C3d-Targeted factor H inhibits tissue complement in disease models and reduces glomerular injury without affecting circulating complement.

Complement-mediated diseases can be treated using systemic inhibitors. However, complement components are abundant in circulation, affecting systemic inhibitors' exposure and efficacy. Furthermore, because of complement's essential role in immunity, systemic treatments raise infection risk in patients. To address these challenges, we developed antibody fusion proteins combining the alternative-pathway complement inhibitor factor H (fH1-5) with an anti-C3d monoclonal antibody (C3d-mAb-2fH). Because C3d is deposited at sites of complement activity, this molecule localizes to tissue complement while minimizing circulating complement engagement. These fusion proteins bind to deposited complement in diseased human skin sections and localize to activated complement in a primate skin injury model. We further explored the pharmacology of C3d-mAb-2fH proteins in rodent models with robust tissue complement activation. Doses of C3d-mAb-2fH >1 mg/kg achieved >75% tissue complement inhibition in mouse and rat injury models while avoiding circulating complement blockade. Glomerular-specific complement inhibition reduced proteinuria and preserved podocyte foot-process architecture in rat membranous nephropathy, indicating disease-modifying efficacy. These data indicate that targeting local tissue complement results in durable and efficacious complement blockade in skin and kidney while avoiding systemic inhibition, suggesting broad applicability of this approach in treating a range of complement-mediated diseases.

Discovery of Autoantibodies Targeting Nephrin in Minimal Change Disease Supports a Novel Autoimmune Etiology.

BACKGROUND: Failure of the glomerular filtration barrier, primarily by loss of slit diaphragm architecture, underlies nephrotic syndrome in minimal change disease. The etiology remains unknown. The efficacy of B cell-targeted therapies in some patients, together with the known proteinuric effect of anti-nephrin antibodies in rodent models, prompted us to hypothesize that nephrin autoantibodies may be present in patients with minimal change disease. METHODS: We evaluated sera from patients with minimal change disease, enrolled in the Nephrotic Syndrome Study Network (NEPTUNE) cohort and from our own institutions, for circulating nephrin autoantibodies by indirect ELISA and by immunoprecipitation of full-length nephrin from human glomerular extract or a recombinant purified extracellular domain of human nephrin. We also evaluated renal biopsies from our institutions for podocyte-associated punctate IgG colocalizing with nephrin by immunofluorescence. RESULTS: In two independent patient cohorts, we identified circulating nephrin autoantibodies during active disease that were significantly reduced or absent during treatment response in a subset of patients with minimal change disease. We correlated the presence of these autoantibodies with podocyte-associated punctate IgG in renal biopsies from our institutions. We also identified a patient with steroid-dependent childhood minimal change disease that progressed to end stage kidney disease; she developed a massive post-transplant recurrence of proteinuria that was associated with high pretransplant circulating nephrin autoantibodies. CONCLUSIONS: Our discovery of nephrin autoantibodies in a subset of adults and children with minimal change disease aligns with published animal studies and provides further support for an autoimmune etiology. We propose a new molecular classification of nephrin autoantibody minimal change disease to serve as a framework for instigation of precision therapeutics for these patients.

Collaborative Cohort of Cohorts for COVID-19 Research (C4R) Study: Study Design.

The Collaborative Cohort of Cohorts for COVID-19 Research (C4R) is a national prospective study of adults comprising 14 established US prospective cohort studies. Starting as early as 1971, investigators in the C4R cohort studies have collected data on clinical and subclinical diseases and their risk factors, including behavior, cognition, biomarkers, and social determinants of health. C4R links this pre-coronavirus disease 2019 (COVID-19) phenotyping to information on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and acute and postacute COVID-related illness. C4R is largely population-based, has an age range of 18-108 years, and reflects the racial, ethnic, socioeconomic, and geographic diversity of the United States. C4R ascertains SARS-CoV-2 infection and COVID-19 illness using standardized questionnaires, ascertainment of COVID-related hospitalizations and deaths, and a SARS-CoV-2 serosurvey conducted via dried blood spots. Master protocols leverage existing robust retention rates for telephone and in-person examinations and high-quality event surveillance. Extensive prepandemic data minimize referral, survival, and recall bias. Data are harmonized with research-quality phenotyping unmatched by clinical and survey-based studies; these data will be pooled and shared widely to expedite collaboration and scientific findings. This resource will allow evaluation of risk and resilience factors for COVID-19 severity and outcomes, including postacute sequelae, and assessment of the social and behavioral impact of the pandemic on long-term health trajectories.

A conceptual framework linking immunology, pathology, and clinical features in primary membranous nephropathy.

Primary membranous nephropathy is a leading cause of adult nephrotic syndrome. The field took a major step forward with the identification of phospholipase A2 receptor (PLA2R) as a target antigen in the majority of cases and with the ability to measure circulating autoantibodies to PLA2R. Since then, the existence of additional target antigens such as thrombospondin type-1 domain-containing 7A, exostosin 1 and 2, neural EGFL like 1, and semaphorin 3B has been demonstrated. The ability to detect and monitor levels of circulating autoantibodies has opened a new window onto the humoral aspect of primary membranous nephropathy. Clinicians now rely on clinical parameters such as proteinuria, as well as levels of circulating autoantibodies against PLA2R and the results of immunofluorescence staining for PLA2R within kidney biopsy tissue, to guide the management of this disease. The relationship between immunologic and clinical disease course is consistent, but not necessarily intuitive. In addition, kidney biopsy provides only a single snapshot of disease that needs to be interpreted in light of changing clinical and serological findings. A clear understanding of these dynamic parameters is essential for staging, treatment, and management of this disease. This review aims to shed light on current knowledge regarding the development and time course of changes in the serum levels of autoantibodies against PLA2R, proteinuria, and histological findings that underlie the pathophysiology of primary membranous nephropathy.

Loss of Roundabout Guidance Receptor 2 (Robo2) in Podocytes Protects Adult Mice from Glomerular Injury by Maintaining Podocyte Foot Process Structure.

Roundabout guidance receptor 2 (ROBO2) plays an important role during early kidney development. ROBO2 is expressed in podocytes, inhibits nephrin-induced actin polymerization, down-regulates nonmuscle myosin IIA activity, and destabilizes kidney podocyte adhesion. However, the role of ROBO2 during kidney injury, particularly in mature podocytes, is not known. Herein, we report that loss of ROBO2 in podocytes [Robo2 conditional knockout (cKO) mouse] is protective from glomerular injuries. Ultrastructural analysis reveals that Robo2 cKO mice display less foot process effacement and better-preserved slit-diaphragm density compared with wild-type littermates injured by either protamine sulfate or nephrotoxic serum (NTS). The Robo2 cKO mice also develop less proteinuria after NTS injury. Further studies reveal that ROBO2 expression in podocytes is up-regulated after glomerular injury because its expression levels are higher in the glomeruli of NTS injured mice and passive Heymann membranous nephropathy rats. Moreover, the amount of ROBO2 in the glomeruli is also elevated in patients with membranous nephropathy. Finally, overexpression of ROBO2 in cultured mouse podocytes compromises cell adhesion. Taken together, these findings suggest that kidney injury increases glomerular ROBO2 expression that might compromise podocyte adhesion and, thus, loss of Robo2 in podocytes could protect from glomerular injury by enhancing podocyte adhesion that helps maintain foot process structure. Our findings also suggest that ROBO2 is a therapeutic target for podocyte injury and podocytopathy.

T95 nucleophosmin phosphorylation as a novel mediator and marker of regulated cell death in acute kidney injury.

The function of site-specific phosphorylation of nucleophosmin (NPM), an essential Bax chaperone, in stress-induced cell death is unknown. We hypothesized that NPM threonine 95 (T95) phosphorylation both signals and promotes cell death. In resting cells, NPM exclusively resides in the nucleus and T95 is nonphosphorylated. In contrast, phosphorylated T95 NPM (pNPM T95) accumulates in the cytosol after metabolic stress, in multiple human cancer cell lines following γ-radiation, and in postischemic human kidney tissue. Based on the T95 phosphorylation consensus sequence, we hypothesized that glycogen synthase kinase-3ß (GSK-3ß) regulates cytosolic NPM translocation by phosphorylating T95 NPM. In a cell-free system, GSK-3ß phosphorylated a synthetic NPM peptide containing T95. In vitro, bidirectional manipulation of GSK-3ß activity substantially altered T95 phosphorylation, cytosolic NPM translocation, and cell survival during stress, mechanistically linking these lethal events. Furthermore, GSK-3ß inhibition in vivo decreased cytosolic pNPM T95 accumulation in kidney tissue after experimental ischemia. In patients with acute kidney injury, both cytosolic NPM accumulation in proximal tubule cells and NPM-rich intratubular casts were detected in frozen renal biopsy tissue. These observations show, for the first time, that GSK-3ß promotes cell death partly by phosphorylating NPM at T95, to promote cytosolic NPM accumulation. T95 NPM is also a rational therapeutic target to ameliorate ischemic renal cell injury and may be a universal injury marker in mammalian cells.

Lymphoproliferative disease and autoimmunity in mice with increased miR-17-92 expression in lymphocytes.

The genomic region encoding the miR-17-92 microRNA (miRNA) cluster is often amplified in lymphoma and other cancers, and cancer cells carrying this amplification have higher expression of miRNA in this cluster. Retroviral expression of miR-17-92 accelerates c-Myc-induced lymphoma development, but precisely how higher expression of miR-17-92 promotes lymphomagenesis remains unclear. Here we generated mice with higher expression of miR-17-92 in lymphocytes. These mice developed lymphoproliferative disease and autoimmunity and died prematurely. Lymphocytes from these mice showed more proliferation and less activation-induced cell death. The miR-17-92 miRNA suppressed expression of the tumor suppressor PTEN and the proapoptotic protein Bim. This mechanism probably contributed to the lymphoproliferative disease and autoimmunity of miR-17-92-transgenic mice and contributes to lymphoma development in patients with amplifications of the miR-17-92 coding region.

Coincidental crystalline light chain cast nephropathy, light chain proximal tubulopathy, and urine crystallopathy: A case report and review of the literature
.

Plasma cell dyscrasias, including multiple myeloma (MM), are associated with diverse forms of pathology in the kidney. Some pathologic lesions, including light chain (myeloma) cast nephropathy (LCCN), are relatively common, while others, such as light chain proximal tubulopathy (LCPT), are less so. Both LCCN and LCPT are associated with clinical manifestations of acute kidney injury. Rare instances of coincidental LCPT and LCCN have been reported, but none to our knowledge of coincidental crystalline forms of these diseases, with similar forms appearing in the urine. While LCPT is usually associated with intracytoplasmic deposition of crystallized light chains, the intraluminal light chain casts in LCCN are typically amorphous and do not form crystals. We report here the co-occurrence of these two monoclonal crystalline forms of acute kidney injury in a 66-year-old woman with known history of κ-restricted multiple myeloma. Additionally, forms suggestive of a crystalline morphology were observed in the urine sediment. Clinicians who observe similar crystalline structures on renal biopsy or in urine sediment should have a high index of suspicion for underlying multiple myeloma as a unifying diagnosis.

KIM-1-/TIM-1-mediated phagocytosis links ATG5-/ULK1-dependent clearance of apoptotic cells to antigen presentation.

Phagocytosis of apoptotic cells by both professional and semi-professional phagocytes is required for resolution of organ damage and maintenance of immune tolerance. KIM-1/TIM-1 is a phosphatidylserine receptor that is expressed on epithelial cells and can transform the cells into phagocytes. Here, we demonstrate that KIM-1 phosphorylation and association with p85 results in encapsulation of phagosomes by lipidated LC3 in multi-membrane organelles. KIM-1-mediated phagocytosis is not associated with increased ROS production, and NOX inhibition does not block LC3 lipidation. Autophagy gene expression is required for efficient clearance of apoptotic cells and phagosome maturation. KIM-1-mediated phagocytosis leads to pro-tolerogenic antigen presentation, which suppresses CD4 T-cell proliferation and increases the percentage of regulatory T cells in an autophagy gene-dependent manner. Taken together, these data reveal a novel mechanism of epithelial biology linking phagocytosis, autophagy and antigen presentation to regulation of the inflammatory response.

Similar Biophysical Abnormalities in Glomeruli and Podocytes from Two Distinct Models.

Background FSGS is a pattern of podocyte injury that leads to loss of glomerular function. Podocytes support other podocytes and glomerular capillary structure, oppose hemodynamic forces, form the slit diaphragm, and have mechanical properties that permit these functions. However, the biophysical characteristics of glomeruli and podocytes in disease remain unclear.Methods Using microindentation, atomic force microscopy, immunofluorescence microscopy, quantitative RT-PCR, and a three-dimensional collagen gel contraction assay, we studied the biophysical and structural properties of glomeruli and podocytes in chronic (Tg26 mice [HIV protein expression]) and acute (protamine administration [cytoskeletal rearrangement]) models of podocyte injury.Results Compared with wild-type glomeruli, Tg26 glomeruli became progressively more deformable with disease progression, despite increased collagen content. Tg26 podocytes had disordered cytoskeletons, markedly abnormal focal adhesions, and weaker adhesion; they failed to respond to mechanical signals and exerted minimal traction force in three-dimensional collagen gels. Protamine treatment had similar but milder effects on glomeruli and podocytes.Conclusions Reduced structural integrity of Tg26 podocytes causes increased deformability of glomerular capillaries and limits the ability of capillaries to counter hemodynamic force, possibly leading to further podocyte injury. Loss of normal podocyte mechanical integrity could injure neighboring podocytes due to the absence of normal biophysical signals required for podocyte maintenance. The severe defects in podocyte mechanical behavior in the Tg26 model may explain why Tg26 glomeruli soften progressively, despite increased collagen deposition, and may be the basis for the rapid course of glomerular diseases associated with severe podocyte injury. In milder injury (protamine), similar processes occur but over a longer time.

TMIGD1 is a novel adhesion molecule that protects epithelial cells from oxidative cell injury.

Oxidative damage to renal tubular epithelial cells is a fundamental pathogenic mechanism implicated in both acute kidney injury and chronic kidney diseases. Because epithelial cell survival influences the outcome of acute kidney injury and chronic kidney diseases, identifying its molecular regulators could provide new insight into pathobiology and possible new therapeutic strategies for these diseases. We have identified transmembrane and immunoglobulin domain-containing 1 (TMIGD1) as a novel adhesion molecule, which is highly conserved in humans and other species. TMIGD1 is expressed in renal tubular epithelial cells and promotes cell survival. The extracellular domain of TMIGD1 contains two putative immunoglobulin domains and mediates self-dimerization. Our data suggest that TMIGD1 regulates transepithelial electric resistance and permeability of renal epithelial cells. TMIGD1 controls cell migration, cell morphology, and protects renal epithelial cells from oxidative- and nutrient-deprivation-induced cell injury. Hydrogen peroxide-induced oxidative cell injury downregulates TMIGD1 expression and targets it for ubiquitination. Moreover, TMIGD1 expression is significantly affected in both acute kidney injury and in deoxy-corticosterone acetate and sodium chloride (deoxy-corticosterone acetate salt)-induced chronic hypertensive kidney disease mouse models. Taken together, we have identified TMIGD1 as a novel cell adhesion molecule expressed in kidney epithelial cells that protects kidney epithelial cells from oxidative cell injury to promote cell survival.

Pregnancy in a Patient With Primary Membranous Nephropathy and Circulating Anti-PLA2R Antibodies: A Case Report.

There is little information about pregnancy outcomes in patients with active membranous nephropathy (MN), especially those with circulating autoantibodies to M-type phospholipase A2receptor (PLA2R), the major autoantigen in primary MN. We present what we believe to be the first known case of successful pregnancy in a 39-year-old woman with PLA2R-associated MN. In the year prior to pregnancy, the patient developed anasarca, hypoalbuminemia (albumin, 1.3-2.2g/dL), and proteinuria (protein excretion, 29.2 g/d). Kidney biopsy revealed MN with staining for PLA2R, and the patient was seropositive for anti-PLA2R autoantibodies. She did not respond to conservative therapy and was treated with intravenous rituximab (2 doses of 1 g each). Several weeks after presentation, she was found to be 6 weeks pregnant and was closely followed up without further immunosuppressive treatment. Proteinuria remained with protein excretion in the 8- to 12-g/d range. Circulating anti-PLA2R levels declined but were still detectable. At 38 weeks, a healthy baby girl was born, without proteinuria at birth or at her subsequent 6-month postnatal visit. At the time of delivery, the mother still had detectable circulating anti-PLA2R of immunoglobulin G1 (IgG1), IgG3, and IgG4 subclasses, although at low titers. Only trace amounts of IgG4 anti-PLA2R were found in cord blood. Potential reasons for the discrepancy between anti-PLA2R levels in the maternal and fetal circulation are discussed.

Renal Interstitial Fibrosis: An Imperfect Predictor of Kidney Disease Progression in Some Patient Cohorts.

BACKGROUND: The extent of interstitial fibrosis on kidney biopsy is regarded as a prognostic indicator and guide to treatment. Patients with extensive fibrosis are assigned to supportive treatments with the expectation that they have advanced beyond the point at which immunosuppressive or other disease-modifying therapies would be of benefit. Our study highlights some of the limitations of using interstitial fibrosis to predict who will develop end-stage renal disease (ESRD). METHODS: Analysis of 434 consecutive renal biopsies performed between 2001 and 2012 at a single center. We assessed the influence of various clinical factors along with fibrosis as predictors of ESRD and dialysis-free survival in various patient groups. RESULTS: Interstitial fibrosis performed well overall as a predictor of progression to dialysis. On average, patients with >50% fibrosis progressed more rapidly than those with either 25-49 or 0-24% fibrosis with a median time to dialysis of 1.2, 6.5 and >10 years, respectively. In contrast, interstitial fibrosis was of less value as a predictor of disease progression in a subset of cases that included patients over the age of 70 and those with diabetic nephropathy on biopsy. Surprisingly, 13.9% of patients with normal renal function had 25-49% fibrosis and 5% had more than 50% fibrosis on biopsy, and 5 years after undergoing biopsy 21% of patients with >50% fibrosis still remained dialysis free. CONCLUSION: Renal fibrosis is an imperfect prognostic indicator for the development of ESRD and caution should be exercised in applying it too rigidly, especially in elderly or diabetic patients.

From man to fish: What can Zebrafish tell us about ApoL1 nephropathy?

BACKGROUND: Risk variant Apolipoprotein L1 (G1/G2) are strongly associated with a spectrum of kidney disease in people of recent African descent. The mechanism of ApoL1 nephropathy is unknown. Podocytes and/or endothelial cells are the presumed target kidney cells. Given the close homology in structure and function of zebrafish (ZF) pronephros and human nephron, we studied the effect of podocyte-specific or endothelium-specific expression of ApoL1 (G0, G1, or G2) on the structure and function of ZF pronephros. METHODS: Wild type (G0) or risk variant ApoL1 (G1/G2) were expressed in podocyte-specific or endothelium-specific under podocin/Flk promoters, respectively, using Gal4-UAS system. Structural pronephric changes were studied with light and electron microscopy (EM). Proteinuria was assayed by measuring renal excretion of GFP-vitamin D binding protein. Puromycin aminonucleoside (PAN) was used as inducer of podocyte injury. RESULTS: Endothelial-specific transgenic expression of G1/G2 is associated with endothelial injury indicated by endothelial cell swelling, segmental early double contours, and loss of endothelium fenestrae. Podocyte specific expression of G1 is associated with segmental podocyte foot process effacement and irregularities relative to G0. Despite the histological changes, the expression of G1/G2 alone in podocyte or endothelium compartment is not associated with edema, proteinuria, or gross whole fish phenotype. Moreover, PAN produced equal pericardial edema in all transgenic fish as well as nontransgenic controls. CONCLUSIONS: Transgenic expression human ApoL1 (G1/G2) is associated with histologic abnormalities in ZF glomeruli but is insufficient to cause quantifiable renal dysfunction. This finding supports the necessity of a "second hit" in the pathogenesis/progression of ApoL1-associated nephropathy.

The differentiation and stress response factor XBP-1 drives multiple myeloma pathogenesis.

Multiple myeloma (MM) evolves from a highly prevalent premalignant condition termed MGUS. The factors underlying the malignant transformation of MGUS are unknown. We report a MGUS/MM phenotype in transgenic mice with Emu-directed expression of the XBP-1 spliced isoform (XBP-1s), a factor governing unfolded protein/ER stress response and plasma-cell development. Emu-XBP-1s elicited elevated serum Ig and skin alterations. With age, Emu-xbp-1s transgenics develop features diagnostic of human MM, including bone lytic lesions and subendothelial Ig deposition. Furthermore, transcriptional profiles of Emu-xbp-1s lymphoid and MM cells show aberrant expression of known human MM dysregulated genes. The similarities of this model with the human disease, coupled with documented frequent XBP-1s overexpression in human MM, serve to implicate XBP-1s dysregulation in MM pathogenesis.

Distinct renal pathology and a chemotactic phenotype after enterohemorrhagic Escherichia coli shiga toxins in non-human primate models of hemolytic uremic syndrome.

Enterohemorrhagic Escherichia coli cause approximately 1.5 million infections globally with 176,000 cases occurring in the United States annually from ingesting contaminated food, most frequently E. coli O157:H7 in ground beef or fresh produce. In severe cases, the painful prodromal hemorrhagic colitis is complicated by potentially lethal hemolytic uremic syndrome (HUS), particularly in children. Bacterial Shiga-like toxins (Stx1, Stx2) are primarily responsible for HUS and the kidney and neurologic damage that ensue. Small animal models are hampered by the inability to reproduce HUS with thrombotic microangiopathy, hemolytic anemia, and acute kidney injury. Earlier, we showed that nonhuman primates (Papio) recapitulated clinical HUS after Stx challenge and that novel therapeutic intervention rescued the animals. Here, we present detailed light and electron microscopic pathology examination of the kidneys from these Stx studies. Stx1 challenge resulted in more severe glomerular endothelial injury, whereas the glomerular injury after Stx2 also included prominent mesangiolysis and an eosinophilic inflammatory infiltration. Both toxins induced glomerular platelet-rich thrombi, interstitial hemorrhage, and tubular injury. Analysis of kidney and other organs for inflammation biomarkers showed a striking chemotactic profile, with extremely high mRNA levels for IL-8, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1α and elevated urine chemokines at 48 hours after challenge. These observations give unique insight into the pathologic consequences of each toxin in a near human setting and present potential pathways for therapeutic intervention.

Eculizumab Use in Scleroderma Renal Crisis With Thrombotic Microangiopathy: A Case Report.

A Black woman in her 40s with past medical history significant for obesity treated with Roux-en-Y bypass surgery and a history of Raynaud's phenomenon, presented with acute pulmonary edema secondary to severe malignant hypertension and critically accelerated acute kidney injury, with evidence of systemic microangiopathic hemolytic anemia in the setting of clinical suspicion of systemic sclerosis sine scleroderma. Renin-angiotensin system blockade (angiotensin-converting enzyme inhibitor) was immediately started at the maximum possible dose in the setting of scleroderma renal crisis. Despite better control of blood pressure and volume status, kidney function continued to rapidly decline, thus a decision was made to go ahead with a kidney biopsy on day 3 of admission, which revealed severe features of scleroderma renal crisis with active thrombotic microangiopathy. The multidisciplinary team elected to treat the patient with terminal complement blockade using eculizumab in addition to high dose lisinopril and blood pressure control. Her serum creatinine peaked at 9.3 mg/dL shortly after eculizumab initiation, but improved soon after, dropping to 2.8 mg/dL after completion of the final eculizumab dose and 1.8 mg/dL 3 years later.

Glomerular Elasticity and Gene Expression Patterns Define Two Phases of Alport Nephropathy.

RATIONALE: Early steps in glomerular injury are poorly understood in collagen IV nephropathies. OBJECTIVES: We characterized structural, functional, and biophysical properties of glomerular capillaries and podocytes in Col4α3-/- mice and analyzed kidney cortex transcriptional profiles at various disease stages. We investigated the effects of TUDCA (suppresses ER stress) on these parameters and used human FSGS transcriptomic data to identify pathways rescued by TUDCA. FINDINGS: In Col4α3-/- mice, podocyte injury develops by 3 months, with maximum glomerular deformability and 40% podocyte loss at 4 months. This period is followed is followed by glomerular capillary stiffening, proteinuria, reduced renal function, inflammatory infiltrates, and fibrosis. Bulk RNA sequencing at sequential time points revealed progressive increases in inflammatory and injury gene expression, and activation of the TNF pathway. Mapping Podocyte-enriched genes from FSGS patients to mice showed that TUDCA, which mitigated renal injury suppressed molecular pathways associated with podocyte stress, hypertrophy and tubulo-interstitial injury. CONCLUSIONS: Col4α3-/- nephropathy progresses in two phases. The first is characterized by podocytopathy, increased glomerular capillary deformability and accelerated podocyte loss, and the second by increased capillary wall stiffening and renal inflammatory and profibrotic pathway activation. The response of podocytes to TUDCA treatment provides insights into signaling pathways in Alport and related nephropathies.

Digital pathology assessment of kidney glomerular filtration barrier ultrastructure in an animal model of podocytopathy.

Background: Transmission electron microscopy (TEM) images can visualize kidney glomerular filtration barrier ultrastructure, including the glomerular basement membrane (GBM) and podocyte foot processes (PFP). Podocytopathy is associated with glomerular filtration barrier morphological changes observed experimentally and clinically by measuring GBM or PFP width. However, these measurements are currently performed manually. This limits research on podocytopathy disease mechanisms and therapeutics due to labor intensiveness and inter-operator variability. Methods: We developed a deep learning-based digital pathology computational method to measure GBM and PFP width in TEM images from the kidneys of Integrin-Linked Kinase (ILK) podocyte-specific conditional knockout (cKO) mouse, an animal model of podocytopathy, compared to wild-type (WT) control mouse. We obtained TEM images from WT and ILK cKO littermate mice at 4 weeks old. Our automated method was composed of two stages: a U-Net model for GBM segmentation, followed by an image processing algorithm for GBM and PFP width measurement. We evaluated its performance with a 4-fold cross-validation study on WT and ILK cKO mouse kidney pairs. Results: Mean (95% confidence interval) GBM segmentation accuracy, calculated as Jaccard index, was 0.73 (0.70-0.76) for WT and 0.85 (0.83-0.87) for ILK cKO TEM images. Automated and manual GBM width measurements were similar for both WT (p=0.49) and ILK cKO (p=0.06) specimens. While automated and manual PFP width measurements were similar for WT (p=0.89), they differed for ILK cKO (p<0.05) specimens. WT and ILK cKO specimens were morphologically distinguishable by manual GBM (p<0.05) and PFP (p<0.05) width measurements. This phenotypic difference was reflected in the automated GBM (p<0.05) more than PFP (p=0.06) widths. Conclusions: These results suggest that certain automated measurements enabled via deep learning-based digital pathology tools could distinguish healthy kidneys from those with podocytopathy. Our proposed method provides high-throughput, objective morphological analysis and could facilitate podocytopathy research and translate into clinical diagnosis.

Plasma proteomics of acute tubular injury.

The kidney tubules constitute two-thirds of the cells of the kidney and account for the majority of the organ's metabolic energy expenditure. Acute tubular injury (ATI) is observed across various types of kidney diseases and may significantly contribute to progression to kidney failure. Non-invasive biomarkers of ATI may allow for early detection and drug development. Using the SomaScan proteomics platform on 434 patients with biopsy-confirmed kidney disease, we here identify plasma biomarkers associated with ATI severity. We employ regional transcriptomics and proteomics, single-cell RNA sequencing, and pathway analysis to explore biomarker protein and gene expression and enriched biological pathways. Additionally, we examine ATI biomarker associations with acute kidney injury (AKI) in the Kidney Precision Medicine Project (KPMP) (n = 44), the Atherosclerosis Risk in Communities (ARIC) study (n = 4610), and the COVID-19 Host Response and Clinical Outcomes (CHROME) study (n = 268). Our findings indicate 156 plasma proteins significantly linked to ATI with osteopontin, macrophage mannose receptor 1, and tenascin C showing the strongest associations. Pathway analysis highlight immune regulation and organelle stress responses in ATI pathogenesis.