronary artery data using CIBERSORT and discuss the challenges and limitations in order to highlight future areas of improvement in the deconvolution framework.

bioinformatics↗

bioRxiv2021

NAD metabolism modulates mitochondrial function and inflammation and prevents progression of diabetic kidney disease

Diabetes mellitus is the leading cause of cardiovascular and renal disease in the United States. In spite of 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 progression of diabetic kidney disease.

pathology↗