Natural History and Clinicopathological Associations of TRPC6-Associated Podocytopathy.

BACKGROUND: Understanding the genetic basis of human diseases has become integral to drug development and precision medicine. Recent advancements have enabled the identification of molecular pathways driving diseases, leading to targeted treatment strategies. The increasing investment in rare diseases by the biotech industry underscores the importance of genetic evidence in drug discovery and approval processes. Here we studied a monogenic Mendelian kidney disease, TRPC6-associated podocytopathy (TRPC6-AP), to present its natural history, genetic spectrum, and clinicopathological associations in a large cohort of patients with causal variants in TRPC6, in order to help define the specific features of disease and further facilitate drug development and clinical trials design. METHODS: the study involved 64 individuals from 39 families with TRPC6 causal missense variants. Clinical data, including age of onset, laboratory results, response to treatment, kidney biopsy findings, and genetic information, were collected from multiple centers nationally and internationally. Exome or targeted sequencing was performed and variant classification was based on strict criteria. Structural and functional analyses of TRPC6 variants were conducted to understand their impact on protein function. In depth re-analysis of light and electron microscopy specimens for 9 available kidney biopsies was conducted to identify pathological features and correlates of TRPC6-AP. RESULTS: Large-scale sequencing data did not support causality for TRPC6 protein-truncating variants. We identified 21 unique TRPC6 missense variants, clustering in three distinct regions of the protein, and with different effects on TRPC6 3D protein structure. Kidney biopsy analysis revealed FSGS patterns of injury in most cases, along with distinctive podocyte features including diffuse foot process effacement and swollen cell bodies. The majority of patients presented in adolescence or early adulthood but with ample variation (average 22, SD ± 14 years), with frequent progression to kidney failure but with variability in time between presentation and ESKD. CONCLUSIONS: This study provides insights into the genetic spectrum, clinicopathological associations, and natural history of TRPC6-AP.

A newly identified protective role of C5aR1 in kidney tubules against toxin-induced acute kidney injury.

Acute kidney injury (AKI) remains a major reason for hospitalization with limited therapeutic options. While complement activation is implicated in AKI, the role of C5a receptor 1 (C5aR1) in kidney tubular cells is unclear. We used aristolochic acid nephropathy (AAN) and folic acid nephropathy (FAN) models to establish the role of C5aR1 in kidney tubules during AKI in germline C5ar1-/- mice, myeloid cell-specific, and kidney tubule-specific C5ar1 knockout mice. After aristolochic acid and folic acid injection, C5ar1-/- mice had increased AKI severity and a higher degree of tubular injury. Macrophage depletion in C5ar1-/- mice or myeloid cell-specific C5ar1 deletion did not affect the outcomes of AA-induced AKI. RNA-sequencing data from RTECs showed that C5ar1 deletion was associated with the downregulation of mitochondrial metabolism and ATP production transcriptional pathways. Metabolic studies confirmed reduced mitochondrial membrane potential at baseline and increased mitochondrial oxidative stress after injury in C5ar1-/- RTECs. Moreover, C5ar1-/- RTECs had enhanced glycolysis, glucose uptake, and lactate production upon injury, corroborated by metabolomics analysis of kidneys from AAN mice. Kidney tubule-specific C5ar1 knockout mice recapitulated exacerbated AKI observed in C5ar1-/- mice in AAN and FAN. Our data indicate that C5aR1 signaling in kidney tubules exerts renoprotective effects against toxin-induced AKI by limiting overt glycolysis and maintaining mitochondrial function, revealing a novel link between the complement system and tubular cell metabolism.

Macrophage WDFY3, a protector against autoimmunity.

Efficient efferocytosis is essential for maintaining homeostasis. Excessive apoptotic cell (AC) death and impaired macrophage efferocytosis lead to autoantigen release and autoantibody production, immune activation, and organ damage. It remains unclear whether these immunogenic autoantigens are the sole cause of increased autoimmunity or if efferocytosis of ACs directly influences macrophage function, impacting their ability to activate T cells and potentially amplifying autoimmune responses. Additionally, it has not been established if enhancing macrophage efferocytosis or modulating macrophage responses to AC engulfment can be protective in autoimmune-like disorders. Our previous work showed WDFY3 is crucial for efficient macrophage efferocytosis. This study reveals that myeloid knockout of Wdfy3 exacerbates autoimmunity in young mice with increased AC burden by systemic injections of ACs and in middle-aged mice developing spontaneous autoimmunity, whereas ectopic overexpression of WDFY3 suppresses autoimmunity in these models. Macrophages, as efferocytes, can activate T cells and the inflammasome upon engulfing ACs, which are suppressed by overexpressing WDFY3. This work uncovered the role of WDFY3 as a protector against autoimmunity by promoting macrophage efferocytosis thus limiting autoantigen production, as well as mitigating T cell activation and inflammasome activation.

Mitigation of checkpoint inhibitor-induced autoimmune hemolytic anemia through modulation of purinergic signaling.

ABSTRACT: Immune checkpoint inhibitors (ICPis) have revolutionized cancer immunotherapy but also can induce autoimmune hemolytic anemia (AIHA), a severe disease with high mortality. However, the cellular and molecular mechanism(s) of AIHA secondary to ICPi therapy (ICPi-AIHA) are unclear, other than being initiated through decreased checkpoint inhibition. Herein, we report ICPi-AIHA in a novel mouse model that shows similar characteristics of known human ICPi-AIHA (eg, autoantibodies, hemolysis, and increased mortality). During ICPi-AIHA, there is the simultaneous reduction of 2 regulatory T-cell populations (FoxP3+ and Tr1 [type 1 regulatory cells]) and an increase in inflammatory T helper cell 17 (TH17). Moreover, a novel CD39+CD73-FoxP3-CD25- CD4+ T-cell subset (ie, CD39 single positive [CD39SP]) emerges, and early increases in CD39SP predict AIHA development; CD39 is an ectonuclease that breaks down adenosine triphosphate (ATP). Additionally, we found that boosting ATPase activity by injecting recombinant apyrase mitigates AIHA development and significant CD39SP reductions, both suggesting a functional role for CD39 and demonstrating a novel therapeutic approach. Importantly, CD39SP are detectable in multiple mouse models developing AIHA and in patients with AIHA, demonstrating applicability to idiopathic and secondary AIHA. Highlighting broader autoimmunity relevance, ICPi-treated NZB mice experienced accelerated onset and severity of lupus, including AIHA. Moreover, ICPi treatment of healthy B6 animals led to detectable CD39SP and development of autoantibodies against multiple autoantigens including those on red blood cells and platelets. Together, our findings provide further insight into the cellular and molecular mechanisms of ICPi-AIHA, leading to novel diagnostic and therapeutic approaches with translational potential for use in humans being treated with ICPi.

Podocyte density as a predictor of long-term kidney outcome in obesity-related glomerulopathy.

Glomerulomegaly and focal segmental glomerulosclerosis are histopathological hallmarks of obesity-related glomerulopathy (ORG). Podocyte injury and subsequent depletion are regarded as key processes in the development of these glomerular lesions in patients with ORG, but their impact on long-term kidney outcome is undetermined. Here, we correlated clinicopathological findings and podocyte depletion retrospectively in patients with ORG. Relative (podocyte density) and absolute (podocyte number per glomerulus) measures of podocyte depletion were estimated using model-based stereology in 46 patients with ORG. The combined endpoint of kidney outcomes was defined as a 30% decline in estimated glomerular filtration rate (eGFR) or kidney failure. Patients with lower podocyte density were predominantly male and had larger body surface area, greater proteinuria, fewer non-sclerotic glomeruli, larger glomeruli and higher single-nephron eGFR. During a median follow-up of 4.1 years, 18 (39%) patients reached endpoint. Kidney survival in patients with lower podocyte density was significantly worse than in patients with higher podocyte density. However, there was no difference in kidney survival between patient groups based on podocyte number per glomerulus. Cox hazard analysis showed that podocyte density, but not podocyte number per glomerulus, was associated with the kidney outcomes after adjustment for clinicopathological confounders. Thus, our study demonstrates that a relative depletion of podocytes better predicts long-term kidney outcomes than does absolute depletion of podocytes. Hence, the findings implicate mismatch between glomerular enlargement and podocyte number as a crucial determinant of disease progression in ORG.

Clinicopathologic Characteristics of Crystalglobulin-Induced Nephropathy: A Case Series.

RATIONALE & OBJECTIVE: Crystalglobulinemia is a rare syndrome characterized by intravascular crystallization of monoclonal immunoglobulins (MIg). Data on kidney involvement are limited to case reports. This series characterizes the clinicopathologic spectrum of crystalglobulin-induced nephropathy (CIN). STUDY DESIGN: Case series. SETTING & PARTICIPANTS: Nineteen CIN cases identified from the nephropathology archives of Mayo Clinic and Columbia University. CIN was defined by intravascular (extracellular) MIg crystals visible by light microscopy (LM) and electron microscopy (EM). RESULTS: Among the cases, 68% were male, and 65% were Caucasian (median age, 56 years). Most patients presented with severe acute kidney injury (AKI) (median creatinine, 3.5mg/dL), hematuria, and mild proteinuria (median, 1.1g/day). Common extrarenal manifestations were constitutional (67%), cutaneous (56%), and rheumatologic (50%). Fifty percent of cases had hypocomplementemia. The hematologic disorders were monoclonal gammopathy of renal significance (MGRS) (72%), lymphoma (17%), or myeloma (11%), with 65% of these disorders discovered concomitantly with CIN. All patients had MIg identified on serum protein electrophoresis/immunofixation (IgGκ in 65%). The serum free light chain ratio was outside the renal range in 40%, and bone marrow biopsy detected the responsible clone in 67%. On LM, crystals involved glomeruli (100%) and vessels (47%), often with an inflammatory reaction (89%) and fibrin (58%). All cases exhibited crystal substructures (mostly paracrystalline) by EM. Immunofluorescence on paraffin-embedded tissue was more sensitive than frozen tissue (92% vs 47%) for demonstrating the crystal composition (IgGκ in 63%). Follow-up observation (median, 20 months) was available in 16 patients. Eighty-one percent received steroids, 44% plasmapheresis, 38% hemodialysis, and 69% chemotherapy. Ninety-percent of patients who received clone-directed therapy achieved kidney recovery versus 20% of those who did not (P=0.02). LIMITATIONS: Retrospective design, small sample size. CONCLUSIONS: CIN is a rare cause of nephropathy associated with lymphoplasmacytic disorders (mostly MGRS) and typically presents with severe AKI and extrarenal manifestations. Diagnosis often requires immunofluorescence performed on paraffin-embedded kidney tissue. Prompt initiation of clone-directed therapy, coupled with corticosteroids and plasmapheresis, may lead to recovery of kidney function.

Collapsing glomerulopathy is likely a major contributing factor for worse allograft survival in patients receiving kidney transplants from black donors.

Although a few registry-based studies have shown associations between receiving kidney allografts from Black donors and shorter allograft survival, detailed, large, single-center studies accounting for common confounding factors are lacking. Furthermore, pathologic alterations underlying this potential disparity have not been systematically studied. We performed a retrospective clinical-pathological study of kidney transplant recipients who received kidney allografts from either Black (n = 407) or White (n = 1,494) donors at Columbia University Irving Medical Center from 2005 to 2018, with median follow-up of 4.5 years post-transplantation. Black donor race was independently associated with allograft failure (adjusted HR = 1.34, p = 0.02) and recipients of kidney allografts from Black donors had a higher incidence of collapsing glomerulopathy [7.4% vs. 1.9%, OR = 4.17, p < 0.001]. When causes of allograft failure were examined, only allograft failure following development of collapsing glomerulopathy was more frequent in recipients of allografts from Black donors [15% vs. 5%, OR = 3.16, p = 0.004]. Notably, when patients who developed collapsing glomerulopathy were excluded from analysis, receiving kidney allografts from Black donors was not independently associated with allograft failure (adjusted HR = 1.24, p = 0.10). These findings revealed that, compared with recipients of kidney allografts from White donors, recipients of kidneys from Black donors have modestly shorter allograft survival and a higher probability of developing collapsing glomerulopathy, which negatively impacts allograft outcome. Identification of collapsing glomerulopathy risk factors may help decrease this complication and improve allograft survival, which optimally may reduce racial disparities post-transplantation.

Kidney Dysfunction and Pathology in the Setting of Hemophagocytic Lymphohistiocytosis.

Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a clinicopathologic syndrome produced by dysregulated activation of the immune system. Acute kidney injury (AKI) and proteinuria have been infrequently described in the setting of HLH, and investigations of underlying histopathologic changes in the kidney are limited. Methods: To characterize kidney pathology in HLH, a retrospective review of 30 patients' clinical and laboratory data, and kidney tissue was performed (18 from autopsy, and 12 biopsied patients). Results: HLH was associated with infection (83%), autoimmune disease (37%), and malignancy (20%), including 30% with concurrent autoimmune disease and infection. Nephrological presentations included subnephrotic range proteinuria (63%), AKI (63%), hematuria (33%), chronic kidney disease (CKD, 20%), nephrotic range proteinuria (13%), and nephrotic syndrome (7%); and 40% of patients required hemodialysis (HD). Among the 12 patients who underwent kidney biopsy, 6 subsequently showed improved kidney function and the remainder had progressive CKD with most progressing to end-stage kidney disease. Autopsy patients had a median terminal admission of 1 month, and 33% of the biopsied patients died (ranging from 0.3-5 months post-biopsy). Variable pathologies were identified, including acute tubular injury (ATI, 43%), lupus nephritis (LN, 23%), collapsing glomerulopathy (17%), thrombotic microangiopathy (TMA, 17%), and cortical necrosis (10%). Most autopsied patients had significant kidney pathology other than ATI that likely contributed to kidney function decline. A majority of patients with HLH exhibited kidney dysfunction that likely contributed to the poor prognosis. Conclusion: Kidney dysfunction in HLH should not be assumed to be solely attributable to ATI, and in certain scenarios a kidney biopsy may be warranted.

Targeting WIP1 phosphatase promotes partial remission in experimental collapsing glomerulopathy.

Collapsing focal segmental glomerulosclerosis (FSGS), also known as collapsing glomerulopathy (CG), is the most aggressive variant of FSGS and is characterized by a rapid progression to kidney failure. Understanding CG pathogenesis represents a key step for the development of targeted therapies. Previous work implicated the telomerase protein component TERT in CG pathogenesis, as transgenic TERT expression in adult mice resulted in a CG resembling that seen in human primary CG and HIV-associated nephropathy (HIVAN). Here, we used the telomerase-induced mouse model of CG (i-TERTci mice) to identify mechanisms to inhibit CG pathogenesis. Inactivation of WIP1 phosphatase, a p53 target acting in a negative feedback loop, blocked disease initiation in i-TERTci mice. Repression of disease initiation upon WIP1 deficiency was associated with senescence enhancement and required transforming growth factor-ß functions. The efficacy of a pharmacologic treatment to reduce disease severity in both i-TERTci mice and in a mouse model of HIVAN (Tg26 mice) was then assessed. Pharmacologic inhibition of WIP1 enzymatic activity in either the telomerase mice with CG or in the Tg26 mice promoted partial remission of proteinuria and ameliorated kidney histopathologic features. Histological as well as high-throughput sequencing methods further showed that selective inhibition of WIP1 does not promote kidney fibrosis or inflammation. Thus, our findings suggest that targeting WIP1 may be an effective therapeutic strategy for patients with CG.

Chronic kidney disease in a murine model of non-alcoholic steatohepatitis (NASH).

Clinical studies suggest that non-alcoholic steatohepatitis (NASH) is an independent risk factor for chronic kidney disease (CKD), but causality and mechanisms linking these two major diseases are lacking. To assess whether NASH can induce CKD, we have characterized kidney function, histological features, transcriptomic and lipidomic profiles in a well-validated murine NASH model. Mice with NASH progressively developed significant podocyte foot process effacement, proteinuria, glomerulosclerosis, tubular epithelial cell injury, lipid accumulation, and interstitial fibrosis. The progression of kidney fibrosis paralleled the severity of the histologic NASH-activity score. Significantly, we confirmed the causal link between NASH and CKD by orthotopic liver transplantation, which attenuated proteinuria, kidney dysfunction, and fibrosis compared with control sham operated mice. Transcriptomic analysis of mouse kidney cortices revealed differentially expressed genes that were highly enriched in mitochondrial dysfunction, lipid metabolic process, and insulin signaling pathways in NASH-induced CKD. Lipidomic analysis of kidney cortices further revealed that phospholipids and sphingolipids were the most significantly changed lipid species. Notably, we found similar kidney histological changes in human NASH and CKD. Thus, our results confirm a causative role of NASH in the development of CKD, reveal potential pathophysiologic mechanisms of NASH-induced kidney injury, and established a valuable model to study the pathogenesis of NASH-associated CKD. This is an important feature of fatty liver disease that has been largely overlooked but has clinical and prognostic importance.

Cure Glomerulonephropathy Pathology Classification and Core Scoring Criteria, Reproducibility, and Clinicopathologic Correlations.

Introduction: Cure Glomerulonephropathy (CureGN) is an observational cohort study of patients with minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN), or IgA nephropathy. We developed a conventional, consensus-based scoring system to document pathologic features for application across multiple pathologists and herein describe the protocol, reproducibility, and correlation with clinical parameters at biopsy. Methods: Definitions were established for glomerular, tubular, interstitial, and vascular lesions evaluated semiquantitatively using digitized light microscopy slides and electron micrographs, and reported immunofluorescence. Cases with curated pathology materials as of April 2019 were scored by a randomly assigned pathologist, with at least 10% of cases scored by a second pathologist. Scoring reproducibility was assessed using Gwet's agreement coefficient (AC)1 statistic and correlations with clinical variables were performed. Results: Of 800 scored biopsies (134 MCD, 194 FSGS, 206 MN, 266 IgA), 94 were scored twice (11.8%). Of 60 pathology features, 46 (76.7%) demonstrated excellent (AC1>0.8), and 12 (20.0%) had good (AC1 0.6-0.8) reproducibility. Mesangial hypercellularity scored as absent, focal, or diffuse had moderate reproducibility (AC1 = 0.58), but good reproducibility (AC1 = 0.71) when scored as absent or focal versus diffuse. The percent glomeruli scored as no lesions had fair reproducibility (AC1 = 0.34). Strongest correlations between pathologic features and clinical characteristics at biopsy included interstitial inflammation, interstitial fibrosis, and tubular atrophy with estimated glomerular filtration rate, foot process effacement with urine protein/creatinine ratio, and active crescents with hematuria. Conclusions: Most scored pathology features showed excellent reproducibility, demonstrating consistency for these features across multiple pathologists. Correlations between certain pathologic features and expected clinical characteristics show the value of this approach for future studies on clinicopathologic correlations and biomarker discovery.

Immunofluorescence Staining for IgG Subclass: Cause for Discrepancy in the Detection of IgG1.

Introduction: Immunofluorescence (IF) staining for IgG subclasses plays an important role in the classification of kidney disease. However, widely used IgG subclass-specific antibodies are now commercially unavailable. Thus, we compared alternative antibodies for performing IgG subclass staining. Methods: A total of 21 cases were stained by 3 different methods: direct IF using fluorescein isothiocyanate (FITC)-conjugated polyclonal antibodies against IgG1-4 (commercially unavailable method), direct IF using FITC-conjugated monoclonal antibodies (clones HP-6091, 6014, 6050, and 6025), indirect IF using monoclonal antibodies (clones HP-6069, 6002, 6050, and 6025), and FITC-conjugated polyclonal secondary antibody. For cases with discrepancy in IgG1 staining, additional direct IF using FITC-conjugated monoclonal antibody (clone 4E3) was performed. Results: Of 21 cases, 11 (52%) had no staining for IgG1 by direct IF using the clone HP-6091 despite ≥1+ staining by the direct IF using polyclonal antibodies. Similarly, direct IF for IgG1 using the clone 4E3 had negative result in all 10 cases with available tissue. However, indirect IF for IgG1 using the clone HP-6069 had similar staining intensity (within 1 order of magnitude) as direct IF using the polyclonal antibodies (10 of 10). Results of IF for IgG2, IgG3, and IgG4 were similar in most cases. Conclusion: The choice of antibodies influences the result of IgG subclass staining, especially for anti-IgG1 antibodies, in which 2 monoclonal antibodies (HP6091 and 4E3) appear less sensitive. Although this may be due to unaccounted variables and requires confirmation, our results may partially explain the difference in IgG1 staining in the literature and underscore the need for careful validation.

Mayo Clinic Consensus Report on Membranous Nephropathy: Proposal for a Novel Classification.

Membranous nephropathy (MN) is a pattern of injury caused by autoantibodies binding to specific target antigens, with accumulation of immune complexes along the subepithelial region of glomerular basement membranes. The past 20 years have brought revolutionary advances in the understanding of MN, particularly via the discovery of novel target antigens and their respective autoantibodies. These discoveries have challenged the traditional classification of MN into primary and secondary forms. At least 14 target antigens have been identified, accounting for 80%-90% of cases of MN. Many of the forms of MN associated with these novel MN target antigens have distinctive clinical and pathologic phenotypes. The Mayo Clinic consensus report on MN proposes a 2-step classification of MN. The first step, when possible, is identification of the target antigen, based on a multistep algorithm and using a combination of serology, staining of the kidney biopsy tissue by immunofluorescence or immunohistochemistry, and/or mass spectrometry methodology. The second step is the search for a potential underlying disease or associated condition, which is particularly relevant when knowledge of the target antigen is available to direct it. The meeting acknowledges that the resources and equipment required to perform the proposed testing may not be generally available. However, the meeting consensus was that the time has come to adopt an antigen-based classification of MN because this approach will allow for accurate and specific MN diagnosis, with significant implications for patient management and targeted treatment.

Mayo Clinic consensus report on membranous nephropathy: proposal for a novel classification.

Membranous nephropathy (MN) is a pattern of injury caused by autoantibodies binding to specific target antigens, with accumulation of immune complexes along the subepithelial region of glomerular basement membranes. The past 20 years have brought revolutionary advances in the understanding of MN, particularly via the discovery of novel target antigens and their respective autoantibodies. These discoveries have challenged the traditional classification of MN into primary and secondary forms. At least 14 target antigens have been identified, accounting for 80%-90% of cases of MN. Many of the forms of MN associated with these novel MN target antigens have distinctive clinical and pathologic phenotypes. The Mayo Clinic consensus report on MN proposes a 2-step classification of MN. The first step, when possible, is identification of the target antigen, based on a multistep algorithm and using a combination of serology, staining of the kidney biopsy tissue by immunofluorescence or immunohistochemistry, and/or mass spectrometry methodology. The second step is the search for a potential underlying disease or associated condition, which is particularly relevant when knowledge of the target antigen is available to direct it. The meeting acknowledges that the resources and equipment required to perform the proposed testing may not be generally available. However, the meeting consensus was that the time has come to adopt an antigen-based classification of MN because this approach will allow for accurate and specific MN diagnosis, with significant implications for patient management and targeted treatment.

Mouse and human studies support DSTYK loss of function as a low-penetrance and variable expressivity risk factor for congenital urinary tract anomalies.

PURPOSE: Previous work identified rare variants in DSTYK associated with human congenital anomalies of the kidney and urinary tract (CAKUT). Here, we present a series of mouse and human studies to clarify the association, penetrance, and expressivity of DSTYK variants. METHODS: We phenotypically characterized Dstyk knockout mice of 3 separate inbred backgrounds and re-analyzed the original family segregating the DSTYK c.654+1G>A splice-site variant (referred to as "SSV" below). DSTYK loss of function (LOF) and SSVs were annotated in individuals with CAKUT, epilepsy, or amyotrophic lateral sclerosis vs controls. A phenome-wide association study analysis was also performed using United Kingdom Biobank (UKBB) data. RESULTS: Results demonstrate ∼20% to 25% penetrance of obstructive uropathy, at least, in C57BL/6J and FVB/NJ Dstyk-/- mice. Phenotypic penetrance increased to ∼40% in C3H/HeJ mutants, with mild-to-moderate severity. Re-analysis of the original family segregating the rare SSV showed low penetrance (43.8%) and no alternative genetic causes for CAKUT. LOF DSTYK variants burden showed significant excess for CAKUT and epilepsy vs controls and an exploratory phenome-wide association study supported association with neurological disorders. CONCLUSION: These data support causality for DSTYK LOF variants and highlights the need for large-scale sequencing studies (here >200,000 cases) to accurately assess causality for genes and variants to lowly penetrant traits with common population prevalence.

Clinicopathologic Spectrum of Lysozyme-Associated Nephropathy.

Introduction: Lysozyme-associated nephropathy (LyN), a rare cause of kidney injury in patients with chronic myelomonocytic leukemia (CMML), has not been well described to date. We report the clinicopathologic spectrum of LyN from a multi-institutional series. Method: We identified 37 native kidney biopsies with LyN and retrospectively obtained clinicopathologic data. Results: Thirty-seven patients had a median age of 74 years and included 78% males. Their most common presentation was acute kidney injury (AKI) or AKI on chronic kidney disease (CKD) (66%) with median estimated glomerular filtration rate (eGFR) of 21.7 ml/min per 1.73 m2, and proteinuria of 1.7 g. A minority (15%) had partial Fanconi syndrome. Serum lysozyme levels were elevated in all tested. Hematologic disorder (n = 28, 76%) was the most common etiology, including CMML (n = 15), acute myeloid leukemia (n = 5), and myelodysplastic syndrome (MDS) (n = 5). Nonhematologic causes (n = 5, 14%), included metastatic neuroendocrine carcinoma (n = 3), sarcoidosis, and leprosy. Etiology was unknown in 4 (11%). Pathology showed proximal tubulopathy with abundant hypereosinophilic intracytoplasmic inclusions, with characteristic staining pattern by lysozyme immunostain. Mortality was high (8/30). However, among the 22 alive, including 85% treated, 7 had improved kidney function, including 1 who discontinued dialysis and 6 with increase in eGFR >15 ml/min per 1.73 m2 compared with eGFR at the time of biopsy. Conclusion: Increased awareness of the full clinicopathologic spectrum of LyN may lead to prompt diagnosis, earlier treatment, and potentially improved outcome of this rare entity.

Implementation and Feasibility of Clinical Genome Sequencing Embedded Into the Outpatient Nephrology Care for Patients With Proteinuric Kidney Disease.

Introduction: The diagnosis and management of proteinuric kidney diseases such as focal segmental glomerulosclerosis (FSGS) are challenging. Genetics holds the promise to improve clinical decision making for these diseases; however, it is often performed too late to enable timely clinical action and it is not implemented within routine outpatient nephrology visits. Methods: We sought to test the implementation and feasibility of clinical rapid genome sequencing (GS) in guiding decision making in patients with proteinuric kidney disease in real-time and embedded in the outpatient nephrology setting. Results: We enrolled 10 children or young adults with biopsy-proven FSGS (9 cases) or minimal change disease (1 case). The mean age at enrollment was 16.2 years (range 2-30). The workflow did not require referral to external genetics clinics but was conducted entirely during the nephrology standard-of-care appointments. The total turn-around-time from enrollment to return-of-results and clinical decision averaged 21.8 days (12.4 for GS), which is well within a time frame that allows clinically relevant treatment decisions. A monogenic or APOL1-related form of kidney disease was diagnosed in 5 of 10 patients. The genetic findings resulted in a rectified diagnosis in 6 patients. Both positive and negative GS findings determined a change in pharmacological treatment. In 3 patients, the results were instrumental for transplant evaluation, donor selection, and the immunosuppressive treatment. All patients and families received genetic counseling. Conclusion: Clinical GS is feasible and can be implemented in real-time in the outpatient care to help guiding clinical management. Additional studies are needed to confirm the cost-effectiveness and broader utility of clinical GS across the phenotypic and demographic spectrum of kidney diseases.

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

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