Collagen IVα345 dysfunction in glomerular basement membrane diseases. I. Discovery of a COL4A3 variant in familial Goodpasture's and Alport diseases.

Diseases of the glomerular basement membrane (GBM), such as Goodpasture's disease (GP) and Alport syndrome (AS), are a major cause of chronic kidney failure and an unmet medical need. Collagen IVα345 is an important architectural element of the GBM that was discovered in previous research on GP and AS. How this collagen enables GBM to function as a permselective filter and how structural defects cause renal failure remain an enigma. We found a distinctive genetic variant of collagen IVα345 in both a familial GP case and four AS kindreds that provided insights into these mechanisms. The variant is an 8-residue appendage at the C-terminus of the α3 subunit of the α345 hexamer. A knock-in mouse harboring the variant displayed GBM abnormalities and proteinuria. This pathology phenocopied AS, which pinpointed the α345 hexamer as a focal point in GBM function and dysfunction. Crystallography and assembly studies revealed underlying hexamer mechanisms, as described in Boudko et al. and Pedchenko et al. Bioactive sites on the hexamer surface were identified where pathogenic pathways of GP and AS converge and, potentially, that of diabetic nephropathy (DN). We conclude that the hexamer functions include signaling and organizing macromolecular complexes, which enable GBM assembly and function. Therapeutic modulation or replacement of α345 hexamer could therefore be a potential treatment for GBM diseases, and this knock-in mouse model is suitable for developing gene therapies.

Genetic testing and glomerular hematuria-A nephrologist's perspective.

Alport syndrome is an inherited disorder of the kidneys that results from variants in three collagen IV genes-COL4A3, COL4A4, and COL4A5. Early diagnosis and pharmacologic intervention can delay the progression of chronic kidney disease and the onset of kidney failure in patients with Alport syndrome. This article describes the evolution of approaches to the diagnosis and early treatment of Alport syndrome.

Acute kidney injury in pediatric hematopoietic cell transplantation: critical appraisal and consensus.

Hematopoietic cell transplantation (HCT) is a common therapy for the treatment of neoplastic and metabolic disorders, hematological diseases, and fatal immunological deficiencies. HCT can be subcategorized as autologous or allogeneic, with each modality being associated with their own benefits, risks, and post-transplant complications. One of the most common complications includes acute kidney injury (AKI). However, diagnosing HCT patients with AKI early on remains quite difficult. Therefore, this evidence-based guideline, compiled by the Pediatric Continuous Renal Replacement Therapy (PCRRT) working group, presents the various factors that contribute to AKI and recommendations regarding optimization of therapy with minimal complications in HCT patients.

Racial disparities and trends in kidney transplant outcomes in patients with Alport syndrome.

BACKGROUND: Registry data from Europe has shown an increase in age at end-stage kidney disease for patients with Alport syndrome in recent years. Whether a similar delay in transplant age has occurred in the United States for Alport patients across all racial/ethnic groups is unknown. MATERIALS AND METHODS: We used data from the Scientific Registry of Transplant Recipients (SRTR) to identify 3,794 Alport patients transplanted between 12/1987 and 12/2017. We divided the study period into five equal eras to assess temporal trends in age at transplant, graft survival, and patient survival across racial groups using linear regression and Cox regression models. RESULTS: The mean age at transplant for Blacks (28.3 years; difference (Black vs. White): 8.9 years; p < 0.0001) and Hispanics (28.7 years; difference (Hispanics vs. White): 8.7 years; p < 0.0001) was significantly younger compared with that of Whites. We observed a temporal increase in age at transplant for Whites but not for Blacks and Hispanics (p-value for interaction: 0.001). Black recipients were at a higher risk of graft loss (aHR: 1.78; 95% CI: 1.47, 2.15; p < 0.0001) and death (aHR: 1.73; 95% CI: 1.11, 2.69; p = 0.02) compared with White recipients. We observed significant improvements in graft survival with each successive era (p < 0.01). Temporal trends in graft survival (interaction p = 0.46) were not modified by race. CONCLUSION: We found racial disparities in age at transplant and long-term graft survival for patients with Alport syndrome in the United States. The age at transplant increased over time for Whites but not Black and Hispanic patients.

Alport Syndrome: Achieving Early Diagnosis and Treatment.

Alport syndrome is a genetically and phenotypically heterogeneous disorder of glomerular, cochlear, and ocular basement membranes resulting from mutations in the collagen IV genes COL4A3, COL4A4, and COL4A5. Alport syndrome can be transmitted as an X-linked, autosomal recessive, or autosomal dominant disorder. Individuals with Alport syndrome have a significant lifetime risk for kidney failure, as well as sensorineural deafness and ocular abnormalities. The availability of effective intervention for Alport syndrome-related kidney disease makes early diagnosis crucial, but this can be impeded by the genotypic and phenotypic complexity of the disorder. This review presents an approach to enhancing early diagnosis and achieving optimal outcomes.

Clinical practice recommendations for the diagnosis and management of Alport syndrome in children, adolescents, and young adults-an update for 2020.

In 2013, we published a set of clinical practice recommendations for the treatment of Alport syndrome in this journal. We recommended delaying the initiation of angiotensin-converting enzyme inhibition until the onset of overt proteinuria or, in some cases, microalbuminuria. Developments that have occurred over the past 7 years have prompted us to revise these recommendations. We now recommend the initiation of treatment at the time of diagnosis in males with X-linked Alport syndrome and in males and females with autosomal recessive Alport syndrome. We further recommend starting treatment at the onset of microalbuminuria in females with X-linked Alport syndrome and in males and females with autosomal dominant Alport syndrome. This article presents the rationale for these revisions as well as recommendations for diagnostic tactics intended to ensure the early diagnosis of Alport syndrome.

Clinical trial recommendations for potential Alport syndrome therapies.

Alport syndrome is experiencing a remarkable increase in preclinical investigations. To proactively address the needs of the Alport syndrome community, as well as offer clarity for future clinical research sponsors, the Alport Syndrome Foundation hosted a workshop to generate consensus recommendations for prospective trials for conventional drugs. Opinions of key stakeholders were carefully considered, including those of the biopharmaceutical industry representatives, academic researchers, clinicians, regulatory agency representatives, and-most critically-patients with Alport syndrome. Recommendations were established for preclinical researchers, the use and selection of biomarkers, standards of care, clinical trial designs, trial eligibility criteria and outcomes, pediatric trial considerations, and considerations for patient engagement, recruitment, and treatment. This paper outlines their recommendations.

A multicenter, randomized, placebo-controlled, double-blind phase 3 trial with open-arm comparison indicates safety and efficacy of nephroprotective therapy with ramipril in children with Alport's syndrome.

Children with Alport syndrome develop renal failure early in life. Since the safety and efficacy of preemptive nephroprotective therapy are uncertain we conducted a randomized, placebo-controlled, double-blind trial in 14 German sites of pediatric patients with ramipril for three to six years plus six months follow-up to determine these parameters. Pretreated children and those whose parents refused randomization became an open-arm control, which were compared to prospective real-world data from untreated children. The co-primary endpoints were safety (adverse drug reactions) and efficacy (time to progression). Out of 66 oligosymptomatic children, 22 were randomized and 44 joined the open-arm comparison. Ramipril therapy showed no safety issues (total of 216.4 patient-years on ramipril; adverse event rate-ratio 1.00; 95% confidence interval 0.66-1.53). Although not significant, our results cautiously showed that ramipril therapy was effective: in the randomized arm, Ramipril decreased the risk of disease progression by almost half (hazard ratio 0.51 (0.12-2.20)), diminished the slope of albuminuria progression and the decline in glomerular filtration. In adjusted analysis, indications of efficacy were supported by prospective data from participants treated open label compared with untreated children, in whom ramipril again seemed to reduce progression by almost half (0.53 (0.22-1.29)). Incorporating these results into the randomized data by Bayesian evidence synthesis resulted in a more precise estimate of the hazard-ratio of 0.52 (0.19-1.39). Thus, our study shows the safety of early initiation of therapy and supports the hope to slow renal failure by many years, emphasizing the value of preemptive therapy. Hence, screening programs for glomerular hematuria in children and young adults could benefit from inclusion of genetic testing for Alport-related gene-variants.

Alport syndrome: a unified classification of genetic disorders of collagen IV α345: a position paper of the Alport Syndrome Classification Working Group.

Mutations in the genes COL4A3, COL4A4, and COL4A5 affect the synthesis, assembly, deposition, or function of the collagen IV α345 molecule, the major collagenous constituent of the mature mammalian glomerular basement membrane. These mutations are associated with a spectrum of nephropathy, from microscopic hematuria to progressive renal disease leading to ESRD, and with extrarenal manifestations such as sensorineural deafness and ocular anomalies. The existing nomenclature for these conditions is confusing and can delay institution of appropriate nephroprotective therapy. Herein we propose a new classification of genetic disorders of the collagen IV α345 molecule with the goal of improving renal outcomes through regular monitoring and early treatment.

Advances and unmet needs in genetic, basic and clinical science in Alport syndrome: report from the 2015 International Workshop on Alport Syndrome.

Alport syndrome (AS) is a genetic disease characterized by haematuric glomerulopathy variably associated with hearing loss and anterior lenticonus. It is caused by mutations in the COL4A3, COL4A4 or COL4A5 genes encoding the α3α4α5(IV) collagen heterotrimer. AS is rare, but it accounts for >1% of patients receiving renal replacement therapy. Angiotensin-converting enzyme inhibition slows, but does not stop, the progression to renal failure; therefore, there is an urgent requirement to expand and intensify research towards discovering new therapeutic targets and new therapies. The 2015 International Workshop on Alport Syndrome targeted unmet needs in basic science, genetics and diagnosis, clinical research and current clinical care. In three intensive days, more than 100 international experts including physicians, geneticists, researchers from academia and industry, and patient representatives from all over the world participated in panel discussions and breakout groups. This report summarizes the most important priority areas including (i) understanding the crucial role of podocyte protection and regeneration, (ii) targeting mutations by new molecular techniques for new animal models and potential gene therapy, (iii) creating optimal interaction between nephrologists and geneticists for early diagnosis, (iv) establishing standards for mutation screening and databases, (v) improving widespread accessibility to current standards of clinical care, (vi) improving collaboration with the pharmaceutical/biotech industry to investigate new therapies, (vii) research in hearing loss as a huge unmet need in Alport patients and (viii) the need to evaluate the risk and benefit of novel (including 'repurposing') therapies on an international basis.

Acute Kidney Injury and the Risk of Mortality in Children Undergoing Hematopoietic Stem Cell Transplantation.

Acute kidney injury (AKI) is a well-documented complication of pediatric hematopoietic stem cell transplantation (HSCT). Dialysis after HSCT is associated with a lower overall survival (OS); however, the association between less severe AKI and OS is unclear. We retrospectively studied 205 consecutive pediatric HSCT patients to determine the incidence and impact of all stages of AKI on OS in pediatric HSCT recipients. We used the peak pRIFLE grade during the first 100 days to classify AKI (ie, R = risk, I = injury, F = failure, L = loss of function, E = end-stage renal disease) and used the modified Schwartz formula to estimate glomerular filtration rate. AKI was observed in 173 of 205 patients (84%). The 1-year OS rate decreased significantly with an increasing severity of pRIFLE grades (P < .01). There was no difference in the OS between patients without AKI and the R/I group. Regardless of the dialysis status, stages F/L/E had significantly lower rates of OS compared with patients without AKI or R/I (P < .01). There was no difference in OS among patients with dialysis and F/L/E without dialysis (P = .65). Stages F/L/E predicted mortality independent of acute graft-versus-host disease, gender, and malignancy. The OS of children after HSCT decreases significantly with an increasing severity of AKI within the first 100 days post-transplant. Although our data did not show an increased risk of mortality with stages R/I, stages F/L/E predicted mortality regardless of dialysis. Prevention and minimization of AKI may improve survival after pediatric HSCT.

The 2014International Workshop on Alport Syndrome.

Alport syndrome, historically referred to as hereditary glomerulonephritis with sensorineural deafness and anterior lenticonus, is a genetic disease of collagen α3α4α5(IV) resulting in renal failure. The collagen α3α4α5(IV) heterotrimer forms a network that is a major component of the kidney glomerular basement membrane (GBM) and basement membranes in the cochlea and eye. Alport syndrome, estimated to affect 1 in 5000-10,000 individuals, is caused by mutations in any one of the three genes that encode the α chain components of the collagen α3α4α5(IV) heterotrimer: COL4A3, COL4A4, and COL4A5. Although angiotensin-converting enzyme inhibition is effective in Alport syndrome patients for slowing progression to end-stage renal disease, it is neither a cure nor an adequate long-term protector. The 2014 International Workshop on Alport Syndrome, held in Oxford, UK, from January 3-5, was organized by individuals and families living with Alport syndrome, in concert with international experts in the clinical, genetic, and basic science aspects of the disease. Stakeholders from diverse communities-patient families, physicians, geneticists, researchers, Pharma, and funding organizations-were brought together so that they could meet and learn from each other and establish strategies and collaborations for the future, with the overall aim of discovering much needed new treatments to prolong kidney function.

Expert guidelines for the management of Alport syndrome and thin basement membrane nephropathy.

Few prospective, randomized controlled clinical trials address the diagnosis and management of patients with Alport syndrome or thin basement membrane nephropathy. Adult and pediatric nephrologists and geneticists from four continents whose clinical practice focuses on these conditions have developed the following guidelines. The 18 recommendations are based on Level D (Expert opinion without explicit critical appraisal, or based on physiology, bench research, or first principles-National Health Service category) or Level III (Opinions of respected authorities, based on clinical experience, descriptive studies, or reports of expert committees-U.S. Preventive Services Task Force) evidence. The recommendations include the use of genetic testing as the gold standard for the diagnosis of Alport syndrome and the demonstration of its mode of inheritance; the need to identify and follow all affected members of a family with X-linked Alport syndrome, including most mothers of affected males; the treatment of males with X-linked Alport syndrome and individuals with autosomal recessive disease with renin-angiotensin system blockade, possibly even before the onset of proteinuria; discouraging the affected mothers of males with X-linked Alport syndrome from renal donation because of their own risk of kidney failure; and consideration of genetic testing to exclude X-linked Alport syndrome in some individuals with thin basement membrane nephropathy. The authors recognize that as evidence emerges, including data from patient registries, these guidelines will evolve further.

Quaternary epitopes of α345(IV) collagen initiate Alport post-transplant anti-GBM nephritis.

Alport post-transplant nephritis (APTN) is an aggressive form of anti-glomerular basement membrane disease that targets the allograft in transplanted patients with X-linked Alport syndrome. Alloantibodies develop against the NC1 domain of α5(IV) collagen, which occurs in normal kidneys, including renal allografts, forming distinct α345(IV) and α1256(IV) networks. Here, we studied the roles of these networks as antigens inciting alloimmunity and as targets of nephritogenic alloantibodies in APTN. We found that patients with APTN, but not those without nephritis, produce two kinds of alloantibodies against allogeneic collagen IV. Some alloantibodies targeted alloepitopes within α5NC1 monomers, shared by α345NC1 and α1256NC1 hexamers. Other alloantibodies specifically targeted alloepitopes that depended on the quaternary structure of α345NC1 hexamers. In Col4a5-null mice, immunization with native forms of allogeneic collagen IV exclusively elicited antibodies to quaternary α345NC1 alloepitopes, whereas alloimmunogens lacking native quaternary structure elicited antibodies to shared α5NC1 alloepitopes. These results imply that quaternary epitopes within α345NC1 hexamers may initiate alloimmune responses after transplant in X-linked Alport patients. Thus, α345NC1 hexamers are the culprit alloantigen and primary target of all alloantibodies mediating APTN, whereas α1256NC1 hexamers become secondary targets of anti-α5NC1 alloantibodies. Reliable detection of alloantibodies by immunoassays using α345NC1 hexamers may improve outcomes by facilitating early, accurate diagnosis.

Improved survival over the last decade in pediatric patients requiring dialysis after hematopoietic cell transplantation.

It is not unusual for children to require dialysis for fluid and electrolyte management after hematopoietic cell transplantation (HCT). Previous studies have documented high mortality in children who require dialysis after HCT, but recent data are lacking. The purpose of this study was to compare the incidence of dialysis after pediatric HCT and the survival of patients who received dialysis in 2 decades, 1990-1999 and 2000-2009. A total of 1427 patients age <21 years who underwent a first HCT at the University of Minnesota between January 1990 and December 2009 were reviewed using prospectively collected data from the institutional HCT database. The incidence of dialysis during the first 100 days post-HCT and survival at 1 year post-HCT in the 2 cohorts were determined. Comparisons between patients who did and did not require dialysis were made using the χ(2) and Fisher exact tests as appropriate. Kaplan-Meier estimates and 95% confidence intervals for 1-year post-HCT overall survival were reported by dialysis group and compared using the log-rank test. Predictors of overall survival among patients requiring dialysis were assessed using univariate and multivariate Cox regression analyses. The incidence of dialysis was not significantly different in the 2 cohorts (8.2% for 1990-1999 versus 8.9% for 2000-2009; P = .6326). Patients requiring dialysis were significantly more likely to survive to or past 1 year in the 2000-2009 cohort compared with the 1990-1999 cohort (23% versus 11%; P < .0001). Multivariate analyses found that older age at the time of HCT, primary disease type, pulmonary hemorrhage, and HCT in 1990-1999 were associated with increased mortality in the dialyzed population. The use of cyclosporine was associated with increased survival in the patients who received dialysis. Dialysis is an important complication of pediatric HCT with an incidence that has remained constant over the last 2 decades. Survival was improved in the 2000-2009 cohort regardless of dialysis status. Despite a recent significant reduction in mortality in patients requiring dialysis, mortality remains higher in these patients than in those who do not need dialysis.

Molecular architecture of the Goodpasture autoantigen in anti-GBM nephritis.

BACKGROUND: In Goodpasture's disease, circulating autoantibodies bind to the noncollagenous-1 (NC1) domain of type IV collagen in the glomerular basement membrane (GBM). The specificity and molecular architecture of epitopes of tissue-bound autoantibodies are unknown. Alport's post-transplantation nephritis, which is mediated by alloantibodies against the GBM, occurs after kidney transplantation in some patients with Alport's syndrome. We compared the conformations of the antibody epitopes in Goodpasture's disease and Alport's post-transplantation nephritis with the intention of finding clues to the pathogenesis of anti-GBM glomerulonephritis. METHODS: We used an enzyme-linked immunosorbent assay to determine the specificity of circulating autoantibodies and kidney-bound antibodies to NC1 domains. Circulating antibodies were analyzed in 57 patients with Goodpasture's disease, and kidney-bound antibodies were analyzed in 14 patients with Goodpasture's disease and 2 patients with Alport's post-transplantation nephritis. The molecular architecture of key epitope regions was deduced with the use of chimeric molecules and a three-dimensional model of the alpha345NC1 hexamer. RESULTS: In patients with Goodpasture's disease, both autoantibodies to the alpha3NC1 monomer and antibodies to the alpha5NC1 monomer (and fewer to the alpha4NC1 monomer) were bound in the kidneys and lungs, indicating roles for the alpha3NC1 and alpha5NC1 monomers as autoantigens. High antibody titers at diagnosis of anti-GBM disease were associated with ultimate loss of renal function. The antibodies bound to distinct epitopes encompassing region E(A) in the alpha5NC1 monomer and regions E(A) and E(B) in the alpha3NC1 monomer, but they did not bind to the native cross-linked alpha345NC1 hexamer. In contrast, in patients with Alport's post-transplantation nephritis, alloantibodies bound to the E(A) region of the alpha5NC1 subunit in the intact hexamer, and binding decreased on dissociation. CONCLUSIONS: The development of Goodpasture's disease may be considered an autoimmune "conformeropathy" that involves perturbation of the quaternary structure of the alpha345NC1 hexamer, inducing a pathogenic conformational change in the alpha3NC1 and alpha5NC1 subunits, which in turn elicits an autoimmune response. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases.)

Clinical practice recommendations for the treatment of Alport syndrome: a statement of the Alport Syndrome Research Collaborative.

We present clinical practice recommendations for the treatment of children with Alport syndrome who are not enrolled in clinical trials. Our goal is to promote early initiation of a standard therapeutic approach that will facilitate assessment of the safety and efficacy of the protocol. The treatment protocol is based on the reduction of proteinuria, intraglomerular pressure, and renal fibrosis via interference with the renin-angiotensin-aldosterone system.