Protein N-glycans in Healthy and Sclerotic Glomeruli in Diabetic Kidney Disease.

BACKGROUND: Diabetes is expected to directly impact renal glycosylation, yet to date, there has not been a comprehensive evaluation of alterations in N-glycan composition in the glomeruli of patients with diabetic kidney disease (DKD). METHODS: We used untargeted mass spectrometry imaging to identify N-glycan structures in healthy and sclerotic glomeruli in FFPE sections from needle biopsies of five patients with DKD and three healthy kidney samples. Regional proteomics was performed on glomeruli from additional biopsies from the same patients to compare the abundances of enzymes involved in glycosylation. Secondary analysis of single nuclei transcriptomics (snRNAseq) data was used to inform on transcript levels of glycosylation machinery in different cell types and states. RESULTS: We detected 120 N-glycans, and among them identified twelve of these protein post-translated modifications that were significantly increased in glomeruli. All glomeruli-specific N-glycans contained an N-acetyllactosamine (LacNAc) epitope. Five N-glycan structures were highly discriminant between sclerotic and healthy glomeruli. Sclerotic glomeruli had an additional set of glycans lacking fucose linked to their core, and they did not show tetra-antennary structures that are common in healthy glomeruli. Orthogonal omics analyses revealed lower protein abundance and lower gene expression involved in synthesizing fucosylated and branched N-glycans in sclerotic podocytes. In snRNAseq and regional proteomics analyses, we observed that genes and/or proteins involved in sialylation and LacNAc synthesis were also downregulated in DKD glomeruli, but this alteration remained undetectable by our spatial N-glycomics assay. CONCLUSIONS: Integrative spatial glycomics, proteomics, and transcriptomics revealed protein N-glycosylation characteristic of sclerotic glomeruli in DKD.

Characteristics of Potential and Actual Living Kidney Donors: A Single-center Experience.

BACKGROUND: There are limited data and no national capture of barriers associated with initiating and completing the donation process for potential living kidney donors (LKDs). METHODS: We performed a retrospective analysis of 3001 intake forms completed by prospective LKDs from 2016 to 2019 at a single transplant center. We analyzed data from all potential donors who completed the intake until they became ineligible or withdrew or donation was complete. We used univariate and multivariate models to evaluate independent factors associated with donation at various stages in the donation process. RESULTS: The donation process was deconstructed into 5 steps: intake form, immunologic compatibility testing, clinic evaluation, selection committee review, and donation. The highest percentage of potential donors dropped out after completing the intake form, primarily because of not responding to the follow-up phone call (22.6%). Of 455 potential LKDs that completed immunologic compatibility testing, 36% were ABO or crossmatch incompatible. One-hundred eighty-eight (7.5%) of all LKD applicants reached donation, the majority of whom were White (91.0%) and female (63.8%). CONCLUSIONS: A minority of LKD applicants make it to donation. Our ability to track all potential LKDs from the initial touch point to the transplant center will help us develop interventions to address barriers to a successful donation.

T-cell receptor diversity in minimal change disease in the NEPTUNE study.

BACKGROUND: Minimal change disease (MCD) is the major cause of childhood idiopathic nephrotic syndrome, which is characterized by massive proteinuria and debilitating edema. Proteinuria in MCD is typically rapidly reversible with corticosteroid therapy, but relapses are common, and children often have many adverse events from the repeated courses of immunosuppressive therapy. The pathobiology of MCD remains poorly understood. Prior clinical observations suggest that abnormal T-cell function may play a central role in MCD pathogenesis. Based on these observations, we hypothesized that T-cell responses to specific exposures or antigens lead to a clonal expansion of T-cell subsets, a restriction in the T-cell repertoire, and an elaboration of specific circulating factors that trigger disease onset and relapses. METHODS: To test these hypotheses, we sequenced T-cell receptors in fourteen MCD, four focal segmental glomerulosclerosis (FSGS), and four membranous nephropathy (MN) patients with clinical data and blood samples drawn during active disease and during remission collected by the Nephrotic Syndrome Study Network (NEPTUNE). We calculated several T-cell receptor diversity metrics to assess possible differences between active disease and remission states in paired samples. RESULTS: Median productive clonality did not differ between MCD active disease (0.0083; range: 0.0042, 0.0397) and remission (0.0088; range: 0.0038, 0.0369). We did not identify dominant clonotypes in MCD active disease, and few clonotypes were shared with FSGS and MN patients. CONCLUSIONS: While these data do not support an obvious role of the adaptive immune system T-cells in MCD pathogenesis, further study is warranted given the limited sample size. A higher resolution version of the Graphical abstract is available as Supplementary information.

Essential role of Wtip in mouse development and maintenance of the glomerular filtration barrier.

Wilms' tumor interacting protein (Wtip) has been implicated in cell junction assembly and cell differentiation and interacts with proteins in the podocyte slit diaphragm, where it regulates podocyte phenotype. To define Wtip expression and function in the kidney, we created a Wtip-deleted mouse model using ß-galactosidase-neomycin (ß-geo) gene trap technology. Wtip gene trap mice were embryonic lethal, suggesting additional developmental roles outside kidney function. Using ß-geo heterozygous and normal mice, Wtip expression was identified in the developing kidneys, heart, and eyes. In the kidney, expression was restricted to podocytes, which appeared initially at the capillary loop stage coinciding with terminal podocyte differentiation. Heterozygous mice had an expected lifespan and showed no evidence of proteinuria or glomerular pathology. However, heterozygous mice were more susceptible to glomerular injury than wild-type littermates and developed more significant and prolonged proteinuria in response to lipopolysaccharide or adriamycin. In normal human kidneys, WTIP expression patterns were consistent with observations in mice and were lost in glomeruli concurrent with loss of synaptopodin expression in disease. Mechanistically, we identified the Rho guanine nucleotide exchange factor 12 (ARHGEF12) as a binding partner for WTIP. ARHGEF12 was expressed in human podocytes and formed high-affinity interactions through their LIM- and PDZ-binding domains. Our findings suggest that Wtip is essential for early murine embryonic development and maintaining normal glomerular filtration barrier function, potentially regulating slit diaphragm and foot process function through Rho effector proteins.NEW & NOTEWORTHY This study characterized dynamic expression patterns of Wilms' tumor interacting protein (Wtip) and demonstrates the novel role of Wtip in murine development and maintenance of the glomerular filtration barrier.

Urinary Proteomics Identifies Cathepsin D as a Biomarker of Rapid eGFR Decline in Type 1 Diabetes.

OBJECTIVE: Understanding mechanisms underlying rapid estimated glomerular filtration rate (eGFR) decline is important to predict and treat kidney disease in type 1 diabetes (T1D). RESEARCH DESIGN AND METHODS: We performed a case-control study nested within four T1D cohorts to identify urinary proteins associated with rapid eGFR decline. Case and control subjects were categorized based on eGFR decline ≥3 and <1 mL/min/1.73 m2/year, respectively. We used targeted liquid chromatography-tandem mass spectrometry to measure 38 peptides from 20 proteins implicated in diabetic kidney disease. Significant proteins were investigated in complementary human cohorts and in mouse proximal tubular epithelial cell cultures. RESULTS: The cohort study included 1,270 participants followed a median 8 years. In the discovery set, only cathepsin D peptide and protein were significant on full adjustment for clinical and laboratory variables. In the validation set, associations of cathepsin D with eGFR decline were replicated in minimally adjusted models but lost significance with adjustment for albuminuria. In a meta-analysis with combination of discovery and validation sets, the odds ratio for the association of cathepsin D with rapid eGFR decline was 1.29 per SD (95% CI 1.07-1.55). In complementary human cohorts, urine cathepsin D was associated with tubulointerstitial injury and tubulointerstitial cathepsin D expression was associated with increased cortical interstitial fractional volume. In mouse proximal tubular epithelial cell cultures, advanced glycation end product-BSA increased cathepsin D activity and inflammatory and tubular injury markers, which were further increased with cathepsin D siRNA. CONCLUSIONS: Urine cathepsin D is associated with rapid eGFR decline in T1D and reflects kidney tubulointerstitial injury.

A Participant-Centered Approach to Understanding Risks and Benefits of Participation in Research Informed by the Kidney Precision Medicine Project.

An understanding of the ethical underpinnings of human subjects research that involves some risk to participants without anticipated direct clinical benefit-such as the kidney biopsy procedure as part of the Kidney Precision Medicine Project (KPMP)-requires a critical examination of the risks as well as the diverse set of countervailing potential benefits to participants. This kind of deliberation has been foundational to the development and conduct of the KPMP. Herein, we use illustrative features of this research paradigm to develop a more comprehensive conceptualization of the types of benefits that may be important to research participants, including respecting pluralistic values, supporting the opportunity to act altruistically, and enhancing benefits to a participant's community. This approach may serve as a model to help researchers, ethicists, and regulators to identify opportunities to better respect and support participants in future research that entails some risk to these participants as well as to improve the quality of research for people with kidney disease.

Lack of APOL1 in proximal tubules of normal human kidneys and proteinuric APOL1 transgenic mouse kidneys.

The mechanism of pathogenesis associated with APOL1 polymorphisms and risk for non-diabetic chronic kidney disease (CKD) is not fully understood. Prior studies have minimized a causal role for the circulating APOL1 protein, thus efforts to understand kidney pathogenesis have focused on APOL1 expressed in renal cells. Of the kidney cells reported to express APOL1, the proximal tubule expression patterns are inconsistent in published reports, and whether APOL1 is synthesized by the proximal tubule or possibly APOL1 protein in the blood is filtered and reabsorbed by the proximal tubule remains unclear. Using both protein and mRNA in situ methods, the kidney expression pattern of APOL1 was examined in normal human and APOL1 bacterial artificial chromosome transgenic mice with and without proteinuria. APOL1 protein and mRNA was detected in podocytes and endothelial cells, but not in tubular epithelia. In the setting of proteinuria, plasma APOL1 protein did not appear to be filtered or reabsorbed by the proximal tubule. A side-by-side examination of commercial antibodies used in prior studies suggest the original reports of APOL1 in proximal tubules likely reflects antibody non-specificity. As such, APOL1 expression in podocytes and endothelia should remain the focus for mechanistic studies in the APOL1-mediated kidney diseases.

Apolipoprotein L1 and mechanisms of kidney disease susceptibility.

PURPOSE OF REVIEW: Allelic variants in the gene for apolipoprotein L1 (APOL1), found only in individuals of African ancestry, explain a majority of the excess risk of kidney disease in African Americans. However, a clear understanding how the disease-associated APOL1 variants cause kidney injury and the identity of environmental stressors that trigger the injury process have not been determined. RECENT FINDINGS: Basic mechanistic studies of APOL1 biochemistry and cell biology, bolstered by new antibody reagents and inducible pluripotent stem cell-derived cell systems, have focused on the cytotoxic effect of the risk variants when APOL1 gene expression is induced. Since the APOL1 variants evolved to alter a key protein-protein interaction with the trypanosome serum resistance-associated protein, additional studies have begun to address differences in APOL1 interactions with other proteins expressed in podocytes, including new observations that APOL1 variants may alter podocyte cytoskeleton dynamics. SUMMARY: A unified mechanism of pathogenesis for the various APOL1 nephropathies still remains unclear and controversial. As ongoing studies have consistently implicated the pathogenic gain-of-function effects of the variant proteins, novel therapeutic development inhibiting the synthesis or function of APOL1 proteins is moving toward clinical trials.

Rationale and design of the Kidney Precision Medicine Project.

Chronic kidney disease (CKD) and acute kidney injury (AKI) are common, heterogeneous, and morbid diseases. Mechanistic characterization of CKD and AKI in patients may facilitate a precision-medicine approach to prevention, diagnosis, and treatment. The Kidney Precision Medicine Project aims to ethically and safely obtain kidney biopsies from participants with CKD or AKI, create a reference kidney atlas, and characterize disease subgroups to stratify patients based on molecular features of disease, clinical characteristics, and associated outcomes. An additional aim is to identify critical cells, pathways, and targets for novel therapies and preventive strategies. This project is a multicenter prospective cohort study of adults with CKD or AKI who undergo a protocol kidney biopsy for research purposes. This investigation focuses on kidney diseases that are most prevalent and therefore substantially burden the public health, including CKD attributed to diabetes or hypertension and AKI attributed to ischemic and toxic injuries. Reference kidney tissues (for example, living-donor kidney biopsies) will also be evaluated. Traditional and digital pathology will be combined with transcriptomic, proteomic, and metabolomic analysis of the kidney tissue as well as deep clinical phenotyping for supervised and unsupervised subgroup analysis and systems biology analysis. Participants will be followed prospectively for 10 years to ascertain clinical outcomes. Cell types, locations, and functions will be characterized in health and disease in an open, searchable, online kidney tissue atlas. All data from the Kidney Precision Medicine Project will be made readily available for broad use by scientists, clinicians, and patients.

Podocyte density is reduced in kidney allografts with high-risk APOL1 genotypes at transplantation.

Variants in apolipoprotein L1 (APOL1) gene are associated with nondiabetic kidney diseases in black subjects and reduced kidney transplant graft survival. Living and deceased black kidney donors (n = 107) were genotyped for APOL1 variants. To determine whether allografts from high-risk APOL1 donors have reduced podocyte densities contributing to allograft failure, we morphometrically estimated podocyte number, glomerular volume, and podocyte density. We compared allograft loss and eGFR trajectories stratified by APOL1 high-risk and low-risk genotypes. Demographic characteristics were similar in high-risk (n = 16) and low-risk (n = 91) donors. Podocyte density was significantly lower in high-risk than low-risk donors (108 ± 26 vs 127 ± 40 podocytes/106 um3 , P = .03). Kaplan-Meier graft survival (high-risk 61% vs. low-risk 91%, p-value = 0.049) and multivariable Cox models (hazard ratio = 2.6; 95% CI, 0.9-7.8) revealed higher graft loss in recipients of APOL1 high-risk allografts over 48 months. More rapid eGFR decline was seen in recipients of high-risk APOL1 allografts (P < .001). At 60 months, eGFR was 27 vs. 51 mL/min/1.73 min2 in recipients of APOL1 high-risk vs low-risk kidney allografts, respectively. Kidneys from high-risk APOL1 donors had worse outcomes versus low-risk APOL1 genotypes. Lower podocyte density in kidneys from high-risk APOL1 donors may increase susceptibility to CKD from subsequent stresses in both the recipients and donors.

Modelling kidney disease using ontology: insights from the Kidney Precision Medicine Project.

An important need exists to better understand and stratify kidney disease according to its underlying pathophysiology in order to develop more precise and effective therapeutic agents. National collaborative efforts such as the Kidney Precision Medicine Project are working towards this goal through the collection and integration of large, disparate clinical, biological and imaging data from patients with kidney disease. Ontologies are powerful tools that facilitate these efforts by enabling researchers to organize and make sense of different data elements and the relationships between them. Ontologies are critical to support the types of big data analysis necessary for kidney precision medicine, where heterogeneous clinical, imaging and biopsy data from diverse sources must be combined to define a patient's phenotype. The development of two new ontologies - the Kidney Tissue Atlas Ontology and the Ontology of Precision Medicine and Investigation - will support the creation of the Kidney Tissue Atlas, which aims to provide a comprehensive molecular, cellular and anatomical map of the kidney. These ontologies will improve the annotation of kidney-relevant data, and eventually lead to new definitions of kidney disease in support of precision medicine.

Association of preeclampsia with infant APOL1 genotype in African Americans.

BACKGROUND: Black women in the United States and Africa are at an increased risk for preeclampsia. Allelic variants in the gene for apolipoprotein LI, APOL1, are found only in populations of African ancestry, and have been shown to contribute significant risk for kidney disease. Recent studies suggest these APOL1 variants also may contribute risk for preeclampsia. METHODS: The association of preeclampsia with carriage of APOL1 risk alleles was evaluated in a case-control study of deliveries from black women at a single center in Cleveland, Ohio that included gross and histopathologic evaluations of placental tissues (395 cases and 282 controls). Using logistic regression models, associations between fetal APOL1 genotype and preeclampsia were evaluated using several case definitions based on prematurity and severity of preeclampsia, with uncomplicated term pregnancies as controls. Associations between APOL1 genotype and pathological features were also examined. RESULTS: The infant APOL1 genotype was significantly associated with preeclampsia in a dominant inheritance pattern with odds ratio of 1.41 (P=0.029, 95% CI 1.037, 1.926). Stratifying preeclampsia cases by preterm birth, significant associations were detected for both recessive (O.R.=1.70, P=0.038) and additive (O.R.=1.33, P=0.028) inheritance patterns. APOL1 genotype, however, was not significantly associated with pathological changes or other perinatal observations. CONCLUSIONS: Preeclampsia appears to be another disease associated with APOL1 variants, however, further studies are needed to increase confidence in the mode of inheritance. By understanding the association of APOL1 variants with preeclampsia, genetic screening tests for APOL1 may be useful to predict at-risk pregnancies and targeted interventions may be developed to improve pregnancy outcomes.

Frequency of ClinVar Pathogenic Variants in Chronic Kidney Disease Patients Surveyed for Return of Research Results at a Cleveland Public Hospital.

Return of results is not common in research settings as standards are not yet in place for what to return, how to return, and to whom. As a pioneer of large-scale of return of research results, the Precision Medicine Initiative Cohort now known of All of Us plans to return pharmacogenomic results and variants of clinical significance to its participants starting late 2019. To better understand the local landscape of possibilities regarding return of research results, we assessed the frequency of pathogenic variants and APOL1 renal risk variants in a small diverse cohort of chronic kidney disease patients (CKD) ascertained from a public hospital in Cleveland, Ohio genotyped on the Illumina Infinium MegaEX. Of the 23,720 ClinVar-designated variants directly assayed by the MegaEX, 8,355 (35%) had at least one alternate allele in the 130 participants genotyped. Of these, 18 ClinVar variants deemed pathogenic by multiple submitters with no conflicts in interpretation were distributed across 27 participants. The majority of these pathogenic ClinVar variants (14/18) were associated with autosomal recessive disorders. Of note were four African American carriers of TTR rs76992529 associated with amyloidogenic transthyretin amyloidosis, otherwise known as familial transthyretin amyloidosis (FTA). FTA, an autosomal dominant disorder with variable penetrance, is more common among African-descent populations compared with European-descent populations. Also common in this CKD population were APOL1 renal risk alleles G1 (rs73885319) and G2 (rs71785313) with 60% of the study population carrying at least one renal risk allele. Both pathogenic ClinVar variants and APOL1 renal risk alleles were distributed among participants who wanted actionable genetic results returned, wanted genetic results returned regardless of actionability, and wanted no results returned. Results from this local genetic study highlight challenges in which variants to report, how to interpret them, and the participant's potential for follow-up, only some of the challenges in return of research results likely facing larger studies such as All of Us.

APOL1-G0 protects podocytes in a mouse model of HIV-associated nephropathy.

African polymorphisms in the gene for Apolipoprotein L1 (APOL1) confer a survival advantage against lethal trypanosomiasis but also an increased risk for several chronic kidney diseases (CKD) including HIV-associated nephropathy (HIVAN). APOL1 is expressed in renal cells, however, the pathogenic events that lead to renal cell damage and kidney disease are not fully understood. The podocyte function of APOL1-G0 versus APOL1-G2 in the setting of a known disease stressor was assessed using transgenic mouse models. Transgene expression, survival, renal pathology and function, and podocyte density were assessed in an intercross of a mouse model of HIVAN (Tg26) with two mouse models that express either APOL1-G0 or APOL1-G2 in podocytes. Mice that expressed HIV genes developed heavy proteinuria and glomerulosclerosis, and had significant losses in podocyte numbers and reductions in podocyte densities. Mice that co-expressed APOL1-G0 and HIV had preserved podocyte numbers and densities, with fewer morphologic manifestations typical of HIVAN pathology. Podocyte losses and pathology in mice co-expressing APOL1-G2 and HIV were not significantly different from mice expressing only HIV. Podocyte hypertrophy, a known compensatory event to stress, was increased in the mice co-expressing HIV and APOL1-G0, but absent in the mice co-expressing HIV and APOL1-G2. Mortality and renal function tests were not significantly different between groups. APOL1-G0 expressed in podocytes may have a protective function against podocyte loss or injury when exposed to an environmental stressor. This was absent with APOL1-G2 expression, suggesting APOL1-G2 may have lost this protective function.

APOL1 polymorphisms and kidney disease: loss-of-function or gain-of-function?

The mechanism that explains the association of APOL1 variants with nondiabetic kidney diseases in African Americans remains unclear. Kidney disease risk is inherited as a recessive trait, and many studies investigating the intracellular function of APOL1 have indicated the APOL1 variants G1 and G2 are associated with cytotoxicity. Whether cytotoxicity results from the absence of a protective effect conferred by the G0 allele or is induced by a deleterious effect of variant allele expression has not be conclusively established. A central issue hampering basic biology studies is the lack of model systems that authentically replicate APOL1 expression patterns. APOL1 is present in humans and a few other primates and appears to have important functions in the kidney, as the kidney is the primary target for disease associated with the genetic variance. There have been no studies to date assessing the function of untagged APOL1 protein under native expression in human or primate kidney cells, and no studies have examined the heterozygous state, a disease-free condition in humans. A second major issue is the chronic kidney disease (CKD)-associated APOL1 variants are conditional mutations, where the disease-inducing function is only evident under the appropriate environmental stimulus. In addition, it is possible there may be more than one mechanism of pathogenesis that is dependent on the nature of the stressor or other genetic variabilities. Studies addressing the function of APOL1 and how the CKD-associated APOL1 variants cause kidney disease are challenging and remain to be fully investigated under conditions that faithfully model known human genetics and physiology.

Willingness to Participate in a National Precision Medicine Cohort: Attitudes of Chronic Kidney Disease Patients at a Cleveland Public Hospital.

Multiple ongoing, government-funded national efforts longitudinally collect health data and biospecimens for precision medicine research with ascertainment strategies increasingly emphasizing underrepresented groups in biomedical research. We surveyed chronic kidney disease patients from an academic, public integrated tertiary care system in Cleveland, Ohio, to examine local attitudes toward participation in large-scale government-funded studies. Responses (n = 103) indicate the majority (71%) would participate in a hypothetical national precision medicine cohort and were willing to send biospecimens to a national repository and share de-identified data, but <50% of respondents were willing to install a phone app to track personal data. The majority of participants (62%) indicated that return of research results was very important, and the majority (54%) also wanted all of their research-collected health and genetic data returned. Response patterns did not differ by race/ethnicity. Overall, we found high willingness to participate among this Cleveland patient population already participating in a local genetic study. These data suggest that despite common perceptions, subjects from communities traditionally underrepresented in genetic research will participate and agree to store samples and health data in repositories. Furthermore, most participants want return of research results, which will require a plan to provide these data in a secure, accessible, and understandable manner.