Genetic Disorders of the Extracellular Matrix: From Cell and Gene Therapy to Future Applications in Regenerative Medicine.

Metazoans have evolved to produce various types of extracellular matrix (ECM) that provide structural support, cell adhesion, cell-cell communication, and regulated exposure to external cues. Epithelial cells produce and adhere to a specialized sheet-like ECM, the basement membrane, that is critical for cellular homeostasis and tissue integrity. Mesenchymal cells, such as chondrocytes in cartilaginous tissues and keratocytes in the corneal stroma, produce a pericellular matrix that presents optimal levels of growth factors, cytokines, chemokines, and nutrients to the cell and regulates mechanosensory signals through specific cytoskeletal and cell surface receptor interactions. Here, we discuss laminins, collagen types IV and VII, and perlecan, which are major components of these two types of ECM. We examinegenetic defects in these components that cause basement membrane pathologies such as epidermolysis bullosa, Alport syndrome, rare pericellular matrix-related chondrodysplasias, and corneal keratoconus and discuss recent advances in cell and gene therapies being developed for some of these disorders.

Ramipril therapy in integrin α1-null, autosomal recessive Alport mice triples lifespan: mechanistic clues from RNA-seq analysis.

The standard of care for patients with Alport syndrome (AS) is angiotensin-converting enzyme (ACE) inhibitors. In autosomal recessive Alport (ARAS) mice, ACE inhibitors double lifespan. We previously showed that deletion of Itga1 in Alport mice [double-knockout (DKO) mice] increased lifespan by 50%. This effect seemed dependent on the prevention of laminin 211-mediated podocyte injury. Here, we treated DKO mice with vehicle or ramipril starting at 4 weeks of age. Proteinuria and glomerular filtration rates were measured at 5-week intervals. Glomeruli were analyzed for laminin 211 deposition in the glomerular basement membrane (GBM) and GBM ultrastructure was analyzed using transmission electron microscopy (TEM). RNA sequencing (RNA-seq) was performed on isolated glomeruli at all time points and the results were compared with cultured podocytes overlaid (or not) with recombinant laminin 211. Glomerular filtration rate declined in ramipril-treated DKO mice between 30 and 35 weeks. Proteinuria followed these same patterns with normalization of foot process architecture in ramipril-treated DKO mice. RNA-seq revealed a decline in the expression of Foxc2, nephrin (Nphs1), and podocin (Nphs2) mRNAs, which was delayed in the ramipril-treated DKO mice. GBM accumulation of laminin 211 was delayed in ramipril-treated DKO mice, likely due to a role for α1ß1 integrin in CDC42 activation in Alport mesangial cells, which is required for mesangial filopodial invasion of the subendothelial spaces of the glomerular capillary loops. Ramipril synergized with Itga1 knockout, tripling lifespan compared with untreated ARAS mice. © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Collagen IV of basement membranes: II. Emergence of collagen IVα345 enabled the assembly of a compact GBM as an ultrafilter in mammalian kidneys.

The collagen IVα345 (Col-IVα345) scaffold, the major constituent of the glomerular basement membrane (GBM), is a critical component of the kidney glomerular filtration barrier. In Alport syndrome, affecting millions of people worldwide, over two thousand genetic variants occur in the COL4A3, COL4A4, and COL4A5 genes that encode the Col-IVα345 scaffold. Variants cause loss of scaffold, a suprastructure that tethers macromolecules, from the GBM or assembly of a defective scaffold, causing hematuria in nearly all cases, proteinuria, and often progressive kidney failure. How these variants cause proteinuria remains an enigma. In a companion paper, we found that the evolutionary emergence of the COL4A3, COL4A4, COL4A5, and COL4A6 genes coincided with kidney emergence in hagfish and shark and that the COL4A3 and COL4A4 were lost in amphibians. These findings opened an experimental window to gain insights into functionality of the Col-IVα345 scaffold. Here, using tissue staining, biochemical analysis and TEM, we characterized the scaffold chain arrangements and the morphology of the GBM of hagfish, shark, frog, and salamander. We found that α4 and α5 chains in shark GBM and α1 and α5 chains in amphibian GBM are spatially separated. Scaffolds are distinct from one another and from the mammalian Col-IVα345 scaffold, and the GBM morphologies are distinct. Our findings revealed that the evolutionary emergence of the Col-IVα345 scaffold enabled the genesis of a compact GBM that functions as an ultrafilter. Findings shed light on the conundrum, defined decades ago, whether the GBM or slit diaphragm is the primary filter.

Tauroursodeoxycholic acid ameliorates renal injury induced by COL4A3 mutation.

COL4A3/A4/A5 mutations have been identified as critical causes of Alport syndrome and other genetic chronic kidney diseases. However, the underlying pathogenesis remains unclear, and specific treatments are lacking. Here, we constructed a transgenic Alport syndrome mouse model by generating a mutation (Col4a3 p.G799R) identified previously from one large Alport syndrome family into mice. We observed that the mutation caused a pathological decrease in intracellular and secreted collagen IV α3α4α5 heterotrimers. The mutant collagen IV α3 chains abnormally accumulated in the endoplasmic reticulum and exhibited defective secretion, leading to persistent endoplasmic reticulum stress in vivo and in vitro. RNA-seq analysis revealed that the MyD88/p38 MAPK pathway plays key roles in mediating subsequent inflammation and apoptosis signaling activation. Treatment with tauroursodeoxycholic acid, a chemical chaperone drug that functions as an endoplasmic reticulum stress inhibitor, effectively suppressed endoplasmic reticulum stress, promoted secretion of the α3 chains, and inhibited the activation of the MyD88/p38 MAPK pathway. Tauroursodeoxycholic acid treatment significantly improved kidney function in vivo. These results partly clarified the pathogenesis of kidney injuries associated with Alport syndrome, especially in glomeruli, and suggested that tauroursodeoxycholic acid might be useful for the early clinical treatment of Alport syndrome.

Reassuring pregnancy outcomes in women with mild COL4A3-5-related disease (Alport syndrome) and genetic type of disease can aid personalized counseling.

Individualized pre-pregnancy counseling and antenatal care for women with chronic kidney disease (CKD) require disease-specific data. Here, we investigated pregnancy outcomes and long-term kidney function in women with COL4A3-5 related disease (Alport Syndrome, (AS)) in a large multicenter cohort. The ALPART-network (mAternaL and fetal PregnAncy outcomes of women with AlpoRT syndrome), an international collaboration of 17 centers, retrospectively investigated COL4A3-5 related disease pregnancies after the 20th week. Outcomes were stratified per inheritance pattern (X-Linked AS (XLAS)), Autosomal Dominant AS (ADAS), or Autosomal Recessive AS (ARAS)). The influence of pregnancy on estimated glomerular filtration rate (eGFR)-slope was assessed in 192 pregnancies encompassing 116 women (121 with XLAS, 47 with ADAS, and 12 with ARAS). Median eGFR pre-pregnancy was over 90ml/min/1.73m2. Neonatal outcomes were favorable: 100% live births, median gestational age 39.0 weeks and mean birth weight 3135 grams. Gestational hypertension occurred during 23% of pregnancies (reference: 'general' CKD G1-G2 pregnancies incidence is 4-20%) and preeclampsia in 20%. The mean eGFR declined after pregnancy but remained within normal range (over 90ml/min/1.73m2). Pregnancy did not significantly affect eGFR-slope (pre-pregnancy ß=-1.030, post-pregnancy ß=-1.349). ARAS-pregnancies demonstrated less favorable outcomes (early preterm birth incidence 3/11 (27%)). ARAS was a significant independent predictor for lower birth weight and shorter duration of pregnancy, next to the classic predictors (pre-pregnancy kidney function, proteinuria, and chronic hypertension) though missing proteinuria values and the small ARAS-sample hindered analysis. This is the largest study to date on AS and pregnancy with reassuring results for mild AS, though inheritance patterns could be considered in counseling next to classic risk factors. Thus, our findings support personalized reproductive care and highlight the importance of investigating kidney disease-specific pregnancy outcomes.

Clinical Significance of the Cystic Phenotype in Alport Syndrome.

RATIONALE & OBJECTIVE: Alport syndrome (AS) is the most common genetic glomerular disease caused by mutations that affect type IV collagen. However, the clinical characteristics and significance of AS with kidney cysts are not well defined. This study investigated the prevalence and clinical significance of cystic kidney phenotype in AS. STUDY DESIGN: Retrospective cohort study. SETTING & PARTICIPANTS: One hundred-eight patients with AS and a comparison cohort of 79 patients with IgA nephropathy (IgAN). Clinical, genetic, and imaging data were collected from medical records. EXPOSURE: Cystic kidney phenotype evaluated by ultrasonography and defined as the presence of≥3 cysts in each kidney; demographic characteristics and estimated glomerular filtration rate (eGFR) at disease onset. OUTCOME: Cystic kidney phenotype in the AS and IgAN cohorts; time to chronic kidney disease (CKD) stage 3b and longitudinal changes in eGFR in the AS cohort. ANALYTICAL APPROACH: Logistic regression analysis to test independent strengths of associations of clinical/demographic features with the binary outcome of cystic phenotype. Survival analysis for the outcome of reaching CKD stage 3b and linear mixed models for changes in eGFR over time in the AS cohort. RESULTS: We studied 108 patients with AS; 76 (70%) had a genetic diagnosis. Autosomal dominant AS was prevalent, accounting for 68% of patients with a genetic diagnosis. Cystic kidney phenotype was observed in 38% of patients with AS and was associated with normal-sized kidneys in all but 3 patients, who showed increased total kidney volume, mimicking autosomal dominant polycystic kidney disease. The prevalence of cystic kidney phenotype was significantly higher in patients with AS when compared with the group of patients with IgAN (42% vs 19%; P=0.002). Patients with the cystic kidney phenotype were older and had more marked reduction in eGFR than patients without cystic changes. Among patients with AS, the cystic phenotype was associated with older age and a faster decline eGFR. LIMITATIONS: Retrospective, single-center study. CONCLUSIONS: Cystic kidney phenotype is a common finding in AS. The cystic kidney phenotype is a common finding in AS, suggesting a possible role in cystogenesis for the genetic variants that cause this disease. PLAIN-LANGUAGE SUMMARY: Hematuria is the classic renal presentation of Alport syndrome (AS), a hereditary glomerulopathy caused by pathogenic variants of the COL4A3-5 genes. An atypical kidney cystic phenotype has been rarely reported in individuals with these variants. To determine the prevalence of kidney cysts, we performed abdominal ultrasonography in a large group of patients with AS and a comparison group of patients with another glomerular kidney disease, IgA nephropathy (IgAN). Multiple kidney cysts, usually with normal kidney volume, were found in 38% of patients with AS. A few patients' kidney volumes were large enough to mimic a different hereditary cystic kidney disease, autosomal dominant polycystic kidney disease. The overall prevalence of kidney cysts in AS was more than double that observed in the well-matched comparison group with IgAN. These findings emphasize the high prevalence of cystic kidney phenotype in AS, suggesting a likely association between the genetic variants that cause this disease and the development of kidney cysts.

Is there a dominant-negative effect in individuals with heterozygous disease-causing variants in COL4A3/COL4A4?

Alport syndrome (AS) shows a broad phenotypic spectrum ranging from isolated microscopic hematuria (MH) to end-stage kidney disease (ESKD). Monoallelic disease-causing variants in COL4A3/COL4A4 have been associated with autosomal dominant AS (ADAS) and biallelic variants with autosomal recessive AS (ARAS). The aim of this study was to analyze clinical and genetic data regarding a possible genotype-phenotype correlation in individuals with disease-causing variants in COL4A3/COL4A4. Eighty-nine individuals carrying at least one COL4A3/COL4A4 variant classified as (likely) pathogenic according to the American College of Medical Genetics guidelines and current amendments were recruited. Clinical data concerning the prevalence and age of first reported manifestation of MH, proteinuria, ESKD, and extrarenal manifestations were collected. Individuals with monoallelic non-truncating variants reported a significantly higher prevalence and earlier diagnosis of MH and proteinuria than individuals with monoallelic truncating variants. Individuals with biallelic variants were more severely affected than those with monoallelic variants. Those with biallelic truncating variants were more severely affected than those with compound heterozygous non-truncating/truncating variants or individuals with biallelic non-truncating variants. In this study an association of heterozygous non-truncating COL4A3/COL4A4 variants with a more severe phenotype in comparison to truncating variants could be shown indicating a potential dominant-negative effect as an explanation for this observation. The results for individuals with ARAS support the, still scarce, data in the literature.

Cystic phenotype and chronic kidney disease in autosomal dominant Alport syndrome.

BACKGROUND AND HYPOTHESIS: Autosomal Dominant Alport Syndrome (ADAS), also known as Thin Basement Membrane Disease (TBMD), is caused by pathogenic variants in COL4A3 and COL4A4 genes. A cystic phenotype has been described in some patients with TBMD, but no genetic studies were performed. We conducted a genetic and radiologic investigation in a cohort of ADAS patients to analyze the prevalence of multicystic kidney disease (MKD) and its association with Chronic Kidney Disease (CKD). METHODS: Retrospective single-center cohort study. Thirty-one patients showing pathogenic or likely pathogenic variants in COL4A3 or COL4A4 from a cohort of 79 patients with persistent microscopic hematuria were included. Mean follow-up was 9.4±9.6 years. The primary objective of the study was to determine the prevalence of MKD in the cohort of ADAS patients. Secondary objectives were to determine risk factors associated with an eGFR<45 ml/min/1.73m2 at the time of genetic and radiologic evaluation and to investigate the coexistence of other genetic abnormalities associated with familial hematuria and cystic kidney disease. RESULTS: MKD was found in 16 patients (52%). Mean number of cysts per kidney was 12.7±5.5. No genetic abnormalities were found in a panel of 101 other genes related to familial hematuria, focal segmental glomerulosclerosis and cystic kidney disease. A greater number of patients with MKD had an eGFR<45 ml/min/1.73m2 (63% vs 7%, p=0.006) and more advanced CKD than patients without MKD. The annual rate of eGFR decline was greater in patients with MKD: -1.8 vs 0.06 ml/min/1.73m2/year (p=0.009). By multivariable linear regression analysis, the main determinants of eGFR change per year were time-averaged proteinuria (p=0.002) and MKD (p=0.02). CONCLUSION: MKD is commonly found in ADAS and is associated with a worse kidney outcome. No pathogenic variants were found in genes other than COL4A3/COL4A4.

Increased prevalence of Kidney cysts in individuals carrying heterozygous COL4A3 or COL4A4 pathogenic variants.

BACKGROUND: Clinical variability among individuals with heterozygous pathogenic/likely pathogenic (P/LP) variants in the COL4A3/COL4A4 genes (also called autosomal dominant Alport syndrome or COL4A3/COL4A4 related disorder) is huge; many individuals are asymptomatic or show microhematuria, while others may develop proteinuria and chronic kidney disease (CKD). The prevalence of simple kidney cysts (KC) in the general population varies according to age, and patients with advanced CKD are prone to have them. A possible association between heterozygous COL4A3, COL4A4, and COL4A5 P/LP variants and KC has been described in small cohorts. The presence of KC in a multicenter cohort of individuals with heterozygous P/LP variants in the COL4A3/COL4A4 genes is assessed in this study. METHODS: We evaluated the presence of KC by ultrasound in 157 individuals with P/LP variants in COL4A3 (40.7%) or COL4A4 (53.5%) without kidney replacement therapy. The association between presence of KC and age, proteinuria, eGFR, and causative gene was analyzed. Prevalence of KC was compared with historical case series in the general population. RESULTS: Half of the individuals with P/LP variants in COL4A3/COL4A4 showed KC, which is a significantly higher percentage than in the general population. Only 3.8% (6/157) had cystic nephromegaly. Age and eGFR showed an association with the presence of KC (p<0.001). No association was found between KC and proteinuria, sex, or causative gene. CONCLUSIONS: Individuals with COL4A3/COL4A4 P/LP variants are prone to develop KC more frequently than the general population, and their presence is related to age and to eGFR. Neither proteinuria, sex nor the causative gene influences the presence of KC in these individuals.

Dual inhibition of the endothelin and angiotensin receptor ameliorates renal and inner ear pathologies in Alport mice.

Alport syndrome (AS), a type IV collagen disorder, leads to glomerular disease and, in some patients, hearing loss. AS is treated with inhibitors of the renin-angiotensin system; however, a need exists for novel therapies, especially those addressing both major pathologies. Sparsentan is a single-molecule dual endothelin type-A and angiotensin II type 1 receptor antagonist (DEARA) under clinical development for focal segmental glomerulosclerosis and IgA nephropathy. We report the ability of sparsentan to ameliorate both renal and inner ear pathologies in an autosomal-recessive Alport mouse model. Sparsentan significantly delayed onset of glomerulosclerosis, interstitial fibrosis, proteinuria, and glomerular filtration rate decline. Sparsentan attenuated glomerular basement membrane defects, blunted mesangial filopodial invasion into the glomerular capillaries, increased lifespan more than losartan, and lessened changes in profibrotic/pro-inflammatory gene pathways in both the glomerular and the renal cortical compartments. Notably, treatment with sparsentan, but not losartan, prevented accumulation of extracellular matrix in the strial capillary basement membranes in the inner ear and reduced susceptibility to hearing loss. Improvements in lifespan and in renal and strial pathology were observed even when sparsentan was initiated after development of renal pathologies. These findings suggest that sparsentan may address both renal and hearing pathologies in Alport syndrome patients. © 2023 Travere Therapeutics, Inc and The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

An unusual case of nephrotic syndrome.

BACKGROUND: Alport syndrome is a genetically heterogenous disorder resulting from variants in genes coding for alpha-3/4/5 chains of Collagen IV, which results in defective basement membranes in the kidney, cochlea and eye. The syndrome has different inheritance patterns and historically, was thought of as a disease affecting solely males. CASE: A 15-year-old female presented with pedal oedema, hypertension and proteinuria. She underwent a kidney biopsy which showed findings in keeping with focal segmental glomerulosclerosis. Her condition was refractory to steroids. Steroid-resistant nephrotic syndrome genetics were sent, revealing a rare pathogenic variant in the COL4A5 gene. CONCLUSION: Heterozygous females with X-linked Alport syndrome can develop chronic kidney disease and hearing loss. Clinicians should be mindful when reviewing kidney histology to include Alport syndrome as a differential for female patients. COL4A3-5 genes should be included in all steroid-resistant nephrotic syndrome genetic panels.

Ocular manifestations of the genetic causes of focal and segmental glomerulosclerosis.

Genetic forms of focal and segmental glomerulosclerosis (FSGS) often have extra-renal manifestations. This study examined FSGS-associated genes from the Genomics England Renal proteinuria panel for reported and likely ocular features. Thirty-two of the 55 genes (58%) were associated with ocular abnormalities in human disease, and a further 12 (22%) were expressed in the retina or had an eye phenotype in mouse models. The commonest genes affected in congenital nephrotic syndrome (NPHS1, NPHS2, WT1, LAMB2, PAX2 but not PLCE1) may have ocular manifestations . Many genes affected in childhood-adolescent onset FSGS (NPHS1, NPHS2, WT1, LAMB2, SMARCAL1, NUP107 but not TRPC6 or PLCE1) have ocular features. The commonest genes affected in adult-onset FSGS (COL4A3-COL4A5, GLA ) have ocular abnormalities but not the other frequently affected genes (ACTN4, CD2AP, INF2, TRPC6). Common ocular associations of genetic FSGS include cataract, myopia, strabismus, ptosis and retinal atrophy. Mitochondrial forms of FSGS (MELAS, MIDD, Kearn's Sayre disease) are associated with retinal atrophy and inherited retinal degeneration. Some genetic kidney diseases (CAKUT, ciliopathies, tubulopathies) that result in secondary forms of FSGS also have ocular features. Ocular manifestations suggest a genetic basis for FSGS, often help identify the affected gene, and prompt genetic testing. In general, ocular abnormalities require early evaluation by an ophthalmologist, and sometimes, monitoring or treatment to improve vision or prevent visual loss from complications. In addition, the patient should be examined for other syndromic features and first degree family members assessed.

Trimerization profile of type IV collagen COL4A5 exon deletion in X-linked Alport syndrome.

BACKGROUND: Alport syndrome (AS) is a genetic kidney disease caused by a mutation in type IV collagen α3, α4, and α5, which are normally secreted as heterotrimer α345(IV). Nonsense mutation in these genes causes severe AS phenotype. We previously revealed that the exon-skipping approach to remove a nonsense mutation in α5(IV) ameliorated the AS pathology. However, the effect of removing an exon on trimerization is unknown. Here, we assessed the impact of exon deletion on trimerization to evaluate their possible therapeutic applicability and to predict the severity of mutations associated with exon-skipping. METHODS: We produced exon deletion constructs (ΔExon), nonsense, and missense mutants by mutagenesis and evaluated their trimer formation and secretion activities using a nanoluciferase-based assay that we previously developed. RESULTS: Exon-skipping had differential effects on the trimer secretion of α345(IV). Some ΔExons could form and secrete α345(IV) trimers and had higher activity compared with nonsense mutants. Other ΔExons had low secretion activity, especially for those with exon deletion near the C-terminal end although the intracellular trimerization was normal. No difference was noted in the secretion of missense mutants and their ΔExon counterpart. CONCLUSION: Exon skipping is advantageous for nonsense mutants in AS with severe phenotypes and early onset of renal failure but applications may be limited to ΔExons capable of normal trimerization and secretion. This study provides information on α5(IV) exon-skipping for possible therapeutic application and the prediction of the trimer behavior associated with exon-skipping in Alport syndrome.

Complement-mediated thrombotic microangiopathy on a background of Alport syndrome: A case report.

Here we present a case of complement-mediated thrombotic microangiopathy (TMA) in a patient who has a background of Stage 5 chronic kidney disease secondary to Alport syndrome. We explain our approach to the diagnosis of TMA, especially the reliance on non-renal manifestations of TMA and the role of kidney biopsy given there was a background of advanced kidney impairment at baseline.

Exploring the link between Alport syndrome and multiple intracranial artery stenoses: a case report of COL4A5 mutation (118/120 characters).

BACKGROUND: Alport syndrome is a genetic disorder caused by mutations in the COL4A5 gene, which encodes type IV collagen α5 chain, leading to chronic nephritis, hearing loss, and ocular abnormalities. Recent reports suggest this genetic mutation may also increase the risk of cerebral aneurysms and fibromuscular dysplasia, indicating a potential association with vascular vulnerability. CASE PRESENTATION: A 66-year-old woman was admitted with recurrent transient weakness of the left hand, which had gradually worsened in duration over three months. Her medical history included chronic nephritis since childhood. Her two sons had end-stage renal disease and hearing loss since their 20s, and her mother also had chronic kidney disease and hearing loss. One son had a history of traumatic subarachnoid hemorrhage, and the other had spinal epidural hematoma. On admission, she had reduced renal function with proteinuria, acute cerebral infarction in the subcortical white matter of the right fronto-parietal and parieto-occipital lobes, and multiple intracranial arterial stenoses (ICAS), including the right middle and right posterior cerebral artery. Vessel wall imaging of the right middle cerebral artery showed a concentric stenotic pattern. Genetic tests identified a pathogenic missense mutation in exon 24 of COL4A5 (exon 24:c.G1700 >C: p.(Gly567Arg)) that was heterozygous for the patient and hemizygous for her son. She was diagnosed with Alport syndrome. CONCLUSION: It is important to consider Alport syndrome as a possible cause of ICAS in patients with a family history of renal failure or hearing loss and to conduct a genetic analysis of type IV collagen genes. (249/250 words).

[Spontaneous rupture of the anterior lens capsule in Alport syndrome (case study)]. / Samoproizvol'noe vskrytie perednei kapsuly khrustalika pri sindrome Al'porta (klinicheskoe nablyudenie).

Alport syndrome is a hereditary disease characterized by glomerulopathy, manifested by hematuria and/or proteinuria, progressive decline in renal function, often combined with hearing and vision pathology. This article presents a clinical case of spontaneous opening of the anterior lens capsule in a patient with Alport syndrome, accompanied by uveitis and ophthalmic hypertension, and describes the features of the surgical aid and the postoperative period.

Farnesoid X receptor prevents neutrophil extracellular traps via reduced sphingosine-1-phosphate in chronic kidney disease.

Farnesoid X receptor (FXR) activation reduces renal inflammation, but the underlying mechanisms remain elusive. Neutrophil extracellular traps (NETs) are webs of DNA formed when neutrophils undergo specialized programmed cell death (NETosis). The signaling lipid sphingosine-1-phosphate (S1P) stimulates NETosis via its receptor on neutrophils. Here, we identify FXR as a negative regulator of NETosis via repressing S1P signaling. We determined the effects of the FXR agonist obeticholic acid (OCA) in mouse models of adenosine phosphoribosyltransferase (APRT) deficiency and Alport syndrome, both genetic disorders that cause chronic kidney disease. Renal FXR activity is greatly reduced in both models, and FXR agonism reduces disease severity. Renal NETosis and sphingosine kinase 1 (Sphk1) expression are increased in diseased mice, and they are reduced by OCA in both models. Genetic deletion of FXR increases Sphk1 expression, and Sphk1 expression correlates with NETosis. Importantly, kidney S1P levels in Alport mice are two-fold higher than controls, and FXR agonism restores them back to baseline. Short-term inhibition of sphingosine synthesis in Alport mice with severe kidney disease reverses NETosis, establishing a causal relationship between S1P signaling and renal NETosis. Finally, extensive NETosis is present in human Alport kidney biopsies (six male, nine female), and NETosis severity correlates with clinical markers of kidney disease. This suggests the potential clinical relevance of the newly identified FXR-S1P-NETosis pathway. In summary, FXR agonism represses kidney Sphk1 expression. This inhibits renal S1P signaling, thereby reducing neutrophilic inflammation and NETosis.NEW & NOTEWORTHY Many preclinical studies have shown that the farnesoid X receptor (FXR) reduces renal inflammation, but the mechanism is poorly understood. This report identifies FXR as a novel regulator of neutrophilic inflammation and NETosis via the inhibition of sphingosine-1-phosphate signaling. Additionally, NETosis severity in human Alport kidney biopsies correlates with clinical markers of kidney disease. A better understanding of this signaling axis may lead to novel treatments that prevent renal inflammation and chronic kidney disease.

A wave of deep intronic mutations in X-linked Alport syndrome.

X-linked Alport syndrome (XLAS) is an inherited kidney disease caused exclusively by pathogenic variants in the COL4A5 gene. In 10-20% of cases, DNA sequencing of COL4A5 exons or flanking regions cannot identify molecular causes. Here, our objective was to use a transcriptomic approach to identify causative events in a group of 19 patients with XLAS without identified mutation by Alport gene panel sequencing. Bulk RNAseq and/or targeted RNAseq using a capture panel of kidney genes was performed. Alternative splicing events were compared to those of 15 controls by a developed bioinformatic score. When using targeted RNAseq, COL4A5 coverage was found to be 23-fold higher than with bulk RNASeq and revealed 30 significant alternative splicing events in 17 of the 19 patients. After computational scoring, a pathogenic transcript was found in all patients. A causative variant affecting COL4A5 splicing and absent in the general population was identified in all cases. Altogether, we developed a simple and robust method for identification of aberrant transcripts due to pathogenic deep-intronic COL4A5 variants. Thus, these variants, potentially targetable by specific antisense oligonucleotide therapies, were found in a high percentage of patients with XLAS in whom pathogenic variants were missed by conventional DNA sequencing.

Effectiveness of renin-angiotensin-aldosterone system blockers in patients with Alport syndrome: a systematic review and meta-analysis.

BACKGROUND: Although renin-angiotensin-aldosterone system (RAAS) blockers have been considered the primary treatment for patients with Alport syndrome (AS) for a decade, there is no comprehensive review with evidence-based analysis evaluating the effectiveness of RAAS blockers in AS. METHODS: A systematic review and meta-analysis was performed of published studies that compared outcomes related to disease progression between patients with AS receiving RAAS blockers with those taking non-RAAS treatment. Outcomes were meta-analyzed using the random effects models. Cochrane risk-of-bias, Newcastle-Ottawa Scale and Grading of Recommendations Assessment, Development and Evaluation methodology (GRADE) assessment determined the certainty of evidence. RESULTS: A total of eight studies (1182 patients) were included in the analysis. Overall, the risk of bias was low to moderate. Compared with non-RAAS treatment, RAAS blockers could reduce the rate of progression to end-stage kidney disease (ESKD) [four studies; hazard ratio (HR) 0.33, 95% confidence interval (CI) 0.24-0.45; moderate certainty evidence]. After stratified by genetic types, a similar benefit was detected: male X-linked AS (XLAS) (HR 0.32, 95% CI 0.22-0.48), autosomal recessive AS (HR 0.25, 95% CI 0.10-0.62), female XLAS and autosomal dominant AS (HR 0.40, 95% CI 0.21-0.75). In addition, RAAS blockers showed a clear gradient of benefit depending on the stage of disease at the initiation of treatment. CONCLUSION: This meta-analysis suggested that RAAS blockers could be considered as a specific therapy to delay of ESKD for AS with any genetic type, especially at the early stage of the disease, and every further more-effective therapy would be advised to be applied on top of this standard of care.

Glomerular basement membrane deposition of collagen α1(III) in Alport glomeruli by mesangial filopodia injures podocytes via aberrant signaling through DDR1 and integrin α2ß1.

In Alport mice, activation of the endothelin A receptor (ETA R) in mesangial cells results in sub-endothelial invasion of glomerular capillaries by mesangial filopodia. Filopodia deposit mesangial matrix in the glomerular basement membrane (GBM), including laminin 211 which activates NF-κB, resulting in induction of inflammatory cytokines. Herein we show that collagen α1(III) is also deposited in the GBM. Collagen α1(III) localized to the mesangium in wild-type mice and was found in both the mesangium and the GBM in Alport mice. We show that collagen α1(III) activates discoidin domain receptor family, member 1 (DDR1) receptors both in vitro and in vivo. To elucidate whether collagen α1(III) might cause podocyte injury, cultured murine Alport podocytes were overlaid with recombinant collagen α1(III), or not, for 24 h and RNA was analyzed by RNA sequencing (RNA-seq). These same cells were subjected to siRNA knockdown for integrin α2 or DDR1 and the RNA was analyzed by RNA-seq. Results were validated in vivo using RNA-seq from RNA isolated from wild-type and Alport mouse glomeruli. Numerous genes associated with podocyte injury were up- or down-regulated in both Alport glomeruli and cultured podocytes treated with collagen α1(III), 18 of which have been associated previously with podocyte injury or glomerulonephritis. The data indicate α2ß1 integrin/DDR1 co-receptor signaling as the dominant regulatory mechanism. This may explain earlier studies where deletion of either DDR1 or α2ß1 integrin in Alport mice ameliorates renal pathology. © 2022 Boys Town National Research Hospital. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.