Intravital imaging reveals glomerular capillary distension and endothelial and immune cell activation early in Alport syndrome.

Alport syndrome (AS) is a genetic disorder caused by mutations in type IV collagen that lead to defective glomerular basement membrane, glomerular filtration barrier (GFB) damage, and progressive chronic kidney disease. While the genetic basis of AS is well known, the molecular and cellular mechanistic details of disease pathogenesis have been elusive, hindering the development of mechanism-based therapies. Here, we performed intravital multiphoton imaging of the local kidney tissue microenvironment in a X-linked AS mouse model to directly visualize the major drivers of AS pathology. Severely distended glomerular capillaries and aneurysms were found accompanied by numerous microthrombi, increased glomerular endothelial surface layer (glycocalyx) and immune cell homing, GFB albumin leakage, glomerulosclerosis, and interstitial fibrosis by 5 months of age, with an intermediate phenotype at 2 months. Renal histology in mouse or patient tissues largely failed to detect capillary aberrations. Treatment of AS mice with hyaluronidase or the ACE inhibitor enalapril reduced the excess glomerular endothelial glycocalyx and blocked immune cell homing and GFB albumin leakage. This study identified central roles of glomerular mechanical forces and endothelial and immune cell activation early in AS, which could be therapeutically targeted to reduce mechanical strain and local tissue inflammation and improve kidney function.

A disease-causing variant of COL4A5 in a Chinese family with Alport syndrome: a case series.

BACKGROUND: Alport syndrome (AS), which is a rare hereditary disease caused by mutations of genes including COL4A3, COL4A4 and COL4A5, has a wide spectrum of phenotypes. Most disease-causing variants of AS are located in the exons or the conservative splicing sites of these genes, while little is known about the intronic disease-causing variants. METHODS: A Chinese AS family was recruited in this study. All the clinical data of AS patient were collected from medical records. After pedigree analysis, the pathogenic variants were studied by the whole exome sequencing (WES). Minigene assay and in vivo RT-PCR analysis were performed to validate the functions of the variants. RESULTS: Renal biopsy showed a typical histopathology changes of AS. WES revealed compound heterozygous substitution, NM_033380 c.991-14(IVS17) A > G, in the intron 17 of the COL4A5 gene, which were confirmed by Sanger sequencing. Moreover, the variant was co-segregated with the phenotype in this family. Minigene assay in cultured cell lines showed that a splicing error was induced by this intronic variant, which further confirmed by in vivo RT-PCR analysis. CONCLUSION: A novel intronic disease-causing variant in COL4A5 gene was identified by WES, which was the molecular pathogenic basis of AS.

Optical coherence tomography angiography findings in patients with Alport syndrome / Alterações encontradas na angiografia por tomografia de coerência óptica (OCT-A) em pacientes com síndrome de Alport

ABSTRACT Purpose: To describe the findings on optical coherence tomography angiography associated with Alport syndrome. Methods: Descriptive study from a referral ophthalmology service (Hospital Evangélico de Vila Velha, Brazil). Patients diagnosed with Alport syndrome were included. Results: The study group consisted of four patients (one female and three males) diagnosed with Alport syndrome. Visual acuity in the worst eye was between 20/40 and 20/60. All male patients had anterior lenticonus on biomicroscopy. The observed retinal findings included dots and flecks and pigmentary changes in the macula. On optical coherence tomography angiography, the inner retinal layers of all patients displayed thinning (especially in the temporal quadrant of the macula) and an increase in the foveal avascular zone. A thick choroid was observed in both eyes of the two youngest patients. Conclusions: In patients with Alport syndrome, the inner retinal layers suffer changes due to type IV collagen mutations. Optical coherence tomography angiography makes it possible to visualize and document these findings, making it a useful tool in the detection of early retinal findings associated with Alport syndrome.

RESUMO Objetivos: Descrever os achados na angiografia por tomografia de coerência óptica associada à síndrome de Alport. Métodos: Estudo descritivo de um serviço de referência em Oftalmologia (Hospital Evangélico de Vila Velha, Brasil). Os pacientes diagnosticados com síndrome de Alport, foram incluídos. Resultados: O grupo de estudo foi composto por quatro pacientes (um feminino e três homens) com diagnóstico de síndrome de Alport. A acuidade visual no pior olho estava entre 20/40 a 20/60. Todos os pacientes do sexo masculino apresentaram lenticone anterior à biomiscroscopia. Os achados da retina observados incluíram pontos e manchas e alterações pigmentares na mácula. Na angiotomografia de coerência óptica, as camadas internas da retina de todos os pacientes apresentaram afinamento (especialmente na região temporal da mácula) e aumento da zona avascular foveal. Uma coroide espessa foi observada em ambos os olhos dos dois pacientes mais jovens. Conclusões: Em pacientes com síndrome de Alport, as camadas internas da retina sofrem alterações devido à mutação do colágeno tipo IV. A angiotomografia de coerência óptica permite visualizar esses achados, tornando-o uma ferramenta útil na detecção de achados iniciais da retina associados à síndrome de Alport.

Bidirectional, non-necrotizing glomerular crescents are the critical pathology in X-linked Alport syndrome mouse model harboring nonsense mutation of human COL4A5.

X-linked Alport syndrome (XLAS) is a progressive kidney disease caused by genetic abnormalities of COL4A5. Lack of collagen IV α5 chain staining and "basket-weave" by electron microscopy (EM) in glomerular basement membrane (GBM) are its typical pathology. However, the causal relationship between GBM defects and progressive nephropathy is unknown. We analyzed sequential pathology in a mouse model of XLAS harboring a human nonsense mutation of COL4A5. In mutant mice, nephropathy commenced from focal GBM irregularity by EM at 6 weeks of age, prior to exclusive crescents at 13 weeks of age. Low-vacuum scanning EM demonstrated substantial ragged features in GBM, and crescents were closely associated with fibrinoid exudate, despite lack of GBM break and podocyte depletion at 13 weeks of age. Crescents were derived from two sites by different cellular components. One was CD44 + cells, often with fibrinoid exudate in the urinary space, and the other was accumulation of α-SMA + cells in the thickened Bowman's capsule. These changes finally coalesced, leading to global obliteration. In conclusion, vulnerability of glomerular and capsular barriers to the structural defect in collagen IV may cause non-necrotizing crescents via activation of PECs and migration of interstitial fibroblasts, promoting kidney disease in this model.

Optical coherence tomography angiography findings in patients with Alport syndrome.

PURPOSE: To describe the findings on optical coherence tomography angiography associated with Alport syndrome. METHODS: Descriptive study from a referral ophthalmology service (Hospital Evangélico de Vila Velha, Brazil). Patients diagnosed with Alport syndrome were included. RESULTS: The study group consisted of four patients (one female and three males) diagnosed with Alport syndrome. Visual acuity in the worst eye was between 20/40 and 20/60. All male patients had anterior lenticonus on biomicroscopy. The observed retinal findings included dots and flecks and pigmentary changes in the macula. On optical coherence tomography angiography, the inner retinal layers of all patients displayed thinning (especially in the temporal quadrant of the macula) and an increase in the foveal avascular zone. A thick choroid was observed in both eyes of the two youngest patients. CONCLUSIONS: In patients with Alport syndrome, the inner retinal layers suffer changes due to type IV collagen mutations. Optical coherence tomography angiography makes it possible to visualize and document these findings, making it a useful tool in the detection of early retinal findings associated with Alport syndrome.

Alport-leiomyomatosis syndrome requiring subtotal esophagectomy for refractory gastroesophageal reflux disease after childhood partial esophagogastrectomy: a case report.

Alport-leiomyomatosis syndrome is an extremely rare condition occurring at a young age in which Alport syndrome coexists with diffuse leiomyomatosis of the digestive tract (primarily the esophagus). Most patients with diffuse esophageal leiomyomatosis require esophagectomy of variable extents. A 20-year-old man with Alport-leiomyomatosis syndrome was diagnosed with dysphasia and hematuria in childhood. Although he underwent partial esophagogastrectomy at 8 years of age, extremely severe gastroesophageal reflux symptoms were noted postoperatively. He was diagnosed with refractory severe reflux esophagitis associated with diffuse leiomyomatosis and esophagogastric anastomosis, for which he underwent subtotal esophagectomy, gastric tube reconstruction, and esophagogastric anastomosis in the left neck. The postoperative course was generally good, and he had no postoperative reflux symptoms. To achieve long-term control of symptoms, the lesion must be removed completely; nevertheless, unnecessarily extensive esophagectomy should be avoided.

Phacoemulsification in bilateral anterior lenticonus in Alport syndrome: A case report.

RATIONALE: To report the visual status and results of phacoemulsification cataract surgery in a young patient with Alport syndrome associated with bilateral anterior lenticonus. The milestone of this report is the use of anterior segment optical coherence tomography (AS-OCT) to confirm the central protrusion of the anterior surface of the crystalline lens. PATIENT CONCERNS: A 23-year-old young woman presented with severe progressive visual loss in both eyes, which started several years ago. DIAGNOSES: Refractive status was indicative of high myopia with astigmatism and vision was not improved with optimal correction to better than 0.1 in the right eye and 0.2 in the left eye (visual acuities given in decimal notation). Slit-lamp examination showed transparent cornea, anterior lenticonus and posterior sub-capsular cataract in both eyes. The classical appearance of oil droplet was evident using retro-illumination on the slit lamp. INTERVENTIONS: The natural lenses were replaced with intraocular lens (IOL). OUTCOMES: An excellent refractive status achieved associated with an uncorrected distance visual acuity 0.9 and 0.8 in the right and left eye, respectively. LESSONS: AS-OCT is a valuable device for confirming the budging of the anterior crystalline lens surface.

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.

Detection of Choriocapillaris Loss in Alport Syndrome With Swept-Source OCT Angiography.

A patient previously diagnosed with Alport Syndrome was evaluated using multimodal imaging. Optical coherence tomography (OCT) demonstrated significant thinning of the inner retina within the macula, and inner retinal cysts were found in the peripheral macula. OCT angiography demonstrated loss of the choriocapillaris. Abnormal collagen appears to have multiple deleterious effects on the retinal and choroidal structure and vasculature. [Ophthalmic Surg Lasers Imaging Retina. 2018;49:138-141.].

Three-dimensional electron microscopy reveals the evolution of glomerular barrier injury.

Glomeruli are highly sophisticated filters and glomerular disease is the leading cause of kidney failure. Morphological change in glomerular podocytes and the underlying basement membrane are frequently observed in disease, irrespective of the underlying molecular etiology. Standard electron microscopy techniques have enabled the identification and classification of glomerular diseases based on two-dimensional information, however complex three-dimensional ultrastructural relationships between cells and their extracellular matrix cannot be easily resolved with this approach. We employed serial block face-scanning electron microscopy to investigate Alport syndrome, the commonest monogenic glomerular disease, and compared findings to other genetic mouse models of glomerular disease (Myo1e-/-, Ptpro-/-). These analyses revealed the evolution of basement membrane and cellular defects through the progression of glomerular injury. Specifically we identified sub-podocyte expansions of the basement membrane with both cellular and matrix gene defects and found a corresponding reduction in podocyte foot process number. Furthermore, we discovered novel podocyte protrusions invading into the glomerular basement membrane in disease and these occurred frequently in expanded regions of basement membrane. These findings provide new insights into mechanisms of glomerular barrier dysfunction and suggest that common cell-matrix-adhesion pathways are involved in the progression of disease regardless of the primary insult.

[Analysis of diagnosis and treatment of Alport syndrome].

OBJECTIVE: To investigate the clinical characteristics and the status of diagnosis and treatment of patients with Alport syndrome in China. METHOD: Patients with affirmative diagnosis of Alport syndrome from Department of Pedatrics, Peking University First Hospital in the past 20 years (1995-2015) were analyzed retrospectively. The clinical data including initial symptoms, visit reasons, age at onset of disease, family history, hereditary mode, methods of diagnosis, misdiagnosis and mistreatment were collected. RESULT: A total of 398 patients with Alport syndrome were included in this study, 48.2% of patients had the onset of symptoms before age of 3 years. The rate of onset of symptoms and diagnosis before age of 17 years were 95.7%. The initial symptoms included gross hematuria (37.2%), microscopic hematuria and proteinuria (25.1%), microscopic hematuria (14.8%), edema of eyelid and lower limbs (10.3%), increased foam in urine (4.3%), etc.; 39.5% of patients had no symptoms of urinary tract. Only 14.0% of the patients were diagnosed as Alport syndrome for the first time, and 86.0% of the patients were misdiagnosed. Hormones and immunosuppressive agents were used in 19.0% of patients diagnosed as Alport syndrome, and in 43.0% of patients there was misdiagnosis. Skin biopsy and immunofluorescence of type Ⅳ collagen ɑ5 chain in epithelial basement membrane had a detection rate of 77.8%. Electron microscopy of glomerular basement membrane had a detection rate of 92.6%, and genetic testing 96.6%. The time interval of diagnosis was 18.2 months and was gradually shortened in recent years. CONCLUSION: Alport syndrome developed at a very young age. Hematuria was the most frequent initial symptom. There was a high rate of misdiagnosis and mistreatment for Alport syndrome. Genetic testing for Alport syndrome had advantages of high detection rate, genetic consultation and prenatal diagnosis.

Perimacular changes in Alport's syndrome.

From 1962 to 1977, 79 patients with Alport's syndrome underwent serial fundoscopic examination. In 29 patients (24 males, 5 females), symmetric bilateral perimacular changes were found, consisting of bright, whitish or yellowish dense granulations, surrounding the foveal area. Fluorescein angiography was normal in 8/8 studied cases. All 29 patients had bilateral lenticonus and neural hearing loss; ultrastructural lesion of glomerular basement membrane was present in 11/11 studied patients. Chronic renal failure developed in 26 patients, with 20 treated by maintenance hemodialysis. In contrast, perimacular lesions were absent in the other 50 patients (27 males, 23 females). Anterior lenticonus was detected in none; perceptive deafness was demonstrated in only 20; kidney ultrastructural lesions in 16 of 30 studied cases. Patients with perimacular changes had significantly earlier renal failure (P less than 0.001) than patients without. In conclusion, perimacular changes appear as a simple and reliable indicator of Alport's syndrome, often associated with early renal failure.