LRG1 loss effectively restrains glomerular TGF-ß signaling to attenuate diabetic kidney disease.

Transforming growth factor (TGF)-ß signaling is a well-established pathogenic mediator of diabetic kidney disease (DKD). However, owing to its pleiotropic actions, its systemic blockade is not therapeutically optimal. The expression of TGF-ß signaling regulators can substantially influence TGF-ß's effects in a cell- or context-specific manner. Among these, leucine-rich α2-glycoprotein 1 (LRG1) is significantly increased in glomerular endothelial cells (GECs) in DKD. As LRG1 is a secreted molecule that can exert autocrine and paracrine effects, we examined the effects of LRG1 loss in kidney cells in diabetic OVE26 mice by single-cell transcriptomic analysis. Gene expression analysis confirmed a predominant expression of Lrg1 in GECs, which further increased in diabetic kidneys. Loss of Lrg1 led to the reversal of angiogenic and TGF-ß-induced gene expression in GECs, which were associated with DKD attenuation. Notably, Lrg1 loss also mitigated the increased TGF-ß-mediated gene expression in both podocytes and mesangial cells in diabetic mice, indicating that GEC-derived LRG1 potentiates TGF-ß signaling in glomerular cells in an autocrine and paracrine manner. Indeed, a significant reduction in phospho-Smad proteins was observed in the glomerular cells of OVE26 mice with LRG1 loss. These results indicate that specific antagonisms of LRG1 may be an effective approach to curb the hyperactive glomerular TGF-ß signaling to attenuate DKD.

Podocyte-derived soluble RARRES1 drives kidney disease progression through direct podocyte and proximal tubular injury.

Retinoic acid receptor responder protein-1 (RARRES1) is a podocyte-enriched transmembrane protein whose increased expression correlates with human glomerular disease progression. RARRES1 promotes podocytopenia and glomerulosclerosis via p53-mediated podocyte apoptosis. Importantly, the cytopathic actions of RARRES1 are entirely dependent on its proteolytic cleavage into a soluble protein (sRARRES1) and subsequent podocyte uptake by endocytosis, as a cleavage mutant RARRES1 exerted no effects in vitro or in vivo. As RARRES1 expression is upregulated in human glomerular diseases, here we investigated the functional consequence of podocyte-specific overexpression of RARRES1 in mice in the experimental focal segmental glomerulosclerosis and diabetic kidney disease. We also examined the effects of long-term RARRES1 overexpression on slowly developing aging-induced kidney injury. As anticipated, the induction of podocyte overexpression of RARRES1 (Pod-RARRES1WT) significantly worsened glomerular injuries and worsened kidney function in all three models, while overexpression of RARRES1 cleavage mutant (Pod-RARRES1MT) did not. Remarkably, direct uptake of sRARRES1 was also seen in proximal tubules of injured Pod-RARRES1WT mice and associated with exacerbated tubular injuries, vacuolation, and lipid accumulation. Single-cell RNA sequence analysis of mouse kidneys demonstrated RARRES1 led to a marked deregulation of lipid metabolism in proximal tubule subsets. We further identified matrix metalloproteinase 23 (MMP23) as a highly podocyte-specific metalloproteinase and responsible for RARRES1 cleavage in disease settings, as adeno-associated virus 9-mediated knockdown of MMP23 abrogated sRARRES1 uptake in tubular cells in vivo. Thus, our study delineates a previously unrecognized mechanism by which a podocyte-derived protein directly facilitates podocyte and tubular injury in glomerular diseases and suggests that podocyte-specific functions of RARRES1 and MMP23 may be targeted to ameliorate glomerular disease progression in vivo.

A large-scale retrospective study enabled deep-learning based pathological assessment of frozen procurement kidney biopsies to predict graft loss and guide organ utilization.

Lesion scores on procurement donor biopsies are commonly used to guide organ utilization for deceased-donor kidneys. However, frozen sections present challenges for histological scoring, leading to inter- and intra-observer variability and inappropriate discard. Therefore, we constructed deep-learning based models to recognize kidney tissue compartments in hematoxylin & eosin-stained sections from procurement needle biopsies performed nationwide in years 2011-2020. To do this, we extracted whole-slide abnormality features from 2431 kidneys and correlated with pathologists' scores and transplant outcomes. A Kidney Donor Quality Score (KDQS) was derived and used in combination with recipient demographic and peri-transplant characteristics to predict graft loss or assist organ utilization. The performance on wedge biopsies was additionally evaluated. Our model identified 96% and 91% of normal/sclerotic glomeruli respectively; 94% of arteries/arterial intimal fibrosis; 90% of tubules. Whole-slide features of Sclerotic Glomeruli (GS)%, Arterial Intimal Fibrosis (AIF)%, and Interstitial Space Abnormality (ISA)% demonstrated strong correlations with corresponding pathologists' scores of all 2431 kidneys, but had superior associations with post-transplant estimated glomerular filtration rates in 2033 and graft loss in 1560 kidneys. The combination of KDQS and other factors predicted one- and four-year graft loss in a discovery set of 520 kidneys and a validation set of 1040 kidneys. By using the composite KDQS of 398 discarded kidneys due to "biopsy findings", we suggest that if transplanted, 110 discarded kidneys could have had similar survival to that of other transplanted kidneys. Thus, our composite KDQS and survival prediction models may facilitate risk stratification and organ utilization while potentially reducing unnecessary organ discard.

High-dimensional mass cytometry identified circulating natural killer T-cell subsets associated with protection from cytomegalovirus infection in kidney transplant recipients.

Cytomegalovirus (CMV) infection is associated with poor kidney transplant outcomes. While innate and adaptive immune cells have been implicated in its prevention, an in-depth characterization of the in vivo kinetics of multiple cell subsets and their role in protecting against CMV infection has not been achieved. Here, we performed high-dimensional immune phenotyping by mass cytometry, and functional assays, on 112 serially collected samples from CMV seropositive kidney transplant recipients. Advanced unsupervised deep learning analysis was used to assess immune cell populations that significantly correlated with prevention against CMV infection and anti-viral immune function. Prior to infection, kidney transplant recipients who developed CMV infection showed significantly lower CMV-specific cell-mediated immune (CMI) frequencies than those that did not. A broad diversity of circulating cell subsets within innate and adaptive immune compartments were associated with CMV infection or protective CMV-specific CMI. While percentages of CMV (tetramer-stained)-specific T cells associated with high CMI responses and clinical protection, circulating CD3+CD8midCD56+ NK-T cells overall strongly associated with low CMI and subsequent infection. However, three NK-T cell subsets sharing the CD11b surface marker associated with CMV protection and correlated with strong anti-viral CMI frequencies in vitro. These data were validated in two external independent cohorts of kidney transplant recipients. Thus, we newly describe the kinetics of a novel NK-T cell subset that may have a protective role in post-transplantation CMV infection. Our findings pave the way to more mechanistic studies aimed at understanding the function of these cells in protection against CMV infection.

Tubular-specific expression of HIV protein Vpr leads to severe tubulointerstitial damage accompanied by progressive fibrosis and cystic development.

Chronic kidney disease (CKD) is a common cause of morbidity in human immunodeficiency virus (HIV)-positive individuals. HIV infection leads to a wide spectrum of kidney cell damage, including tubular epithelial cell (TEC) injury. Among the HIV-1 proteins, the pathologic effects of viral protein R (Vpr) are well established and include DNA damage response, cell cycle arrest, and cell death. Several in vitro studies have unraveled the molecular pathways driving the cytopathic effects of Vpr in tubular epithelial cells. However, the in vivo effects of Vpr on tubular injury and CKD pathogenesis have not been thoroughly investigated. Here, we use a novel inducible tubular epithelial cell-specific Vpr transgenic mouse model to show that Vpr expression leads to progressive tubulointerstitial damage, interstitial inflammation and fibrosis, and tubular cyst development. Importantly, Vpr-expressing tubular epithelial cells displayed significant hypertrophy, aberrant cell division, and atrophy; all reminiscent of tubular injuries observed in human HIV-associated nephropathy (HIVAN). Single-cell RNA sequencing analysis revealed the Vpr-mediated transcriptomic responses in specific tubular subsets and highlighted the potential multifaceted role of p53 in the regulation of cell metabolism, proliferation, and death pathways in Vpr-expressing tubular epithelial cells. Thus, our study demonstrates that HIV Vpr expression in tubular cells is sufficient to induce HIVAN-like tubulointerstitial damage and fibrosis, independent of glomerulosclerosis and proteinuria. Additionally, as this new mouse model develops progressive CKD with diffuse fibrosis and kidney failure, it can serve as a useful tool to examine the mechanisms of kidney disease progression and fibrosis in vivo.

Kidney single-cell transcriptome profile reveals distinct response of proximal tubule cells to SGLT2i and ARB treatment in diabetic mice.

Angiotensin receptor blockers (ARBs) and sodium-glucose cotransporter 2 inhibitors (SGLT2i) have been used as the standard therapy for patients with diabetic kidney disease (DKD). However, how these two drugs possess additive renoprotective effects remains unclear. Here, we conducted single-cell RNA sequencing to profile the kidney cell transcriptome of db/db mice treated with vehicle, ARBs, SGLT2i, or ARBs plus SGLT2i, using db/m mice as control. We identified 10 distinct clusters of kidney cells with predominant proximal tubular (PT) cells. We found that ARBs had more anti-inflammatory and anti-fibrotic effects, while SGLT2i affected more mitochondrial function in PT. We also identified a new PT subcluster, was increased in DKD, but reversed by the treatments. This new subcluster was also confirmed by immunostaining of mouse and human kidneys with DKD. Together, our study reveals kidney cell-specific gene signatures in response to ARBs and SGLT2i and identifies a new PT subcluster, which provides new insight into the pathogenesis of DKD.

Blood Transcriptomes of SARS-CoV-2-Infected Kidney Transplant Recipients Associated with Immune Insufficiency Proportionate to Severity.

BACKGROUND: Among patients with COVID-19, kidney transplant recipients (KTRs) have poor outcomes compared with non-KTRs. To provide insight into management of immunosuppression during acute illness, we studied immune signatures from the peripheral blood during and after COVID-19 infection from a multicenter KTR cohort. METHODS: We ascertained clinical data by chart review. A single sample of blood was collected for transcriptome analysis. Total RNA was poly-A selected and RNA was sequenced to evaluate transcriptome changes. We also measured cytokines and chemokines of serum samples collected during acute infection. RESULTS: A total of 64 patients with COVID-19 in KTRs were enrolled, including 31 with acute COVID-19 (<4 weeks from diagnosis) and 33 with post-acute COVID-19 (>4 weeks postdiagnosis). In the blood transcriptome of acute cases, we identified genes in positive or negative association with COVID-19 severity scores. Functional enrichment analyses showed upregulation of neutrophil and innate immune pathways but downregulation of T cell and adaptive immune activation pathways. This finding was independent of lymphocyte count, despite reduced immunosuppressant use in most KTRs. Compared with acute cases, post-acute cases showed "normalization" of these enriched pathways after 4 weeks, suggesting recovery of adaptive immune system activation despite reinstitution of immunosuppression. Analysis of the non-KTR cohort with COVID-19 showed significant overlap with KTRs in these functions. Serum inflammatory cytokines followed an opposite trend (i.e., increased with disease severity), indicating that blood lymphocytes are not the primary source. CONCLUSIONS: The blood transcriptome of KTRs affected by COVID-19 shows decreases in T cell and adaptive immune activation pathways during acute disease that, despite reduced immunosuppressant use, associate with severity. These pathways show recovery after acute illness.

LINE-1 promotes tumorigenicity and exacerbates tumor progression via stimulating metabolism reprogramming in non-small cell lung cancer.

BACKGROUND: Long Interspersed Nuclear Element-1 (LINE-1, L1) is increasingly regarded as a genetic risk for lung cancer. Transcriptionally active LINE-1 forms a L1-gene chimeric transcript (LCTs), through somatic L1 retrotransposition (LRT) or L1 antisense promoter (L1-ASP) activation, to play an oncogenic role in cancer progression. METHODS: Here, we developed Retrotransposon-gene fusion estimation program (ReFuse), to identify and quantify LCTs in RNA sequencing data from TCGA lung cancer cohort (n = 1146) and a single cell RNA sequencing dataset then further validated those LCTs in an independent cohort (n = 134). We next examined the functional roles of a cancer specific LCT (L1-FGGY) in cell proliferation and tumor progression in LUSC cell lines and mice. RESULTS: The LCT events correspond with specific metabolic processes and mitochondrial functions and was associated with genomic instability, hypomethylation, tumor stage and tumor immune microenvironment (TIME). Functional analysis of a tumor specific and frequent LCT involving FGGY (L1-FGGY) reveal that the arachidonic acid (AA) metabolic pathway was activated by the loss of FGGY through the L1-FGGY chimeric transcript to promote tumor growth, which was effectively targeted by a combined use of an anti-HIV drug (NVR) and a metabolic inhibitor (ML355). Lastly, we identified a set of transcriptomic signatures to stratify the LUSC patients with a higher risk for poor outcomes who may benefit from treatments using NVR alone or combined with an anti-metabolism drug. CONCLUSIONS: This study is the first to characterize the role of L1 in metabolic reprogramming of lung cancer and provide rationale for L1-specifc prognosis and potential for a therapeutic strategy for treating lung cancer. TRIAL REGISTRATION: Study on the mechanisms of the mobile element L1-FGGY promoting the proliferation, invasion and immune escape of lung squamous cell carcinoma through the 12-LOX/Wnt pathway, Ek2020111. Registered 27 March 2020 - Retrospectively registered.

The single-cell landscape of kidney immune cells reveals transcriptional heterogeneity in early diabetic kidney disease.

The pathogenesis of diabetic kidney disease (DKD) involves multifactorial processes that converge to initiate and advance the disease. Although DKD is not typically classified as an inflammatory glomerular disease, mounting evidence supports the involvement of kidney inflammation as a key contributor in DKD pathogenesis, particularly through macrophages. However, detailed identification and corresponding phenotypic changes of macrophages in DKD remain poorly understood. To capture the gene expression changes in specific macrophage cell subsets in early DKD, we performed single-cell transcriptomic analysis of CD45-enriched kidney immune cells from type 1 diabetic OVE26 mice at two time points during the disease development. We also undertook a focused analysis of mononuclear phagocytes (macrophages and dendritic cells). Our results show increased resident and infiltrating macrophage subsets in the kidneys of mice with diabetes over time, with heightened expression of pro-inflammatory or anti-inflammatory genes in a subset-specific manner. Further analysis of macrophage polarization states in each subset in the kidneys showed changes consistent with the continuum of activation and differentiation states, with gene expression tending to shift toward undifferentiated phenotypes but with increased M1-like inflammatory phenotypes over time. By deconvolution analysis of RNAseq samples and by immunostaining of biopsies from patients with DKD, we further confirmed a differential expression of select genes in specific macrophage subsets essentially recapitulating the studies in mice. Thus, our study provides a comprehensive analysis of macrophage transcriptomic profiles in early DKD that underscores the dynamic macrophage phenotypes in disease progression.

Deep learning identified pathological abnormalities predictive of graft loss in kidney transplant biopsies.

Interstitial fibrosis, tubular atrophy, and inflammation are major contributors to kidney allograft failure. Here we sought an objective, quantitative pathological assessment of these lesions to improve predictive utility and constructed a deep-learning-based pipeline recognizing normal vs. abnormal kidney tissue compartments and mononuclear leukocyte infiltrates. Periodic acid- Schiff stained slides of transplant biopsies (60 training and 33 testing) were used to quantify pathological lesions specific for interstitium, tubules and mononuclear leukocyte infiltration. The pipeline was applied to the whole slide images from 789 transplant biopsies (478 baseline [pre-implantation] and 311 post-transplant 12-month protocol biopsies) in two independent cohorts (GoCAR: 404 patients, AUSCAD: 212 patients) of transplant recipients to correlate composite lesion features with graft loss. Our model accurately recognized kidney tissue compartments and mononuclear leukocytes. The digital features significantly correlated with revised Banff 2007 scores but were more sensitive to subtle pathological changes below the thresholds in the Banff scores. The Interstitial and Tubular Abnormality Score (ITAS) in baseline samples was highly predictive of one-year graft loss, while a Composite Damage Score in 12-month post-transplant protocol biopsies predicted later graft loss. ITASs and Composite Damage Scores outperformed Banff scores or clinical predictors with superior graft loss prediction accuracy. High/intermediate risk groups stratified by ITASs or Composite Damage Scores also demonstrated significantly higher incidence of estimated glomerular filtration rate decline and subsequent graft damage. Thus, our deep-learning approach accurately detected and quantified pathological lesions from baseline or post-transplant biopsies and demonstrated superior ability for prediction of post-transplant graft loss with potential application as a prevention, risk stratification or monitoring tool.

Genome-wide non-HLA donor-recipient genetic differences influence renal allograft survival via early allograft fibrosis.

Donor-recipient (D-R) differences at human leukocyte antigen (HLA) loci are currently incorporated into organ sharing, allocation and immunosuppression decisions. However, while acute rejection episodes have substantially diminished, progressive histologic damage occurs in allografts and improved long-term survival remains an unrealized goal among kidney recipients. Here we tested the hypothesis that non-HLA dependent, genome-wide D-R genetic differences could contribute to unchecked alloimmunity with histologic and functional consequences, culminating in long-term allograft failure. Genome-wide single nucleotide polymorphism (SNP) array data, excluding the HLA region, was utilized from 385 transplants to study the role of D-R differences upon serial histology and allograft survival. ADMIXTURE analysis was performed to quantitatively estimate ancestry in each D-R pair and PLINK was used to estimate the proportion of genome-shared identity-by-descent (pIBD) between D-R pairs. Subsequently, quantitative measures of recipient ancestry based on non-HLA SNPs was associated with death-censored allograft survival in adjusted Cox models. In D-R pairs of similar ancestry, pIBD was significantly associated with allograft survival independent of HLA mismatches in 224 transplants. Surprisingly, pIBD and recipient ancestry were not associated with clinical or subclinical rejection at any time post-transplant. Significantly, in multivariable analysis, pIBD inversely correlated with vascular intimal fibrosis in 160 biopsies obtained less than one year which in turn was significantly associated with allograft survival. Thus, our novel data show that non-HLA D-R differences associate with early vascular intimal fibrosis and allograft survival.

Single-Cell RNA Profiling of Glomerular Cells Shows Dynamic Changes in Experimental Diabetic Kidney Disease.

BACKGROUND: Recent single-cell RNA sequencing (scRNA-seq) analyses have offered much insight into cell-specific gene expression profiles in normal kidneys. However, in diseased kidneys, understanding of changes in specific cells, particularly glomerular cells, remains limited. METHODS: To elucidate the glomerular cell-specific gene expression changes in diabetic kidney disease, we performed scRNA-seq analysis of isolated glomerular cells from streptozotocin-induced diabetic endothelial nitric oxide synthase (eNOS)-deficient (eNOS-/-) mice and control eNOS-/- mice. RESULTS: We identified five distinct cell populations, including glomerular endothelial cells, mesangial cells, podocytes, immune cells, and tubular cells. Using scRNA-seq analysis, we confirmed the expression of glomerular cell-specific markers and also identified several new potential markers of glomerular cells. The number of immune cells was significantly higher in diabetic glomeruli compared with control glomeruli, and further cluster analysis showed that these immune cells were predominantly macrophages. Analysis of differential gene expression in endothelial and mesangial cells of diabetic and control mice showed dynamic changes in the pattern of expressed genes, many of which are known to be involved in diabetic kidney disease. Moreover, gene expression analysis showed variable responses of individual cells to diabetic injury. CONCLUSIONS: Our findings demonstrate the ability of scRNA-seq analysis in isolated glomerular cells from diabetic and control mice to reveal dynamic changes in gene expression in diabetic kidneys, with variable responses of individual cells. Such changes, which might not be apparent in bulk transcriptomic analysis of glomerular cells, may help identify important pathophysiologic factors contributing to the progression of diabetic kidney disease.

A Peripheral Blood Gene Expression Signature to Diagnose Subclinical Acute Rejection.

BACKGROUND: In kidney transplant recipients, surveillance biopsies can reveal, despite stable graft function, histologic features of acute rejection and borderline changes that are associated with undesirable graft outcomes. Noninvasive biomarkers of subclinical acute rejection are needed to avoid the risks and costs associated with repeated biopsies. METHODS: We examined subclinical histologic and functional changes in kidney transplant recipients from the prospective Genomics of Chronic Allograft Rejection (GoCAR) study who underwent surveillance biopsies over 2 years, identifying those with subclinical or borderline acute cellular rejection (ACR) at 3 months (ACR-3) post-transplant. We performed RNA sequencing on whole blood collected from 88 individuals at the time of 3-month surveillance biopsy to identify transcripts associated with ACR-3, developed a novel sequencing-based targeted expression assay, and validated this gene signature in an independent cohort. RESULTS: Study participants with ACR-3 had significantly higher risk than those without ACR-3 of subsequent clinical acute rejection at 12 and 24 months, faster decline in graft function, and decreased graft survival in adjusted Cox analysis. We identified a 17-gene signature in peripheral blood that accurately diagnosed ACR-3, and validated it using microarray expression profiles of blood samples from 65 transplant recipients in the GoCAR cohort and three public microarray datasets. In an independent cohort of 110 transplant recipients, tests of the targeted expression assay on the basis of the 17-gene set showed that it identified individuals at higher risk of ongoing acute rejection and future graft loss. CONCLUSIONS: Our targeted expression assay enabled noninvasive diagnosis of subclinical acute rejection and inflammation in the graft and may represent a useful tool to risk-stratify kidney transplant recipients.

Subpathway-GM: identification of metabolic subpathways via joint power of interesting genes and metabolites and their topologies within pathways.

Various 'omics' technologies, including microarrays and gas chromatography mass spectrometry, can be used to identify hundreds of interesting genes, proteins and metabolites, such as differential genes, proteins and metabolites associated with diseases. Identifying metabolic pathways has become an invaluable aid to understanding the genes and metabolites associated with studying conditions. However, the classical methods used to identify pathways fail to accurately consider joint power of interesting gene/metabolite and the key regions impacted by them within metabolic pathways. In this study, we propose a powerful analytical method referred to as Subpathway-GM for the identification of metabolic subpathways. This provides a more accurate level of pathway analysis by integrating information from genes and metabolites, and their positions and cascade regions within the given pathway. We analyzed two colorectal cancer and one metastatic prostate cancer data sets and demonstrated that Subpathway-GM was able to identify disease-relevant subpathways whose corresponding entire pathways might be ignored using classical entire pathway identification methods. Further analysis indicated that the power of a joint genes/metabolites and subpathway strategy based on their topologies may play a key role in reliably recalling disease-relevant subpathways and finding novel subpathways.

Cholesterol 25-Hydroxylase Protects Against Diabetic Kidney Disease by Regulating ADP Ribosylation Factor 4.

Cholesterol 25-hydroxylase (CH25H), an enzyme involved in cholesterol metabolism, regulates inflammatory responses and lipid metabolism. However, its role in kidney disease is not known.  The author found that CH25H transcript is expressed mostly in glomerular and peritubular endothelial cells and that its expression increased in human and mouse diabetic kidneys.  Global deletion of Ch25h in Leprdb/db mice aggravated diabetic kidney disease (DKD), which is associated with increased endothelial cell apoptosis. Treatment of 25-hydroxycholesterol (25-HC), the product of CH25H, alleviated kidney injury in Leprdb/db mice. Mechanistically, 25-HC binds to GTP-binding protein ADP-ribosylation factor 4 (ARF4), an essential protein required for maintaining protein transport in the Golgi apparatus. Interestingly, ARF4's GTPase-activating protein ASAP1 is also predominantly expressed in endothelial cells and its expression increased in DKD. Suppression of ARF4 activity by deleting ARF4 or overexpressing ASAP1 results in endothelial cell death. These results indicate that 25-HC binds ARF4 to inhibit its interaction with ASAP1, and thereby resulting in enhanced ARF4 activity to confer renoprotection. Therefore, treatment of 25-HC improves kidney injury in DKD in part by restoring ARF4 activity to maintain endothelial cell survival. This study provides a novel mechanism and a potential new therapy for DKD.

Integrated multiomics implicates dysregulation of ECM and cell adhesion pathways as drivers of severe COVID-associated kidney injury.

COVID-19 has been a significant public health concern for the last four years; however, little is known about the mechanisms that lead to severe COVID-associated kidney injury. In this multicenter study, we combined quantitative deep urinary proteomics and machine learning to predict severe acute outcomes in hospitalized COVID-19 patients. Using a 10-fold cross-validated random forest algorithm, we identified a set of urinary proteins that demonstrated predictive power for both discovery and validation set with 87% and 79% accuracy, respectively. These predictive urinary biomarkers were recapitulated in non-COVID acute kidney injury revealing overlapping injury mechanisms. We further combined orthogonal multiomics datasets to understand the mechanisms that drive severe COVID-associated kidney injury. Functional overlap and network analysis of urinary proteomics, plasma proteomics and urine sediment single-cell RNA sequencing showed that extracellular matrix and autophagy-associated pathways were uniquely impacted in severe COVID-19. Differentially abundant proteins associated with these pathways exhibited high expression in cells in the juxtamedullary nephron, endothelial cells, and podocytes, indicating that these kidney cell types could be potential targets. Further, single-cell transcriptomic analysis of kidney organoids infected with SARS-CoV-2 revealed dysregulation of extracellular matrix organization in multiple nephron segments, recapitulating the clinically observed fibrotic response across multiomics datasets. Ligand-receptor interaction analysis of the podocyte and tubule organoid clusters showed significant reduction and loss of interaction between integrins and basement membrane receptors in the infected kidney organoids. Collectively, these data suggest that extracellular matrix degradation and adhesion-associated mechanisms could be a main driver of COVID-associated kidney injury and severe outcomes.

TYRO3 agonist as therapy for glomerular disease.

Podocyte injury and loss are key drivers of primary and secondary glomerular diseases, such as focal segmental glomerulosclerosis (FSGS) and diabetic kidney disease (DKD). We previously demonstrated the renoprotective role of protein S (PS) and its cognate tyrosine-protein kinase receptor, TYRO3, in models of FSGS and DKD and that their signaling exerts antiapoptotic and antiinflammatory effects to confer protection against podocyte loss. Among the 3 TAM receptors (TYRO3, AXL, and MER), only TYRO3 expression is largely restricted to podocytes, and glomerular TYRO3 mRNA expression negatively correlates with human glomerular disease progression. Therefore, we posited that the agonistic PS/TYRO3 signaling could serve as a potential therapeutic approach to attenuate glomerular disease progression. As PS function is not limited to TYRO3-mediated signal transduction but includes its anticoagulant activity, we focused on the development of TYRO3 agonists as an optimal therapeutic approach to glomerular disease. Among the small-molecule TYRO3 agonistic compounds screened, compound 10 (C-10) showed a selective activation of TYRO3 without any effects on AXL or MER. We also confirmed that C-10 directly binds to TYRO3, but not the other receptors. In vivo, C-10 attenuated proteinuria, glomerular injury, and podocyte loss in mouse models of Adriamycin-induced nephropathy and a db/db model of type 2 diabetes. Moreover, these renoprotective effects of C-10 were lost in Tyro3-knockout mice, indicating that C-10 is a selective agonist of TYRO3 activity that mitigates podocyte injury and glomerular disease. Therefore, C-10, a TYRO3 agonist, could be potentially developed as a new therapy for glomerular disease.

Human TLR8 induces inflammatory bone marrow erythromyeloblastic islands and anemia in SLE-prone mice.

Anemia commonly occurs in systemic lupus erythematosus, a disease characterized by innate immune activation by nucleic acids. Overactivation of cytoplasmic sensors by self-DNA or RNA can cause erythroid cell death, while sparing other hematopoietic cell lineages. Whereas chronic inflammation is involved in this mechanism, less is known about the impact of systemic lupus erythematosus on the BM erythropoietic niche. We discovered that expression of the endosomal ssRNA sensor human TLR8 induces fatal anemia in Sle1.Yaa lupus mice. We observed that anemia was associated with a decrease in erythromyeloblastic islands and a block in differentiation at the CFU-E to proerythroblast transition in the BM. Single-cell RNAseq analyses of isolated BM erythromyeloblastic islands from human TLR8-expressing mice revealed that genes associated with essential central macrophage functions including adhesion and provision of nutrients were down-regulated. Although compensatory stress erythropoiesis occurred in the spleen, red blood cell half-life decreased because of hemophagocytosis. These data implicate the endosomal RNA sensor TLR8 as an additional innate receptor whose overactivation causes acquired failure of erythropoiesis via myeloid cell dysregulation.

HCK induces macrophage activation to promote renal inflammation and fibrosis via suppression of autophagy.

Renal inflammation and fibrosis are the common pathways leading to progressive chronic kidney disease (CKD). We previously identified hematopoietic cell kinase (HCK) as upregulated in human chronic allograft injury promoting kidney fibrosis; however, the cellular source and molecular mechanisms are unclear. Here, using immunostaining and single cell sequencing data, we show that HCK expression is highly enriched in pro-inflammatory macrophages in diseased kidneys. HCK-knockout (KO) or HCK-inhibitor decreases macrophage M1-like pro-inflammatory polarization, proliferation, and migration in RAW264.7 cells and bone marrow-derived macrophages (BMDM). We identify an interaction between HCK and ATG2A and CBL, two autophagy-related proteins, inhibiting autophagy flux in macrophages. In vivo, both global or myeloid cell specific HCK-KO attenuates renal inflammation and fibrosis with reduces macrophage numbers, pro-inflammatory polarization and migration into unilateral ureteral obstruction (UUO) kidneys and unilateral ischemia reperfusion injury (IRI) models. Finally, we developed a selective boron containing HCK inhibitor which can reduce macrophage pro-inflammatory activity, proliferation, and migration in vitro, and attenuate kidney fibrosis in the UUO mice. The current study elucidates mechanisms downstream of HCK regulating macrophage activation and polarization via autophagy in CKD and identifies that selective HCK inhibitors could be potentially developed as a new therapy for renal fibrosis.