Alport Disease Research Articles

Endothelin Inhibitors in Chronic Kidney Disease: New Treatment Prospects.

The endothelin system is reported to play a significant role in glomerular and tubulointerstitial kidney disease. In the kidney, endothelins are produced in mesangial cells and the glomerular basement membrane by the endothelium and podocytes. The endothelin system regulates glomerular function by inducing proliferation, increasing permeability and in effect proteinuria, and stimulating inflammation, tubular fibrosis, and glomerular scarring. Endothelin A receptor antagonists have been proven to delay the progression of chronic kidney disease and play a protective role in immunoglobulin A nephropathy, focal segmental glomerulosclerosis, and diabetic nephropathy. There are several ongoing research studies with ETAR antagonists in nondiabetic nephropathy, Alport disease, vasculitis and scleroderma nephropathy, which results are promising. Some reports suggest that the endothelin system might contribute to ischemia-reperfusion injury, acute graft rejection and deterioration of graft function. Endothelin inhibition in renal transplantation and its influence on graft survival is the future direction needing further research. The most frequent side effects associated with ETAR antagonists is fluid retention. Additionally, it should be considered if selective ETAR antagonists therapy needs to be co-administered with sodium-glucose co-transporter 2 inhibitors, renin-angiotensin-aldosterone inhibitors or diuretics and which patients should be recruited to such treatment to minimize the risk of adverse outcomes.

#956 Exome sequencing in adults with secondary thrombotic microangiopathy with kidney involvement

Abstract Background and Aims Currently, genetic testing is recommended in patients with thrombotic microangiopathy (TMA) and renal involvement when a complement-dependent TMA is clinically suspected. Due to a possibly poor phenotype-genotype correlation, this strategy may be suboptimal. We aimed to assess the impact of a strategy using Exome Sequencing (ES) regardless of initial suspicion. Method We conducted a prospective observational study. Data was collected in all consecutive adult patients who received a primary diagnostic ES test as part of routine care for unexplained chronic kidney disease between October 10, 2017, and December 31, 2022, at the Nephrology Department of Assistance Publique-Hôpitaux de Paris/Sorbonne University, Paris, France (Tenon and Pitié-Salpêtrière hospitals). Patients with thrombotic thrombocytopenic purpura or typical hemolytic and uremic syndrome were not included. The association between patients’ phenotypes and genotypes was explored using a validated method relying on Term Frequency-Inverse Document Frequency (TF-IDF). Results 1 413 patients underwent ES, of whom 153 (11%) had biological and/or histological indicators of TMA. ES identified a variant consistent with the renal phenotype in 20/153 (13%) cases. In 13/20 cases (65%), a genetic disorder which was not complement TMA was diagnosed: five patients had nephronophthisis (TTC21B, n = 4, NPHP3, n = 1); two had Alport's disease (COL4A4 and COL4A3, n = 1); one had Cobalamin-C deficiency (MMACHC); five had other distinct conditions (variants in SOX18, IFT140, WT1, TREX1 and UMOD). Direct clinical implications of performing ES were observed in 35/153 (23%) cases. An interactive web-app was developed to enable in-depth exploration of phenotype-genotype association, and no association between patients’ phenotypes and genotypes were observed. Conclusion First-tier ES in patients with TMA and renal involvement, irrespective of clinical suspicion, revealed diagnoses that would not have been initially obtained according to the currently recommended strategy. In patients with TMA and renal involvement, we did not find an association between patients’ phenotypes and genotypes.

#5399 VONAFEXOR AS A NOVEL THERAPY FOR CHRONIC KIDNEY DISEASE

Abstract Background and Aims Chronic kidney diseases (CKD) are characterized by the progressive loss of renal function. The molecular mechanisms underlying this progression are still poorly characterized and probably multifactorial. Therefore, there is a real need to expand the very limited number of therapies able to slow down the rate of CKD progression. Recent findings indicate that NR1H4, a druggable nuclear receptor, might play an important role in CKD. The aim of this study was to investigate the benefit of Vonafexor, a novel small molecule and specific NR1H4 agonist in acquired and genetic models of CKD. Method Two complementary experimental models of CKD (the 75% excision of total renal mass (Subtotal Nephrectomy; Nx) and the Col4a3−/− mice, a model of Alport disease) were studied. NR1H4 and the expression of some of its downstream targets were evaluated by quantitative RT-PCR, western blot and immunohistochemistry. In the Nx model, mice were treated with Vonafexor by daily oral gavage from 5 to 8 weeks after the surgery. The possible beneficial effect was compared to that of Losartan and other NR1H4 agonists. In the Alport model, mice were treated by Vonafexor from 5 to 8 weeks after birth. In both models, group of mice were sacrificed at 5 weeks to evaluate the extent of renal lesions at baseline. Renal lesions were quantified using image J. Renal inflammation, myofibroblast activation and podocyte number were quantified after specific staining. Human biopsies were also studied. RNAseq was performed using Illumina NovaSeq6000. Results We observed that NR1H4 expression and OSTA and OSTB, two downstream targets of NR1H4, were significantly reduced in remnant kidneys of Nx FVB/N mice compared to sham-operated animals 8 weeks after surgery. Co-localization staining revealed that NR1H4 is mainly expressed in proximal tubules and, to a minor extent, in Henle loops. Renal lesions were already present at 5 weeks in both experimental models. Remarkably, Vonafexor stopped the progression of renal lesions, in terms of glomerulosclerosis and tubular dilatations and, more importantly, induced a regression of interstitial fibrosis 8 weeks after nephrectomy as compared to the 5-week baseline. Lymphocytes and macrophage infiltration, as well as activated myofibroblasts, were also significantly reduced. Similarly, podocyte loss was stopped by Vonafexor treatment. Of note, the beneficial effect of Vonafexor was unique compared to other classes of NR1H4 agonists and also higher than that of Losartan. The same beneficial curative effect was found in Col4a3−/− mice. RT-PCR confirmed the activation of several NR1H4 downstream targets in mice treated with Vonafexor, while RNAseq analysis revealed that Vonafexor impacted the pathological activation of pathways involved in cell cycle, cell signaling and metabolism. These results may be relevant to humans, since NR1H4 expression was significantly reduced in biopsies from patients with CKD of different etiologies as compared to control kidneys. Conclusion Altogether our results identified in Vonafexor a novel key drug to control CKD. This is consistent with recent results obtained in LIVIFY phase 2 clinical trial in which Vonafexor has already demonstrated beneficial effects on eGFR in NASH patients with mild to moderate CKD.

A case of Alport syndrome with pregnancy-related atypical hemolytic uremic syndrome and crescentic glomerulonephritis

Kidney function may be impaired during pregnancy due to various reasons, and the physiological changes of pregnancy may unmask or worsen pre-existing kidney disease. Herein, we report a pregnant patient presenting with nephrotic-range proteinuria. She later developed acute kidney injury and pre-eclampsia. However, hemolytic anemia and thrombocytopenia persisted after delivery, and she was diagnosed with atypical hemolytic uremic syndrome (aHUS). Although hematological abnormalities resolved with eculizumab treatment, her renal functions did not improve. Kidney biopsy showed crescentic glomerulonephritis without thrombotic microangiopathy features. Concurrently, she was evaluated for hearing impairment, and a diagnosis of Alport syndrome was confirmed with genetic testing. Kidney function may worsen in patients with Alport syndrome during pregnancy. However, crescentic glomerulonephritis (GN) is a rare finding in Alport disease. Pauci-immune crescentic GN has been shown to be related to dysregulated activation of the alternative complement pathway, which is also the underlying pathophysiological mechanism in aHUS.

Electron microscopy in nephropathology

Non-neoplastic kidney diseases represent abroad spectrum of diseases. Although their pathogenesis differs, the histological findings may be similar in terms of conventional morphology. Aprecise classification of these diseases is a prerequisite for correct therapy and prognostic assessment. In the diagnostic process, the magnification achieved by electron microscopy is essential and cannot be replaced by any other technique. The most frequent diagnostic questions addressed by ultrastructural studies represent (1)alterations of podocytes (e.g., minimal-change disease), (2)changes of the thickness and structure of the glomerular basement membrane (e.g., diabetic glomerulosclerosis or Alport disease), (3)the presence, characteristics and exact localisation of immune complexes (e.g., membranous glomerulonephritis or lupus nephritis), (4)alterations of endothelial cells and capillaries (e.g., thrombotic microangiopathy) and (5)diseases of the tubular cells (e.g., light-chain nephropathy or toxic effects). Therefore, ultrastructural investigations are-together with conventional microscopy and immunohistochemistry (or immunofluorescence)-an integral part of the so-called triple-diagnostics in routine nephropathology.

FC034: Genomic Approach of the Undetermined Kidney Disease

Abstract BACKGROUND AND AIMS Chronic kidney disease (CKD) is considered as a public health problem. Almost 20%–25% of patients with end-stage kidney disease have been reported with an undetermined kidney disease (UKD) in large national registry. Recent data have shown that the number of monogenic disease-causing variants among patients with CKD is about 20%–30%. Monogenic disease-causing variants are underdiagnosed. We performed whole exome sequencing on patients in our centre with UKD. METHOD All patients gave their written informed consent for genetic testing. Whole exome sequencing by next-generation sequencing was proposed in routine care practice to patient with UKD between January 2019 and December 2021. High and reproducible coverage achieved by our sequencing approach enabled copy-number variation (CNV) analysis. All patients with inconclusive biopsy results were included. Only one patient from the same pedigree was included. We excluded patients with a typical presentation of ADPKD, familial IgA nephropathy, patients with typical presentation of Gitelman or Bartter syndrome or established genetic diagnosis in the family. RESULTS A total of 211 adult patients were included (120 males) with a median age of 48 years (range 37–62 years). Of these, 50 patients were treated with haemodialysis, 34 patients had a kidney transplantation. A total of 156 had self-declared Caucasian. Consanguinity was reported by 12 patients and suspected in 11 patients. Family history of kidney disease was present in 108 patients with both first and second degree affected in 38 patients. Kidney biopsy were inconclusive in 62 patients. Kidney biopsy was impossible in 57 incidents patients with CKD during the study period. A WES was performed first to avoid kidney biopsy in nine patients. A negative genetic testing prior to the WES with gene panel has been performed in 31 patients. We detected 27 monogenic renal disorders in 73 patients with either pathogenic or likely pathogenic variants and 5 copy number variants among 211 patients (37%). The glomerular disease represent the first yielded diagnostic subgroup in which Alport diseases (COL4A3/4/5) represent the dominant diagnostic (COL4A4 n = 18; COL4A3 n = 12; COL4A5 n = 7; TTC21B n = 3; APOL1 = 2; INF2 n = 2; PAX2 n = 2; TRPC6 n = 2; TREX1 n = 1; COQ8B n = 1; NPHS1 n = 1). Tubulointerstitial diseases is the second yielded diagnostic subgroup (UMOD n = 7; NPHP1 n = 2; NPHP3 n = 1; CCL5 n = 1; HNF1B n = 1; CNM4 n = 1; KCNJ1 n = 1). Cystic disease and CAKUT is the third yielded diagnostic subgroup (PKHD1 n = 3; DNAJB11 n = 2; VHL n = 1; PKD1 n = 1; GREB1L n = 1; PBX1 n = 1). In addition, we find two patients with pathogenic complement variation (CFH et C3), one patient with TTR amyloidosis and one patient with familial renal glucosuria (SLC5A2). Six of the nine patients with WES first to avoid renal biopsy, have positive diagnostic (five Alport disease and one APOL1 nephropathy). Interestingly, one of these nine patients had a WES for nephrotic syndrome during type-I diabetes and a dialysis sister with undetermined glomerular disease and consanguineous parents, which detected a known variation in TTC21B gene (P209L) at a heterozygote state that did not explain the nephrotic syndrome. We performed Sanger sequencing for her sister and revealed the same variation at a homozygous state and explain her renal phenotype. Among 57 patients with impossible kidney biopsy, the WES was positive for 18 patients. The diagnostic yield of WES was higher in consanguineous patients (P = 0.04) and familial history of kidney disease (P < 0.0001). The diagnostic yielded reach 60.5% when the patient had family history of renal disease in both first and second degree of the pedigree. CONCLUSION WES identified a molecular diagnosis in almost 40% of patients with UKD in our centre. An exome first may have a high diagnostic yielded and avoid an invasive procedure like kidney biopsy or when it is impossible. WES must be included as a standard of care for patients with UKD.

P0086FOCAL SEGMENTAL GLOMERULOSCLEROSIS AND TROMBOCYTOPENIA WITHOUT BLEEDING DIATHESIS: WHEN GENETICS MATTER

Abstract Background and Aims A 45-year old patient presented to our renal unit because of increased serum creatinine (2 mg/dL), proteinuria (2g/24h collection) and haematuria; platelets were 12000/mcL. His family history was negative. He had already undergone cataract surgery and was wearing a hearing device. Alport syndrome was suspected and kidney biopsy performed. Light microscopy showed focal segmental glomerulosclerosis (FSGS), while immunofluorescence was negative. Electron microscopy was not available. No specific treatment was undertaken and he progressed to end-stage renal disease (ESRD) in 10 years. The patient chose peritoneal dialysis. He was considered at high risk of bleeding but the Tenchkoff catheter was inserted without problems. After 3 months he received kidney transplant and again no pathological bleeding occurred. Method A genetic test was requested using next generation sequencing (NGS). Results A mutation of myosin heavy chain 9 (MYH9) gene which encodes the non-muscle myosin heavy chain-IIA (NMMH-IIA) was found. This mutation is associated with thrombocytopenia and proteinuria; platelets are giant and display normal aggregation, even in those patients who have a very low count. Proteinuria can be mild or in the nephrotic range, with or without microhaematuria. Kidney involvement is highly heterogeneous and leads to ESRD in half of the cases. Light microscopy shows FSGS as the prevalent pattern whereas electron microscopy is characterized by podocyte foot process effacement. In fact, NMMH-IIA is expressed in podocytes and controls cytoskeleton and cell migration. The differential diagnosis with Alport disease is challenging because additional features of MYH9 mutation are sensorineural deafness and pre-senile cataract. For all these reasons, accurate kidney biopsy study, family history and genetics are decisive. Conclusion Genetic testing has a pivotal role in understanding kidney diseases and should be more and more used.

A Case of Anti-Glomerular Basement Membrane Crescentic Glomerulonephritis in a Female Renal Allograft Recipient with Unknown Native Disease: Retrospective Molecular Confirmation of Alport Disease

Anti-Glomerular Basement Membrane (anti-GBM) crescentic glomerulonephritis developing in an allograft is a rare phenomenon. A patient with Alport syndrome receiving a renal transplant is at risk of developing anti-GBM glomerulonephritis, due to the absence of normal COL4α3, COL4α4 and COL4α5 trimer of the collagen network. Two unique challenges with planning kidney transplant in such a patient include- ideal donor selection; and risk of developing anti-GBM nephritis. We report a case of post-transplant anti-GBM crescentic glomerulonephritis in a female recipient with unknown native kidney disease who was diagnosed with Alport disease when she presented with hematuria and proteinuria nearly 2 years postrenal transplant. Allograft outcome in our case was unfavourable, patient reaching end-stage kidney disease within 6-month of diagnosis. The patient remains on continuous ambulatory peritoneal dialysis and currently active on the deceased donor transplant waiting list.

Two Specific Sulfatide Species Are Dysregulated during Renal Development in a Mouse Model of Alport Syndrome.

Alport syndrome is caused by mutations in collagen IV that alter the morphology of renal glomerular basement membrane. Mutations result in proteinuria, tubulointerstitial fibrosis, and renal failure but the pathogenic mechanisms are not fully understood. Using imaging mass spectrometry, we aimed to determine whether the spatial and/or temporal patterns of renal lipids are perturbed during the development of Alport syndrome in the mouse model. Our results show that most sulfatides are present at similar levels in both the wild-type (WT) and the Alport kidneys, with the exception of two specific sulfatide species, SulfoHex-Cer(d18:2/24:0) and SulfoHex-Cer(d18:2/16:0). In the Alport but not in WT kidneys, the levels of these species mirror the previously described abnormal laminin expression in Alport syndrome. The presence of these sulfatides in renal tubules but not in glomeruli suggests that this specific aberrant lipid pattern may be related to the development of tubulointerstitial fibrosis in Alport disease.

Contribution of Electron Microscopy to the Clinicopathologic Diagnosis in Childhood Glomerular Renal Diseases

Background: Electron microscopy (EM) provides another diagnostic assessment of glomerular lesions in addition to light and fluorescent microscopy. Objectives: We evaluated the contribution of diagnostic EM in childhood glomerular diseases. Patients and methods: Forty-eight renal biopsies which were assessed by EM between 2000 and 2014 were evaluated. Results: There were 21 (44%) females and 27 (56%) males, ages ranged between 6 and 204 months. EM findings were compatible with light and immunofluorescence microscopy in 65%, made additional contributions to diagnosis in 31% (especially in focal segmental glomerulosclerosis, Alport disease, membranoproliferative glomerulonephritis, dense deposit disease, thin basement membrane disease, and nephronophthisis), and was non-contributory in 4%. Conclusion: Electron microscopic evaluation supports other histopathological diagnoses in most cases, contributes additional diagnostic information in pediatric glomerular disease, especially in FSGS, thin glomerular basement membrane nephropathy, Alport disease, MPGN, and dense deposit disease, and its utilization should clinically justify the increase in cost and testing time.

Development of the Kidney Glomerular Filtration Barrier

The kidney glomerular capillary wall filters blood plasma, yielding a dilute filtrate that is subsequently modified and concentrated by the nephron tubular epithelium during the process of urine formation. The glomerular filtration barrier itself consists of: (a) an inner fenestrated vascular endothelium; (b) outer epithelial podocyte layer with regular, interdigitating cytoplasmic extensions (foot processes) and associated slit diaphragms; and (c) an intervening basement membrane between the endothelial and podocyte layers (glomerular basement membrane [GBM]). This unique three‐layered structure restricts passage of plasma albumin, which, along with larger serum proteins, are retained in the circulation, and only trace amounts are normally found in the proximal filtrate.Exactly how the filtration barrier operates is debated, but damage to any one layer results in loss of albumin and larger proteins into the urine (proteinuria), and, ultimately, can cause renal failure. Our work has focused on examining morphogenesis of the glomerulus and assembly of the GBM. To address these questions, and among other approaches, we grafted embryonic‐day 12 transgenic mouse kidneys bearing transcriptional reporters into newborn wildtype or mutant kidneys (where nephrogenesis is still underway), and examined the resulting hybrid tissues for mutation correction.We and others showed that glomerular endothelial cells derive from angioblasts within metanephroi, podocytes develop from metanephric mesenchyme, and the GBM originates from fusion of subendothelial and sub‐podocyte basement membranes during glomerulogenesis. Further, the GBM of immature glomeruli contains collagen α1α2α1(IV) and laminin α1β1γ1. During glomerular maturation, these GBM isoforms are replaced by collagen α3α4α5(IV) and laminin α5β2γ1. Differentiating endothelial cells and podocytes synthesize collagen α1α2α1(IV) and both laminin isoforms, but collagen α3α4α5(IV) derives solely from maturing podocytes. Although mechanisms for collagen and laminin isoform substitutions are unknown, laminin transitioning is abrupt and precedes that for collagen IV, which occurs slowly. These developmentally distinct timetables suggest that different molecular and/or cellular controls account collagen IV and laminin isoform replacement.Why the GBM undergoes compositional changes during glomerular formation is also uncertain, but this may be important for acquisition of glomerular barrier properties. Patients with mutations to COL4A3, A4, and/or A5, which are genes that encode collagen α3α4α5 (IV), usually develop Alport syndrome, a condition presenting with proteinuria, abnormal GBM composition and GBM delamination, loss of podocyte foot processes, and progressive renal failure. Studies on the Col4a3 knockout mouse, an experimental model of Alport disease, are shedding light on how the GBM informs glomerular barrier development and maintenance.Support or Funding InformationFunds came from NIH grants P01DK065123 and P30GM122731.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Steps on the Alport path to proteinuria

Using a mouse model of Alport disease, Dufek etal. report that endothelial cell-derived endothelin-1 activates mesangial cells, which deposit abnormal laminin isoforms in the Alport glomerular basement membrane. This study extends findings obtained previously by this laboratory implicating mesangial cells in the early pathogenesis of Alport disease. Together with abnormalities in matrix receptor expression, cytoskeletal architecture, and proteinase secretion in podocytes, a clearer picture is emerging on the inception of proteinuria in Alport disease.

V8-05 ROBOTIC KIDNEY TRANSPLANTATION: OUR FIRST CASE

You have accessJournal of UrologyBladder Oncology/Testis/Transplantation/Trauma1 Apr 2016V8-05 ROBOTIC KIDNEY TRANSPLANTATION: OUR FIRST CASE Alberto Breda, Lluís Gausa, Angelo Territo, Ivan Schwartzmann, Oscar Rodríguez, Jorge Caffaratti, Javier Ponce de León, and Humberto Villavicencio Alberto BredaAlberto Breda More articles by this author , Lluís GausaLluís Gausa More articles by this author , Angelo TerritoAngelo Territo More articles by this author , Ivan SchwartzmannIvan Schwartzmann More articles by this author , Oscar RodríguezOscar Rodríguez More articles by this author , Jorge CaffarattiJorge Caffaratti More articles by this author , Javier Ponce de LeónJavier Ponce de León More articles by this author , and Humberto VillavicencioHumberto Villavicencio More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.748AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Kidney transplantation is the preferred treatment for patients with end-stage renal disease. In order to reduce the morbidity of the open surgery, a robotic assisted approach has been recently introduced. According to the literature, the robotic surgery allows the performance of kidney transplantation under optimal operative conditions while maintaining the safety and the functional results of the open approach. Herein, we report our first case of Robotic Assisted Kidney Transplantation (RAKT). METHODS We present the case of a mother donating to her daughter affected by end stage renal disease (ESRD) due to Alport Disease (creatinine: 353 Umol/l; GFR: 13 ml/min per 1.73 m2). RESULTS A RAKT was successfully performed. Surgical time was 120 min with 53 min for vascular suture. The estimated blood loss was < 50 cc. The kidney started to produce urine intra-operatively with a rate of 250 cc/hour, which remained constant over the next hours. During the first postoperative day the patient was ambulating and started oral intake. Pain was minimal, and no analgesia was required after 48 h. Serum creatinine improved progressively to 89 umol/L on postoperative day three. No surgical complications were recorded and the patient was sent home on postoperative day 5. CONCLUSIONS We present our first experience in RAKT from living related donors. We believe that the potential advantages of RAKT are related to the quality of the vascular anastomosis, the possible lower complication rate and the shorter recovery of the recipients. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e772 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Alberto Breda More articles by this author Lluís Gausa More articles by this author Angelo Territo More articles by this author Ivan Schwartzmann More articles by this author Oscar Rodríguez More articles by this author Jorge Caffaratti More articles by this author Javier Ponce de León More articles by this author Humberto Villavicencio More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Podocyturia: A Clue for the Rational Use of Amiloride in Alport Renal Disease.

No specific or efficient treatment exists for Alport syndrome, an X-linked hereditary disease caused by mutations in collagen type IV, a crucial component of the glomerular basement membrane. Kidney failure is usually a major complication of the disease, and patients require renal replacement therapy early in life. Microhematuria and subsequently proteinuria are hallmarks of kidney involvement, which are due to primary basement membrane alterations that mainly cause endothelial thrombosis and podocyte contraction and ulterior irreversible detachment. Commonly drug-based approaches include angiotensin-converting enzyme inhibitors and angiotensin receptor blockers, which are employed to reduce proteinuria and thus retard kidney disease progression and cardiovascular morbidity and mortality. However, as any hereditary disease, it is expressed as early as in the intrauterine life, and usually an index case is helpful to detect family-related cases. As no specific treatment exists, pathophysiologically based approaches are useful. The present case illustrates the reduction rate of urinary podocyte loss and proteinuria after amiloride administration and suggests the molecular pathways involved in Alport renal disease. Finally, podocyturia rather than proteinuria should be considered as an earlier biomarker of kidney involvement and disease progression in Alport disease.

Progression of Alport Kidney Disease in Col4a3 Knock Out Mice Is Independent of Sex or Macrophage Depletion by Clodronate Treatment.

Alport syndrome is a genetic disease of collagen IV (α3, 4, 5) resulting in renal failure. This study was designed to investigate sex-phenotype correlations and evaluate the contribution of macrophage infiltration to disease progression using Col4a3 knock out (Col4a3KO) mice, an established genetic model of autosomal recessive Alport syndrome. No sex differences in the evolution of body mass loss, renal pathology, biomarkers of tubular damage KIM-1 and NGAL, or deterioration of kidney function were observed during the life span of Col4a3KO mice. These findings confirm that, similar to human autosomal recessive Alport syndrome, female and male Col4a3KO mice develop renal failure at the same age and with similar severity. The specific contribution of macrophage infiltration to Alport disease, one of the prominent features of the disease in human and Col4a3KO mice, remains unknown. This study shows that depletion of kidney macrophages in Col4a3KO male mice by administration of clodronate liposomes, prior to clinical onset of disease and throughout the study period, does not protect the mice from renal failure and interstitial fibrosis, nor delay disease progression. These results suggest that therapy targeting macrophage recruitment to kidney is unlikely to be effective as treatment of Alport syndrome.

Collagen (COL4A) mutations are the most frequent mutations underlying adult focal segmental glomerulosclerosis.

Multiple genes underlying focal segmental glomerulosclerosis (FSGS) and/or steroid-resistant nephrotic syndrome (SRNS) have been identified, with the recent inclusion of collagen IV mutations responsible for Alport disease (AD) or thin basement membrane nephropathy (TBMN). We aimed to investigate the distribution of gene mutations in adult patients with primary FSGS/SRNS by targeted next generation sequencing (NGS). Eighty-one adults from 76 families were recruited; 24 families had a history of renal disease. A targeted NGS panel was designed and applied, covering 39 genes implicated in FSGS/SRNS including COL4A3-5. Confirmed pathogenic mutations were found in 10 patients (6 with family history) from 9 families (diagnostic rate 12%). Probably pathogenic mutations were identified in an additional six patients (combined diagnostic rate 20%). Definitely pathogenic mutations were identified in 22% of patients with family history and 10% without. Mutations in COL4A3-5 were present in eight patients from six families, representing 56% of definitely pathogenic mutations, and establishing a diagnosis of AD in six patients and TBMN in two patients. Collagen mutations were identified in 38% of families with familial FSGS, and 3% with sporadic FSGS, with over half the mutations occurring in COL4A5. Patients with collagen mutations were younger at presentation and more likely to have family history, haematuria and glomerular basement membrane abnormalities. We show that collagen IV mutations, including COL4A5, frequently underlie FSGS and should be considered, particularly with a positive family history. Targeted NGS improves diagnostic efficiency by investigating many candidate genes in parallel.

A new mutation in the COL4A3 gene responsible for autosomal dominant Alport syndrome, which only generates hearing loss in some carriers

Bilateral sensorineural hearing loss is a characteristic feature of Alport syndrome, which is always linked to renal manifestations so they have a parallel evolution and prognosis, and deafness helps to identify the renal disease. We report a family that suffers an autosomal dominant Alport syndrome caused by a previously undescribed mutation in the COL4A3 gene, in which several members have hearing impairment as the only clinical manifestation, suggesting that in this family deafness can occur independent of renal disease. This mutation is also present in a patient with anterior lenticonus, an observation only found in families with recessive and sex-linked Alport disease.

Laminations and Microgranule Formation in Pediatric Glomerular Basement Membranes

Glomerular basement membrane (GBM) splitting, laminations, and microgranular formation are classically encountered with Alport disease, but can be found in other glomerular diseases. We found moderate to marked GBM laminations/microgranular formations in 51 of 724 (7%) pediatric diagnostic renal biopsies. These included 12 Alport disease, 12 thin basement membrane disease (TBM), 13 mesangial hypercellularity (MH), 6 focal segmental glomerulosclerosis (FSGS), and 8 other diseases. Follow-up demonstrated progression in most of the Alport disease and FSGS, as expected, but also in 40% of TBM and 30% of MH. Basement membrane laminations/microgranular formations are not specific for Alport disease, may represent a non-specific injury, and may herald a progressive clinical course.

Laminin α2-mediated focal adhesion kinase activation triggers Alport glomerular pathogenesis.

It has been known for some time that laminins containing α1 and α2 chains, which are normally restricted to the mesangial matrix, accumulate in the glomerular basement membranes (GBM) of Alport mice, dogs, and humans. We show that laminins containing the α2 chain, but not those containing the α1 chain activates focal adhesion kinase (FAK) on glomerular podocytes in vitro and in vivo. CD151-null mice, which have weakened podocyte adhesion to the GBM rendering these mice more susceptible to biomechanical strain in the glomerulus, also show progressive accumulation of α2 laminins in the GBM, and podocyte FAK activation. Analysis of glomerular mRNA from both models demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction in Alport disease models, as well as the pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured podocytes significantly reduced expression of MMP-9, MMP-10 and IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small molecule inhibitor of FAK activation, ameliorated fibrosis and glomerulosclerosis, significantly reduced proteinuria and blood urea nitrogen levels, and partially restored GBM ultrastructure. Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was significantly reduced in TAE226 treated animals. Collectively, this work identifies laminin α2-mediated FAK activation in podocytes as an important early event in Alport glomerular pathogenesis and suggests that FAK inhibitors, if safe formulations can be developed, might be employed as a novel therapeutic approach for treating Alport renal disease in its early stages.

Dietary phosphate restriction suppresses phosphaturia but does not prevent FGF23 elevation in a mouse model of chronic kidney disease

Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone that in end-stage renal disease is markedly increased in serum; however, the mechanisms responsible for this increase are unclear. Here, we tested whether phosphate retention in chronic kidney disease (CKD) is responsible for the elevation of FGF23 in serum using Col4α3 knockout mice, a murine model of Alport disease exhibiting CKD. We found a significant elevation in serum FGF23 in progressively azotemic 8 and 12 week-old CKD mice along with an increased fractional excretion of phosphorus. Both moderate and severe phosphate restriction reduced fractional excretion of phosphorus by 8 weeks, yet serum FGF23 levels remained strikingly elevated. By 12 weeks, FGF23 levels were further increased with moderate phosphate restriction, while severe phosphate restriction led to severe bone mineralization defects and decreased FGF23 production in bone. CKD mice on a control diet had low serum 1,25(OH)2D levels and 3-fold higher renal Cyp24α1 gene expression compared to wild-type mice. Severe phosphate restriction increased 1,25(OH)2D levels in CKD mice by 8 weeks and lowered renal Cyp24α1 gene expression despite persistently elevated serum FGF23. Renal klotho gene expression declined in CKD mice on a control diet, but improved with severe phosphate restriction. Thus, dietary phosphate restriction reduces the fractional excretion of phosphorus independent of serum FGF23 levels in mice with CKD.