COL4A3
Collagen alpha-3(IV) chain precursor (Goodpasture antigen) [Contains: Tumstatin]
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Familial glomerular hematuria(s) comprise a genetically heterogeneous group of conditions which include Alport Syndrome (AS) and thin basement membrane nephropathy (TBMN). Here we investigated 57 Greek-Cypriot families presenting glomerular microscopic hematuria (GMH), with or without proteinuria or chronic kidney function decline, but excluded classical AS. We specifically searched the COL4A3/A4 genes and identified 8 heterozygous mutations in 16 families (28,1%). Eight non-related families featured the founder mutation COL4A3-p.(G1334E). Renal biopsies from 8 patients showed TBMN and focal segmental glomerulosclerosis (FSGS). Ten patients (11.5%) reached end-stage kidney disease (ESKD) at ages ranging from 37-69-yo (mean 50,1-yo). Next generation sequencing of the patients who progressed to ESKD failed to reveal a second mutation in any of the COL4A3/A4/A5 genes, supporting that true heterozygosity for COL4A3/A4 mutations predisposes to CRF/ESKD. Although this could be viewed as a milder and late-onset form of autosomal dominant AS, we had no evidence of ultrastructural features or extrarenal manifestations that would justify this diagnosis. Functional studies in cultured podocytes transfected with wild type or mutant COL4A3 chains showed retention of mutant collagens and differential activation of the unfolded protein response (UPR) cascade. This signifies the potential role of the UPR cascade in modulating the final phenotype in patients with collagen IV nephropathies.
MeSH Terms
- Adult
- Aged
- Aging
- Autoantigens
- Base Sequence
- Cell Line
- Collagen Type IV
- Female
- Glomerular Basement Membrane
- Glomerulosclerosis, Focal Segmental
- Hematuria
- High-Throughput Nucleotide Sequencing
- Humans
- Kidney Failure, Chronic
- Male
- Middle Aged
- Mutation
- Nephritis, Hereditary
- Podocytes
- Sequence Analysis, DNA
- Unfolded Protein Response
Alport syndrome is a hereditary type IV collagen disease leading to progressive renal fibrosis, hearing loss and ocular changes. End stage renal failure usually develops during adolescence. COL4A3-/- mice serve as an animal model for progressive renal scarring in Alport syndrome. The present study evaluates the role of Discoidin Domain Receptor 1 (DDR1) in cell-matrix interaction involved in pathogenesis of Alport syndrome including renal inflammation and fibrosis. DDR1/COL4A3 Double-knockouts were compared to COL4A3-/- mice with 50% or 100% expression of DDR1, wildtype controls and to DDR1-/- COL4A3 / controls for over 6years. Double-knockouts lived 47% longer, mice with 50% DDR1 lived 29% longer and showed improved renal function (reduction in proteinuria and blood urea nitrogen) compared to animals with 100% DDR1 expression. Loss of DDR1 reduced proinflammatory, profibrotic cells via signaling of TGFbeta, CTGF, NFkappaB and IL-6 and decreased deposition of extracellular matrix. Immunogold-staining and in-situ hybridisation identified podocytes as major players in DDR1-mediated fibrosis and inflammation within the kidney. In summary, glomerular epithelial cells (podocytes) express DDR1. Loss of DDR1-expression in the kidney delayed renal fibrosis and inflammation in hereditary type IV collagen disease. This supports our hypothesis that podocyte-matrix interaction via collagen receptors plays an important part in progression of renal fibrosis in Alport disease. The blockade of collagen-receptor DDR1 might serve as an important new therapeutic concept in progressive fibrotic and inflammatory diseases in the future.
MeSH Terms
- Animals
- CD3 Complex
- Collagen Type IV
- Connective Tissue Growth Factor
- Discoidin Domain Receptor 1
- Female
- Fibrosis
- Humans
- Immunohistochemistry
- In Situ Hybridization
- Kidney Glomerulus
- Longevity
- Male
- Mice
- Mice, Inbred ICR
- Mice, Knockout
- Microscopy, Electron
- NF-kappa B
- Nephritis, Hereditary
- Proteinuria
- RNA
- Receptor Protein-Tyrosine Kinases
- Reverse Transcriptase Polymerase Chain Reaction
- Transforming Growth Factor beta
- Urea
A heterozygous mutation in autosomal Alport genes COL4A3 and COL4A4 can be found in 20 to 50% of individuals with familial benign hematuria and diffuse glomerular basement membrane thinning (thin basement membrane nephropathy [TBMN]). Approximately 1% of humans are heterozygous carriers of mutations in the autosomal Alport genes and at risk for developing renal failure as a result of TBMN. The incidence and pathogenesis of renal failure in heterozygous COL4A3/4 mutation carriers is still unclear and was examined further in this study using COL4A3 knockout mice. In heterozygous COL4A3( /-) mice lifespan, hematuria and renal function (serum urea and proteinuria) were monitored during a period of 3 yr, and renal tissue was examined by light and electron microscopy, immunohistochemistry, and Western blot. Lifespan of COL4A3( /-) mice was found to be significantly shorter than in healthy controls (21.7 versus 30.3 mo). Persistent glomerular hematuria was detected starting in week 9; proteinuria of > 0.1 g/L started after 3 mo of life and increased to > 3 g/L after 24 mo. The glomerular basement membrane was significantly thinned (167 versus 200 nm in wild type) in 30-wk-old mice, coinciding with focal glomerulosclerosis, tubulointerstitial fibrosis, and increased levels of TGF-beta and connective tissue growth factor. The renal phenotype in COL4A3( /-) mice resembled the clinical and histopathologic phenotype of human cases of TBMN with concomitant progression to chronic renal failure. Therefore, the COL4A3( /-) mouse model will help in the understanding of the pathogenesis of TBMN in humans and in the evaluation of potential therapies.
MeSH Terms
- Aging
- Animals
- Collagen Type IV
- Disease Models, Animal
- Extracellular Matrix
- Glomerular Basement Membrane
- Glomerulonephritis, Membranous
- Kidney
- Kidney Failure, Chronic
- Longevity
- Mice
- Mice, Transgenic
- Nephritis, Hereditary
- Phenotype
- Transforming Growth Factor beta
- Transforming Growth Factor beta1
Chronic renal disease substantially increases the risk of cardiovascular events and death. Vasopeptidase inhibitors are known to show a strong antihypertensive effect. In the present study, we investigated the nephroprotective potential of the vasopeptidase inhibitor AVE7688 beyond its antihypertensive effects in a mouse model of progressive renal fibrosis. COL4A3 -/- mice received 25 mg AVE7688 per kg body weight. Treatment was initiated in week 4 (early) and week 7 (late). Eight mice per group were sacrificed after 7.5 or 9.5 weeks, and serum levels of urea, systemic blood pressure, and proteinuria were measured. Renal tissue was investigated by routine histology, electron microscopy, immunohistochemistry, and Western blotting. Lifespan until death from renal fibrosis was monitored. Lifespan of treated mice increased by 143% (early therapy) and by 53% (late therapy) compared to untreated animals (172 /- 19 vs. 109 /- 15 vs. 71 /- 6 days, P < 0.01). Untreated COL4A3 -/- mice did not develop severe hypertension (mean systolic blood pressure 116 /- 14 vs. 111 /- 9 mm Hg in wild-type mice), and both therapies mildly reduced systemic blood pressure (107 /- 13 and 105 /- 14 mm Hg, data not significant). AVE7688 decreased proteinuria from 12 /- 3 g/L in untreated mice to 2 /- 1 g/L (early) and to 4 /- 1 g/L (late therapy, P < 0.05), as well as serum-urea from 247 /- 27 to 57 /- 10 and to 105 /- 20 mmol/L (P < 0.05). Extent of fibrosis, inflammation, and profibrotic cytokines was reduced by AVE7688 therapy. The results indicate a strong nephroprotective effect of the vasopeptidase inhibitor in this animal model of progressive renal fibrosis. Besides the antihypertensive action of AVE7688, its antifibrotic, anti-inflammatory, and antiproteinuric effects demonstrated in the present study may serve as an important therapeutic option for chronic inflammatory and fibrotic diseases in man.
MeSH Terms
- Animals
- Anti-Inflammatory Agents
- Autoantigens
- Blood Pressure
- Collagen Type IV
- Connective Tissue Growth Factor
- Disease Models, Animal
- Extracellular Matrix
- Fibrosis
- Heterocyclic Compounds, 3-Ring
- Hypertension, Renal
- Immediate-Early Proteins
- Intercellular Signaling Peptides and Proteins
- Life Expectancy
- Mice
- Mice, Inbred Strains
- Mice, Mutant Strains
- Nephritis, Hereditary
- Prodrugs
- Protease Inhibitors
- Proteinuria
- Transforming Growth Factor beta
- Transforming Growth Factor beta1
Little is known about mechanisms regulating gene expression for the alpha chains of basement membrane type IV collagen, arranged head-to-head in transcription units COL4A1-COL4A2, COL4A3-COL4A4, and COL4A5-COL4A6, and implicated broadly in genetic diseases. To investigate these mechanisms, we generated transgenic mouse lines bearing 5'-flanking sequences of COL4A5 and COL4A6, cloned upstream of a lacZ reporter gene. A 3.8-kb fragment upstream of COL4A6 directs reporter gene expression in the esophagus, stomach, and duodenum, whereas a 13.8-kb fragment directs expression in the esophagus only. A 10.6-kb fragment upstream of COL4A5 directs expression in the esophagus. Coupled with evidence of long-range conservation between human and mouse non-coding sequences, described herein, our findings provide the first indication that highly specialized patterns characteristic of COL4A5-COL4A6 expression in vivo arise from effects of distributed cis-acting regulatory elements on a bidirectional proximal promoter, itself transcriptionally competent.
MeSH Terms
- Aging
- Amino Acid Sequence
- Animals
- Collagen Type IV
- Gene Expression Regulation
- Genes, Regulator
- Humans
- Mice
- Mice, Transgenic
- Molecular Sequence Data
- Organ Specificity
- Protein Subunits
- Sequence Homology
- Species Specificity
- Tissue Distribution
- Transcriptional Activation
- Upper Gastrointestinal Tract
Alport syndrome (AS) is a common hereditary cause of end-stage renal failure in adolescence due to defects in type IV collagen genes. Molecular genetics allows early diagnosis, however, no preventive strategy can be offered. Using the COL4A3 -/- mouse, an animal model for human AS, we evaluated therapy with ramipril in mice. One hundred and twenty-two Alport-mice were treated with 10 mg/kg/day ramipril added to drinking water. Proteinuria, serum-urea and lifespan were monitored. Renal matrix was characterized by immunohistochemistry, light- and electron microscopy, and Western blot. Untreated COL4A3 -/- mice died from renal failure after 71 /- 6 days. Early therapy starting at four weeks of age and continuing to death delayed onset and reduced the extent of proteinuria. Uremia was postponed by three weeks in treated animals. Lifespan increased by more than 100% to 150 /- 21 days (P < 0.01). In parallel, decreased deposition of extracellular matrix and lessened interstitial fibrosis as well as reduced amounts of renal transforming growth factor-beta1 (TGF-beta1) could be demonstrated. Late therapy starting at seven weeks decreased proteinuria, however, lifespan did not increase significantly. The results indicate an antiproteinuric and antifibrotic nephroprotective effect of ramipril in COL4A3 -/- mice is mediated by down-regulation of TGF-beta1. This effect in mice is enhanced by initiation of therapy during pre-symptomatic disease. The data in COL4A3 -/- mice as an animal-model for Alport syndrome suggest that ramipril might as well delay renal failure in humans with AS. Early diagnosis and preemptive treatment also may be crucial in humans.
MeSH Terms
- Angiotensin-Converting Enzyme Inhibitors
- Animals
- Autoantigens
- Basement Membrane
- Collagen Type IV
- Disease Models, Animal
- Extracellular Matrix
- Fibrosis
- Kidney
- Kidney Glomerulus
- Longevity
- Mice
- Mice, Knockout
- Nephritis, Hereditary
- Proteinuria
- Ramipril
- Renal Insufficiency
- Transforming Growth Factor beta
- Transforming Growth Factor beta1
- Uremia