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Alport syndrome is a hereditary kidney disease caused by collagen IV mutations that interfere with the formation and deposition of the α3α4α5 protomer into the glomerular basement membrane. In this issue, Yu et al. show that the chemical chaperone tauroursodeoxycholic acid prevented kidney structural changes and function decline in mice with a pathogenic missense Col4a3 mutation by increasing mutant α3α4α5 protomer glomerular basement membrane deposition and preventing podocyte apoptosis induced by endoplasmic reticulum stress., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Antibody-mediated autoimmune pathologies like membranous nephropathy are difficult to model, particularly in the absence of local target antigen expression in model organisms such as mice and rats; as is the case for phospholipase A2 receptor 1 (PLA2R1), the major autoantigen in membranous nephropathy. Here, we generated a transgenic mouse line expressing the full-length human PLA2R1 in podocytes, which has no kidney impairment after birth. Beginning from the age of three weeks, these mice spontaneously developed anti-human PLA2R1 antibodies, a nephrotic syndrome with progressive albuminuria and hyperlipidemia, and the typical morphological signs of membranous nephropathy with granular glomerular deposition of murine IgG in immunofluorescence and subepithelial electron-dense deposits by electron microscopy. Importantly, human PLA2R1-expressing Rag2 -/- mice, which lack mature and functioning B and T lymphocytes, developed neither anti-PLA2R1 antibodies nor proteinuria. Thus, our work demonstrates that podocyte expression of human PLA2R1 can induce membranous nephropathy with an underlying antibody-mediated pathogenesis in mice. Importantly, this antibody-mediated model enables proof-of-concept evaluations of antigen-specific treatment strategies, e.g., targeting autoantibodies or autoantibody-producing cells, and may further help understand the autoimmune pathogenesis of membranous nephropathy., (Copyright © 2022 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

ANCA vasculitis is an autoimmune disease with increased expression of the autoantigen genes, myeloperoxidase (MPO) and proteinase 3 (PRTN3), but the origin and significance of expression is less distinct. To clarify this, we measured MPO and PRTN3 messenger RNA in monocytes, normal-density neutrophils, and in enriched leukocytes from peripheral blood mononuclear cells. Increased autoantigen gene expression was detected in normal-density neutrophils and enriched leukocytes from patients during active disease compared to healthy individuals, with the largest difference in enriched leukocytes. RNA-seq of enriched leukocytes comparing active-remission pairs identified a gene signature for low-density neutrophils. Cell sorting revealed low-density neutrophils contained mature and immature neutrophils depending on the presence or absence of CD10. Both populations contributed to autoantigen expression but the frequency of immature cells in low-density neutrophils did not correlate with low-density neutrophil MPO or PRTN3 expression. Low-density neutrophils were refractory to MPO-ANCA induced oxidative burst, suggesting an alternative role for low-density neutrophils in ANCA vasculitis pathogenesis. In contrast, normal-density neutrophils were activated by MPO-ANCA and monoclonal anti-PR3 antibody. Normal-density neutrophil activation correlated with MPO and PRTN3 mRNA. Increased autoantigen gene expression originating from the mature low-density and normal-density neutrophils suggests transcriptional dysregulation is a hallmark of ANCA vasculitis. Thus, the correlation between autoantigen gene expression and antibody-mediated normal-density neutrophil activation connects autoantigen gene expression with disease pathogenesis., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Iron deficiency, anemia, hyperphosphatemia, and increased fibroblast growth factor 23 (FGF23) are common and interrelated complications of chronic kidney disease (CKD) that are linked to CKD progression, cardiovascular disease and death. Ferric citrate is an oral phosphate binder that decreases dietary phosphate absorption and serum FGF23 concentrations while increasing iron stores and hemoglobin in patients with CKD. Here we compared the effects of ferric citrate administration versus a mineral sufficient control diet using the Col4a3 knockout mouse model of progressive CKD and age-matched wild-type mice. Ferric citrate was given to knockout mice for four weeks beginning at six weeks of age when they had overt CKD, or for six weeks beginning at four weeks of age when they had early CKD. Ten-week-old knockout mice on the control diet showed overt iron deficiency, anemia, hyperphosphatemia, increased serum FGF23, hypertension, decreased kidney function, and left ventricular systolic dysfunction. Ferric citrate rescued iron deficiency and anemia in knockout mice regardless of the timing of treatment initiation. Circulating levels and bone expression of FGF23 were reduced in knockout mice given ferric citrate with more pronounced reductions observed when ferric citrate was initiated in early CKD. Ferric citrate decreased serum phosphate only when it was initiated in early CKD. While ferric citrate mitigated systolic dysfunction in knockout mice regardless of timing of treatment initiation, early initiation of ferric citrate also reduced renal fibrosis and proteinuria, improved kidney function, and prolonged life span. Thus, initiation of ferric citrate treatment early in the course of murine CKD lowered FGF23, slowed CKD progression, improved cardiac function and significantly improved survival., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Studies suggest that altered renal lipid metabolism plays a role in the pathogenesis of diabetic kidney disease and that genetic or pharmacological induction of cholesterol efflux protects from the development of diabetic kidney disease and focal segmental glomerulosclerosis (FSGS). Here we tested whether altered lipid metabolism contributes to renal failure in the Col4a3 knockout mouse model for Alport Syndrome. There was an eight-fold increase in the cholesterol content in renal cortexes of mice with Alport Syndrome. This was associated with increased glomerular lipid droplets and cholesterol crystals. Treatment of mice with Alport Syndrome with hydroxypropyl-β-cyclodextrin (HPβCD) reduced cholesterol content in the kidneys of mice with Alport Syndrome and protected from the development of albuminuria, renal failure, inflammation and tubulointerstitial fibrosis. Cholesterol efflux and trafficking-related genes were primarily affected in mice with Alport Syndrome and were differentially regulated in the kidney cortex and isolated glomeruli. HPβCD also protected from proteinuria and mesangial expansion in a second model of non-metabolic kidney disease, adriamycin-induced nephropathy. Consistent with our experimental findings, microarray analysis confirmed dysregulation of several lipid-related genes in glomeruli isolated from kidney biopsies of patients with primary FSGS enrolled in the NEPTUNE study. Thus, lipid dysmetabolism occurs in non-metabolic glomerular disorders such as Alport Syndrome and FSGS, and HPβCD improves renal function in experimental Alport Syndrome and FSGS., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Monogenic forms of Steroid-Resistant Nephrotic Syndrome (SRNS) have been widely characterized, but genetic screening paradigms preferentially address congenital, infantile onset, and familial cases. Our aim was to characterize the distribution of disease-causing gene mutations in adults with sporadic SRNS or focal segmental glomerulosclerosis (FSGS). We selected adult patients with non-syndromic, biopsy-proven FSGS or SRNS in the absence of known family history. Strict clinical criteria included lack of response to glucocorticoids and cyclosporine, and no recurrence after kidney transplantation. Mutations in SRNS genes were detected using a targeted gene panel. Sixteen of 135 tested participants (11.8%) carried pathogenic mutations in monogenic SRNS genes, and 14 others (10.4%) carried two APOL1 high-risk alleles. Autosomal recessive disease was diagnosed in 5 participants, autosomal dominant disease in 9, and X-linked disease in 2. Four participants carried a de novo heterozygous mutation. Among the 16 participants with identified mutations in monogenic SNRS genes, 7 (43.7%) had type IV collagen mutations. Mutations in monogenic SNRS genes were identified primarily in participants with proteinuria onset before 25 years of age, while the age at disease onset was variable in those with APOL1 high-risk genotype. Mean age at diagnosis was lower and renal survival was worse in participants with identified mutations in SNRS genes than in those without mutations. We found a significant rate of pathogenic mutations in adults with SRNS, with Type IV collagen mutations being the most frequent. These findings may have immediate impact on clinical practice., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Lysyl oxidase like-2 (LOXL2) is an amine oxidase with both intracellular and extracellular functions. Extracellularly, LOXL2 promotes collagen and elastin crosslinking, whereas intracellularly, LOXL2 has been reported to modify histone H3, stabilize SNAIL, and reduce cell polarity. Although LOXL2 promotes liver and lung fibrosis, little is known regarding its role in renal fibrosis. Here we determine whether LOXL2 influences kidney disease in COL4A3 (-/-) Alport mice. These mice were treated with a small molecule inhibitor selective for LOXL2 or with vehicle and assessed for glomerular sclerosis and fibrosis, albuminuria, blood urea nitrogen, lifespan, pro-fibrotic gene expression and ultrastructure of the glomerular basement membrane. Laminin α2 deposition in the glomerular basement membrane and mesangial filopodial invasion of the glomerular capillaries were also assessed. LOXL2 inhibition significantly reduced interstitial fibrosis and mRNA expression of MMP-2, MMP-9, TGF-β1, and TNF-α. LOXL2 inhibitor treatment also reduced glomerulosclerosis, expression of MMP-10, MMP-12, and MCP-1 mRNA in glomeruli, and decreased albuminuria and blood urea nitrogen. Mesangial filopodial invasion of the capillary tufts was blunted, as was laminin α2 deposition in the glomerular basement membrane, and glomerular basement membrane ultrastructure was normalized. There was no effect on lifespan. Thus, LOXL2 plays an important role in promoting both glomerular and interstitial pathogenesis associated with Alport syndrome in mice. Other etiologies of chronic kidney disease are implicated with our observations., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Mutations in the genes COL4A3, COL4A4, and COL4A5 affect the synthesis, assembly, deposition, or function of the collagen IV α345 molecule, the major collagenous constituent of the mature mammalian glomerular basement membrane. These mutations are associated with a spectrum of nephropathy, from microscopic hematuria to progressive renal disease leading to ESRD, and with extrarenal manifestations such as sensorineural deafness and ocular anomalies. The existing nomenclature for these conditions is confusing and can delay institution of appropriate nephroprotective therapy. Herein we propose a new classification of genetic disorders of the collagen IV α345 molecule with the goal of improving renal outcomes through regular monitoring and early treatment., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase in the renin-angiotensin system that catalyzes the breakdown of angiotensin II to angiotensin 1-7. We have reported that ACE2 expression in the kidney is reduced in experimental Alport syndrome but the impact of this finding on disease progression has not been studied. Accordingly, we evaluated effects of murine recombinant ACE2 treatment in Col4a3 knockout mice, a model of Alport syndrome characterized by proteinuria and progressive renal injury. Murine recombinant ACE2 (0.5 mg/kg/day) was administered from four to seven weeks of age via osmotic mini-pump. Pathological changes were attenuated by murine recombinant ACE2 treatment which ameliorated kidney fibrosis as shown by decreased expression of COL1α1 mRNA, less accumulation of extracellular matrix proteins, and inhibition of transforming growth factor-β signaling. Further, increases in proinflammatory cytokine expression, macrophage infiltration, inflammatory signaling pathway activation, and heme oxygenase-1 levels in Col4a3 knockout mice were also reduced by murine recombinant ACE2 treatment. Lastly, murine recombinant ACE2 influenced the turnover of renal ACE2, as it suppressed the expression of tumor necrosis factor-α converting enzyme, a negative regulator of ACE2. Thus, treatment with exogenous ACE2 alters angiotensin peptide metabolism in the kidneys of Col4a3 knockout mice and attenuates the progression of Alport syndrome nephropathy., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Blockers of the renin-angiotensin system are effective in the treatment of experimental and clinical diabetic nephropathy. An approach different from blocking the formation or action of angiotensin II (1-8) that could also be effective involves fostering its degradation. Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase that cleaves angiotensin II (1-8) to form angiotensin (1-7). Therefore, we examined the renal effects of murine recombinant ACE2 in mice with streptozotocin-induced diabetic nephropathy as well as that of amplification of circulating ACE2 using minicircle DNA delivery prior to induction of experimental diabetes. This delivery resulted in a long-term sustained and profound increase in serum ACE2 activity and enhanced ability to metabolize an acute angiotensin II (1-8) load. In mice with streptozotocin-induced diabetes pretreated with minicircle ACE2, ACE2 protein in plasma increased markedly and this was associated with a more than 100-fold increase in serum ACE2 activity. However, minicircle ACE2 did not result in changes in urinary ACE2 activity as compared to untreated diabetic mice. In both diabetic groups, glomerular filtration rate increased significantly and to the same extent as compared to non-diabetic controls. Albuminuria, glomerular mesangial expansion, glomerular cellularity, and glomerular size were all increased to a similar extent in minicircle ACE2-treated and untreated diabetic mice, as compared to non-diabetic controls. Recombinant mouse ACE2 given for 4 weeks by intraperitoneal daily injections in mice with streptozotocin-induced diabetic nephropathy also failed to improve albuminuria or kidney pathology. Thus, a profound augmentation of ACE2 confined to the circulation failed to ameliorate the glomerular lesions and hyperfiltration characteristic of early diabetic nephropathy. These findings emphasize the importance of targeting the kidney rather than the circulatory renin angiotensin system to combat diabetic nephropathy., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Neuropilin1 (Nrp1) is a co-receptor best known to regulate the development of endothelial cells and is a target of anticancer therapies. However, its role in other vascular cells including pericytes is emergent. The kidney is an organ with high pericyte density and cancer patients develop severe proteinuria following administration of NRP1B-neutralizing antibody combined with bevacizumab. Therefore, we investigated whether Nrp1 regulates glomerular capillary integrity after completion of renal development using two mouse models; tamoxifen-inducible NG2Cre to delete Nrp1 specifically in pericytes and administration of Nrp1-neutralizing antibodies. Specific Nrp1 deletion in pericytes did not affect pericyte number but mutant mice developed hematuria with glomerular basement membrane defects. Despite foot process effacement, albuminuria was absent and expression of podocyte proteins remained unchanged upon Nrp1 deletion. Additionally, these mice displayed dilation of the afferent arteriole and glomerular capillaries leading to glomerular hyperfiltration. Nidogen-1 mRNA was downregulated and collagen4α3 mRNA was upregulated with no significant effect on the expression of other basement membrane genes in the mutant mice. These features were phenocopied by treating wild-type mice with Nrp1-neutralizing antibodies. Thus, our results reveal a postdevelopmental role of Nrp1 in renal pericytes as an important regulator of glomerular basement membrane integrity. Furthermore, our study offers novel mechanistic insights into renal side effects of Nrp1 targeting cancer therapies., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Circulating levels of fibroblast growth factor 23 (FGF23) are elevated in patients with chronic kidney disease (CKD), but the mechanisms are poorly understood. Here we tested whether inflammation and iron deficiency regulate FGF23. In wild-type mice, acute inflammation induced by single injections of heat-killed Brucella abortus or interleukin-1β (IL-1β) decreased serum iron within 6 h, and was accompanied by significant increases in osseous Fgf23 mRNA expression and serum levels of C-terminal FGF23, but no changes in intact FGF23. Chronic inflammation induced by repeated bacteria or IL-1β injections decreased serum iron, increased osseous Fgf23 mRNA, and serum C-terminal FGF23, but modestly increased biologically active, intact FGF23 serum levels. Chronic iron deficiency mimicked chronic inflammation. Increased osseous FGF23 cleavage rather than a prolonged half-life of C-terminal FGF23 fragments accounted for the elevated C-terminal FGF23 but near-normal intact FGF23 levels in inflammation. IL-1β injection increased Fgf23 mRNA and C-terminal FGF23 levels similarly in wildtype and Col4a3(ko) mice with CKD but markedly increased intact FGF23 levels only in the CKD mice. Inflammation increased Fgf23 transcription by activating Hif1α signaling. Thus, inflammation and iron deficiency stimulate FGF23 production. Simultaneous upregulation of FGF23 cleavage in osteocytes maintains near-normal levels of biologically active, intact circulating FGF23, whereas downregulated or impaired FGF23 cleavage may contribute to elevated intact serum FGF23 in CKD., (Copyright © 2015 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Focal segmental glomerulosclerosis (FSGS) is a histological lesion with many causes, including inherited genetic defects, with significant proteinuria being the predominant clinical finding at presentation. Mutations in COL4A3 and COL4A4 are known to cause Alport syndrome (AS), thin basement membrane nephropathy, and to result in pathognomonic glomerular basement membrane (GBM) findings. Secondary FSGS is known to develop in classic AS at later stages of the disease. Here, we present seven families with rare or novel variants in COL4A3 or COL4A4 (six with single and one with two heterozygous variants) from a cohort of 70 families with a diagnosis of hereditary FSGS. The predominant clinical finding at diagnosis was proteinuria associated with hematuria. In all seven families, there were individuals with nephrotic-range proteinuria with histologic features of FSGS by light microscopy. In one family, electron microscopy showed thin GBM, but four other families had variable findings inconsistent with classical Alport nephritis. There was no recurrence of disease after kidney transplantation. Families with COL4A3 and COL4A4 variants that segregated with disease represent 10% of our cohort. Thus, COL4A3 and COL4A4 variants should be considered in the interpretation of next-generation sequencing data from such patients. Furthermore, this study illustrates the power of molecular genetic diagnostics in the clarification of renal phenotypes.

Malone et al. performed next-generation sequencing on 70 families with focal segmental glomerulosclerosis (FSGS) and discovered that 10% had variants in surprising 'old' genes, COL4A3 and COL4A4, which are involved in Alport syndrome and thin basement membrane nephropathy. These data show that a subset of renal manifestations associated with COL4A3 or COL4A4 variants cannot be distinguished from FSGS by clinical data or histopathology. Thus, a diagnosis of FSGS may sometimes fall within the spectrum of Alport syndrome.

A spontaneous mutation termed bilateral wasting kidneys (bwk) was identified in a colony of NONcNZO recombinant inbred mice. These mice exhibit a rapid increase of urinary albumin at an early age associated with glomerulosclerosis, interstitial nephritis, and tubular atrophy. The mutation was mapped to a location on chromosome 1 containing the Col4a3 and Col4a4 genes, for which mutations in the human orthologs cause the hereditary nephritis Alport syndrome. DNA sequencing identified a G-to-A mutation in the conserved GT splice donor of Col4a4 intron 30, resulting in skipping of exon 30 but maintaining the mRNA reading frame. Protein analyses showed that mutant collagen α3α4α5(IV) trimers were secreted and incorporated into the glomerular basement membrane (GBM), but levels were low, and GBM lesions typical of Alport syndrome were observed. Moving the mutation into the more renal damage-prone DBA/2J and 129S1/SvImJ backgrounds revealed differences in albuminuria and its rate of increase, suggesting an interaction between the Col4a4 mutation and modifier genes. This novel mouse model of Alport syndrome is the only one shown to accumulate abnormal collagen α3α4α5(IV) in the GBM, as also found in a subset of Alport patients. These mice will be valuable for testing potential therapies, for understanding abnormal collagen IV structure and assembly, and for gaining better insights into the mechanisms leading to Alport syndrome, and to the variability in the age of onset and associated phenotypes.

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-dihydroxyvitamin D (1,25(OH)(2)D) levels and 3-fold higher renal Cyp24α1 gene expression compared to wild-type mice. Severe phosphate restriction increased 1,25(OH)(2)D 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.

Progressive elevations of fibroblastic growth factor 23 (FGF23) in chronic kidney disease may reduce serum 25-hydroxyvitamin D (25(OH)) and 1,25-dihydroxyvitamin D (1,25(OH)(2)D) levels, via stimulation of 24-hydroxylase (Cyp24a1)-mediated catabolism of these vitamin D metabolites. To test this possibility, we measured serum concentrations of 24,25-dihydroxyvitamin D (24,25(OH)(2)D), a product of Cyp24a1 hydroxylation of 25(OH)D, in the Col4a3 knockout mouse, a model of Alport syndrome-derived chronic kidney disease, and in patients with chronic kidney disease of variable severity. There was an inverse correlation between serum FGF23 and both 25(OH)D and 1,25(OH)(2)D in the mouse model, but no significant relationship was observed in the cross-sectional patient cohort. The FGF23-dependent increase in Cyp24a1 mRNA expression in the mouse kidneys was consistent with the possibility that FGF23 induces vitamin D catabolism. There was, however, a reduction in serum 24,25(OH)(2)D levels, rather than the expected elevation, in both the mice and patients with chronic kidney disease. Low 25(OH)D and elevated FGF23 and parathyroid hormone levels were correlated with the reduced serum 24,25(OH)(2)D concentrations of these patients. Thus, we failed to find support for FGF23-mediated catabolism of vitamin D metabolites in chronic kidney disease assessed by 24,25(OH)(2)D levels.

We studied here the clinical course of heterozygous carriers of X-linked Alport syndrome and a subgroup of patients with thin basement membrane disease due to heterozygous autosomal recessive Alport mutations whose prognosis may be worse than formerly thought. We analyzed 234 Alport carriers, including 29 with autosomal recessive mutations. Using Kaplan-Meier estimates and log-rank tests, autosomal and X-linked carriers were found to have similar incidences of renal replacement therapy, proteinuria, and impaired creatinine clearance. Further, age at onset of renal replacement therapy did not differ between X-chromosomal and autosomal carriers. Both groups showed an impaired life expectancy when reaching renal replacement therapy. RAAS inhibition significantly delayed the onset of end-stage renal failure. Not only carriers of X-linked Alport mutations but also heterozygous carriers of autosomal recessive mutations were found to have an increased risk for worse renal function. The risk of end-stage renal disease in both groups affected life expectancy, and this should cause a greater alertness toward patients presenting with what has been wrongly termed 'familial benign hematuria.' Timely therapy can help to delay onset of end-stage renal failure. Thus, yearly follow-up by a nephrologist is advised for X-linked Alport carriers and patients with thin basement membrane nephropathy, microalbuminuria, proteinuria, or hypertension.

The autoimmune form of atypical hemolytic uremic syndrome (HUS) is characterized by circulating autoantibodies against the complement regulator factor H, and is often associated with deficiency of the factor H-related proteins CFHR1 and CFHR3. Here we studied whether anti-factor H autoantibodies crossreact with CFHR1, and determined functional consequences of this. In ELISA, anti-factor H immunoglobulin G (IgG) autoantibodies from 24 atypical HUS patients bound to the short consensus repeat 20 domain of factor H, 21 antibodies also recognized CFHR1, but none CFHR3. Three patients also had anti-factor H IgA autoantibodies crossreacting with CFHR1. Analysis of the IgG fractions in CFHR1-deficient patients found that CFHR1-IgG complexes were formed during plasma exchange treatment, indicating that autoantibodies recognize CFHR1 in vivo. Recombinant CFHR1 prevented hemolysis of sheep erythrocytes caused by patient plasma containing anti-factor H IgG, but it did not inhibit red cell lysis caused by a factor H mutation (W1183 L) in the short consensus repeat 20 domain. Thus, exogenous CFHR1 provided during plasma exchange therapy may neutralize anti-factor H autoantibodies and help in the treatment of autoimmune atypical HUS.

Loss of function mutations in the α3 or α4 chain of type IV collagen cause Alport nephropathy, characterized by progressive glomerulosclerosis. While studying the mechanisms that determine disease progression, we found that the evolution of kidney disease in Col4a3-deficient mice was associated with an influx of immune cell subsets including nonactivated macrophages. This suggested that intrarenal inflammation might accelerate Alport nephropathy. A possible mechanism might be the well-known enhancement of immune recognition by bacterial products. We found that exposure to bacterial endotoxin from 4 to 6 weeks of age did not affect disease progression, whereas an equipotent dose of cytosine-guanine (CpG)-DNA, a synthetic mimic of bacterial DNA, accelerated all aspects of Alport nephropathy and reduced the overall lifespan of Col4a3-deficient mice. This effect was associated with a significant increase of renal CD11b+/Ly6C(hi) macrophages, intrarenal production of inducible nitric oxide synthase, tumor necrosis factor (TNF)-α, interleukin-12, and CXCL10, and loss of podocytes. TNF-α was essential for acceleration of Alport nephropathy, as etanercept (a soluble TNF-α receptor) entirely abrogated the CpG-DNA effect. Thus, systemic exposure to CpG-DNA induces classically activated (M1) macrophages that enhance intrarenal inflammation and disease progression. Hence, factors that modulate the phenotype of renal macrophages can affect the progression of Alport nephropathy and, potentially, other types of chronic kidney diseases.

Cell division autoantigen 1 (CDA1) modulates cell proliferation and transforming growth factor-β (TGF-β) signaling in a number of cellular systems; here we found that its levels were elevated in the kidneys of two animal models of diabetic renal disease. The localization of CDA1 to tubular cells and podocytes in human kidney sections was similar to that seen in the rodent models. CDA1 small interfering RNA knockdown markedly attenuated, whereas its overexpression increased TGF-β signaling, modulating the expression of TGF-β, TGF-β receptors, connective tissue growth factor, collagen types I, III, IV, and fibronectin genes in HK-2 cells. CDA1 and TGF-β together were synergistic in stimulating TGF-β signaling and target gene expression. CDA1 knockdown effectively blocked TGF-β-stimulated expression of collagen genes. This was due to its ability to modulate the TGF-β type I, but not the type II, receptor, leading to increased phosphorylation of Smad3 and extracellular signal-regulated kinase mitogen-activated protein kinase. Furthermore, the Smad3 inhibitor, SIS3, markedly attenuated the activities of CDA1 in stimulating TGF-β signaling as well as gene expression of collagens I, III, and IV. Thus, our in vitro and in vivo findings show that CDA1 has a critical role in TGF-β signaling in the kidney.

Alport syndrome (AS) is a hereditary glomerulopathy due to abnormal composition of the glomerular basement membrane, leading to end-stage renal disease (ESRD). Studies of animal models of AS have suggested a variety of potentially effective therapies, but none of these has been definitely shown to prevent or delay ESRD in human AS. Studies in Alport mice suggest that angiotensin inhibition not only has antiproteinuric effects but suppresses cytokine and collagen production as well as tubulointerstitial fibrogenesis and inflammation. For these reasons, many Alport patients are treated empirically with angiotensin antagonists. Cyclosporine may reduce proteinuria in AS, but the risk of nephrotoxic side effects complicates long-term therapy in children. Current data on the role of HMG-CoA reductase inhibition are sparse, so therapy should be limited to adults with dyslipoproteinemia. Results of some, but not all, studies suggest that bone marrow-derived cells may ameliorate disease in Alport mice. However, until experimental doubts concerning the superiority of bone-marrow transplantation over other treatments are resolved by additional investigation, human research subjects should not be exposed to cell-based therapies that may carry substantial risks. In summary, all potential therapies are off-label use in children. As a consequence, initial therapeutic trials should focus on the safety and efficiency of medical treatment, as well as the optimal timing of therapy.

Alport syndrome (ATS) and benign familial hematuria (BFH) are type IV collagen inherited disorders. Mutations in COL4A5 are generally believed to cause X-linked ATS, whereas mutations in COL4A3 and COL4A4 genes can be associated with the autosomal-recessive and -dominant type of ATS or BFH. In view of the wide spectrum of phenotypes, an exact diagnosis is sometimes difficult to achieve. This study involved screening each exon with boundary intronic sequences of COL4A3, COL4A4, and COL4A5 genes by optimized polymerase chain reaction-single-stranded conformational polymorphism analysis in 17 families with ATS and in 40 families diagnosed as having BFH. Twelve different mutations were found in the COL4A5 gene in ATS patients, comprising nine missense mutations, a splice site mutation, a mutation causing frameshift, and a nonsense mutation. One of the missense mutations (p.G624D) was present not only in one family with ATS but also in five families with suspected BFH. Three heterozygous mutations in the COL4A3 gene (two missense and one frameshift) and four heterozygous mutations in COL4A4 (two splice site, one in-frame deletion, and one missense) were identified in patients with BFH. Sixteen mutations are to the best of our knowledge new and private.

Multipotent mesenchymal stem or stromal cells (MSC) have shown to improve outcome of acute renal injury models, but whether MSC can delay renal failure in chronic kidney disease is not known. We injected primary MSC or saline into mice that lack the alpha3-chain of type IV collagen (COL4A3), a model of chronic kidney disease with close similarities to human Alport disease. Weekly injections of MSC from week 6 to 10 of life prevented the loss of peritubular capillaries and reduced markers of renal fibrosis, that is, interstitial volume, numbers of smooth muscle actin-positive interstitial cells, and interstitial collagen deposits as compared to saline-injected COL4A3-deficient mice. However, renal function, that is, blood urea nitrogen, creatinine levels, proteinuria as well as survival of COL4A3-deficient mice were not affected by MSC injections. Although MSC were found to localize to kidneys of COL4A3-deficient mice after injection, differentiation into renal cells was not detected. However, MSC expressed growth factors, that is, vascular endothelial growth factor (VEGF) and bone morphogenetic protein-7 under basal culture conditions. In fact, VEGF mRNA levels were increased in kidneys of MSC-injected COL4A3-deficient mice and MSC supernatants enhance endothelial cell proliferation in vitro. Thus, weekly injections with MSC prevent loss of peritubular capillaries possibly owing to local production of growth factors rather than by differentiation into renal cells. The maintenance of interstitial vasculature is associated with less interstitial fibrosis but, is insufficient to delay renal failure and survival of COL4A3-deficient mice.

Background: We have demonstrated that a single T-cell epitope pCol(28-40) (SQTTANPSCPEGT) alone, which is derived from NC1 domain of alpha3 chain of type IV collagen (Col4alpha3 NC1), can induce severe glomerulonephritis in Wistar Kyoto rats. This study further characterized this T-cell epitope., Methods: A series of synthetic peptides derived from pCol (28-40) were tested in vivo and in vitro for their T-cell epitope activity and nephritogenicity. Major histocompatability complex (MHC) class II molecules in Wistar Kyoto rats were cloned, and MHC restriction of pCol(28-40) was determined., Results: The T-cell epitope pCol(28-40) was restricted by rat MHC class II RT.1Bl. Ten amino acid residues (29 to 38) were mapped to be the minimum core of the T-cell epitope, which was capable of inducing the T-cell response and severe glomerulonephritis. Only three residues were identified as absolutely critical for the T-cell epitope: position 31 (T) was an anchor residue to the class II molecule, and positions 33 (N) and 34 (P) contributed to the specificity of the T-cell epitope. Thus, only substitution at those positions completely abrogated nephritogenicity of the T-cell epitope. Interestingly, pCol (28-40) also bound to human MHC class II human MHC class II molecule HLA-DRB*1501, which has been linked to human anti-glomerular basement membrane (GBM) disease, suggesting that human homologue of pCol(28-40) could be a potential human T-cell epitope., Conclusion: Our study demonstrated that only few residues in the nephritogenic T-cell epitope pCol(28-40) were critical. Our finding also revealed that pCol(28-40) is a potential nephritogenic T-cell epitope in Goodpasture's syndrome.

Background: 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., Methods: 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., Results: 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., Conclusion: 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.

Background: Alport syndrome is a clinically and genetically heterogeneous nephropathy. The majority of cases are transmitted as an X-linked semidominant condition due to COL4A5 mutations. In this form males are more severely affected than females. Less than 10% of cases are autosomal recessive due to mutation in either COL4A3 or COL4A4. In this rarer form, both males and females are severely affected. Only two cases of autosomal-dominant Alport syndrome have been reported, one due to a COL4A3 mutation and the other due to a COL4A4 mutation. Because of the paucity of the reported families, the natural history of autosomal-dominant Alport syndrome is mostly unknown., Methods: Four families with likely autosomal-dominant Alport syndrome were investigated. COL4A3 and COL4A4 genes were analyzed by denaturing high-performance liquid chromatography (HPLC). Automated sequencing was performed to identify the underlying mutation., Results: Two families had a mutation in the COL4A4 gene and two in the COL4A3. Accurate clinical evaluation of family members showed interesting results. Affected individuals (22 persons) had a wide range of phenotypes from end-stage renal disease (ESRD) in the fifth decade to a nonprogressive isolated microhematuria. Finally, three heterozygous individuals (90, 22 and 11 years old, respectively) were completely asymptomatic., Conclusion: This paper demonstrated that patients affected by autosomal-dominant Alport syndrome have a high clinical variability. Moreover, a reduced penetrance of about 90% (3 of 25) may be considered for the assessment of recurrence risk during genetic counseling of these families.

Background: Thin basement membrane nephropathy (TBMN) is often caused by mutations in the COL4A3 and COL4A4 genes., Methods: We examined 62 unrelated individuals diagnosed with TBMN by renal biopsy (N= 49, 79%) or a positive family history of hematuria but without a biopsy (N= 13, 21%) for mutations in the COL4A3 gene and the COL4A3/COL4A4 promoter. All 52 exons of COL4A3 as well as the COL4A3/COL4A4 promoter were screened with single-stranded conformational polymorphism (SSCP) analysis at 4 degrees C and at room temperature. Amplicons that demonstrated electrophoretic abnormalities were sequenced., Results: Seven mutations were demonstrated in seven patients: G532C and G584C in exon 25, G596R in exon 26, G695R in exon 28, and IVS 2224 - 11C>T, IVS 2980 + 1G>A and IVS 3518 - 7C>G. No mutations were found in the COL4A3/COL4A4 promoter. Four novel polymorphisms or variants (P116T in exon 6, P690P in exon 27, and G895G and A899A in exon 33) were also demonstrated. In addition, P1109S and Q1495R, which had been described previously but whose status was unclear, were shown to be polymorphisms. All seven mutations described here were associated with hematuria. While one mutation (2980 + 1G>A) was found in an individual who also had proteinuria, none of her family members with the same mutation had increased urinary protein. None of the patients with these seven mutations had renal impairment. Hematuria was completely penetrant in families with the G532C, G584C, G596R, and IVS 2980 + 1G>A mutations but not with the G695R and IVS 3518 - 7C>G mutations., Conclusion: COL4A3 mutations are common in TBMN.

Background: The major Goodpasture antibody binding epitopes have been localized to the amino-terminal third of the noncollagenous domain (NC1) of the alpha3 chain of type IV collagen [alpha3(IV)NC1]. The present study determined whether the same epitopes induce glomerulonephritis in rats., Methods: We immunized Wistar Kyoto (WKY) rats with human alpha3(IV)/alpha1(IV)NC1 chimeric proteins or full-length recombinant alpha3(IV)NC1 (alpha3732). Chimeric protein constructs were thirds of alpha3(IV)NC1 (CP333) replaced by corresponding sequences of homologous nonreactive alpha1(IV)NC1 (CP111). All chimeric proteins contained 30 amino acids of type X collagen at the amino terminus except alpha3732. Two other constructs, T195 EA (EA) and T194 EB (EB), were entirely alpha1(IV)NC1, except for antibody-immunodominant amino acids from the first and second thirds of alpha3(IV)NC1., Results: Construct immunized animals developed specific antibody responses to recombinant proteins and native human, bovine and rat NC1. CP311 immunized rats, as well as alpha3732 rats, had glomerular IgG, fibrin, and glomerulonephritis with proteinuria by 3 weeks. CP331 produced more severe disease, comparable to positive controls. CP111 produced no disease. EA, but not EB, induced severe glomerulonephritis. Half-dose each of EA plus EB induced disease identical to full-dose EA alone., Conclusion: The amino third of alpha3(IV)NC1 which contains the major epitope for Goodpasture antibody binding, also induces glomerulonephritis in rats. The middle third of alpha3(IV)NC1 does not induce glomerulonephritis but appears to enhance disease with the amino terminal third. Finally, the presence of the collagen X leader sequence appears to convey greater nephritogenicity. These studies suggest that not only the nephritogenic epitope itself, but flanking sequences and the conformational context of the nephritogenic epitope may influence its ability to cause glomerulonephritis.

Background: Alport syndrome is caused by mutations in type IV collagen alpha3, alpha4, and alpha5 genes. Immunohistochemical analyses of kidney sections from normal individuals and Alport syndrome patients have suggested that the alpha3(IV), alpha4(IV), and alpha5(IV) chains form a heterotrimer in the glomerular basement membrane (GBM) and that a defect in any one of the chains disrupts the assembly of the three chains, resulting in Alport syndrome., Methods: We established stable transformants of HEK293 cells that expressed mouse alpha3(IV) and/or alpha4(IV) and/or alpha5(IV) chains. Using cell extracts and culture media of these cells, experiments were performed to determine whether or not the alpha3(IV) and alpha4(IV) chains were coimmunoprecipitated with the alpha5(IV) chain. Moreover, we examined complex formation of mutant alpha5(IV) chain containing either a deletion or substitution mutation with the alpha3(IV) and alpha4(IV) chains., Results: The established cell strains were named according to their transfected alpha(IV) chains. The alpha3(IV) and alpha4(IV) chains were coimmunoprecipitated with the alpha5(IV) chain in alpha345 cells but not in alpha35 and alpha45 cells. These chains were not coimmunoprecipitated with the alpha5(IV) chain, which lacked either a collagenous domain or NC1 domain. The ability of the alpha5(IV) chain with either a G1182R or C1573R substitution, corresponding to previously reported mutations in Alport syndrome patients, to form a complex with alpha3(IV) and alpha4(IV) chains was diminished., Conclusion: The findings indicate that alpha3(IV), alpha4(IV), and alpha5(IV) chains form a complex, which is a heterotrimer, and that a defect in complex formation might be one of the molecular mechanisms underlying the pathogenesis of Alport syndrome.

Background: Alport disease is a heritable basement membrane disorder caused by mutations in genes encoding the alpha3, alpha4, or alpha5 chains of type IV collagen, which normally comprise the collagenous network of mature glomerular basement membranes (GBMs). In Alport disease, the alpha3(IV), alpha4(IV), alpha5(IV) collagen network is absent and substituted for by alpha1(IV), and alpha2(IV) collagen, which normally is present only in developing, immature GBMs. The disease is marked by progressive GBM thickening and delamination, proteinuria, and renal failure. In addition to collagen IV dysregulation, abnormal GBM laminins also occur and may contribute to the pathogenesis of Alport glomerulopathy., Methods: To investigate laminin dysregulation in a mouse model of Alport disease, we used antibodies specific for laminin-alpha1 and -beta1 chains (to recognize laminin-1), and -alpha5 chain (to recognize laminin-11), and evaluated their distribution during glomerular development in alpha3(IV) collagen-deficient mice., Results: Developing glomeruli of infant alpha3(IV) collagen knockout mice underwent normal down-regulation of laminin-1, but laminin-1 chains were then reexpressed in maturing glomeruli, becoming concentrated in the subepithelial GBM projections typical of Alport disease. Immunoelectron microscopy showed that laminin-1 reexpression took place in both glomerular endothelial cells and podocytes., Conclusions: The absence of a alpha3(IV), alpha4(IV), alpha5(IV) network may stimulate reexpression of laminin-1 by Alport mouse endothelial cells and podocytes. This abnormal GBM, which is more characteristic of immature glomeruli, may promote podocyte foot process effacement and reversion to a less differentiated state.

Background: 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., Methods: 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., Results: 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., Conclusions: 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.

Unlabelled: COL4A3/COL4A4 mutations: From familial hematuria to autosomal-dominant or recessive Alport syndrome., Background: Mutations of the type IV collagen COL4A5 gene cause X-linked Alport syndrome (ATS). Mutations of COL4A3 and COL4A4 have been reported both in autosomal-recessive and autosomal-dominant ATS, as well as in benign familial hematuria (BFH). In the latter conditions, however, clinical features are less defined, few mutations have been reported, and other genes and non-genetic factors may be involved., Methods: We analyzed 36 ATS patients for COL4A3 and COL4A4 mutations by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and direct sequencing. Sporadic patients who had tested negative for COL4A5 mutations were included with typical cases of autosomal recessive ATS to secure a better definition of the phenotype spectrum., Results: We identified seven previously undescribed COL4A3 mutations: in two genetic compounds and three heterozygotes, and one in COL4A4. In agreement with the literature, some of the mutations of compound heterozygotes were associated with microhematuria in healthy heterozygous relatives. The mutations of heterozygous patients are likely dominant, since no change was identified in the second allele even by sequencing, and they are predicted to result in shortened or abnormal chains with a possible dominant-negative effect. In addition, both genes showed rare variants of unclear pathogenicity, and common polymorphisms that are shared in part with other populations., Conclusions: This study extends the mutation spectrum of COL4A3 and COL4A4 genes, and suggests a possible relationship between production of abnormal COL IV chains and dominant expression of a continuous spectrum of phenotypes, from ATS to BFH.

Background: Autoimmunity to kidney antigens causes membranous nephropathy and Goodpasture's disease and very likely is pivotal in many other glomerular diseases. We investigated the potential for central tolerance to the best-characterized kidney autoantigen, the NC1 domain of the alpha3 chain of type IV collagen [alpha3(IV)NC1], which is the target of autoimmune attack in Goodpasture's disease., Methods: Indirect immunofluorescence on human thymus and polymerase chain reaction (PCR) and Southern blot analysis of cDNA reverse transcribed from RNA extracted from human thymus and kidney., Results: Indirect immunofluorescence on human thymus demonstrated the presence of alpha3(IV)NC1 in all six thymus samples examined. The homologous collagen IV chain, alpha5(IV)NC1, also was detected with a similar intra-thymic distribution. Strikingly, thymic alpha3 and alpha5 localized around and within Hassall's corpuscles in the thymic medulla, which are structures implicated in T cell apoptosis and possibly negative selection. In contrast, alpha1(IV)NC1 localized to the basement membranes of interlobular septa and blood vessels, as is typical of collagen IV chains situated outside the thymus. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed the presence of mRNA encoding alpha3(IV)NC1 and alpha5(IV)NC1 in thymic tissue establishing that the antigens were likely to have been synthesized locally., Conclusions: The results demonstrate that alpha3(IV)NC1 is expressed in the human thymus, and therefore should be available for induction of alpha3(IV)NC1-specific tolerance. This observation has the important implication that patients' alpha3(IV)NC1-specific, autoreactive T cells are more likely to recognize cryptic epitopes that are not adequately presented by thymic antigen-presenting cells (APC) than the major antigen-derived epitopes generally identified by conventional approaches.

Background: The characteristic feature of Goodpasture disease is the occurrence of an autoantibody response to the noncollagenous domain of the alpha3 chain of type IV collagen [alpha3(IV)NC1] in the alveolar and glomerular basement membrane. These antibodies are associated with the development of a rapidly progressive glomerulonephritis, with or without lung hemorrhage, whereas autoantibodies specific for the other alpha chains of the heterotrimeric type IV collagen probably do not cause disease. In this study, we have investigated whether differences in fine specificity of autoimmune recognition of the alpha3(IV)NC1 correlate with clinical outcome., Methods: For mapping of antibody binding to type IV collagen, chimeric collagen constructs were generated in which parts of the alpha3(IV)NC1 domain were replaced by the corresponding sequences of homologous nonreactive alpha1(IV). The different recombinant collagen chimeras allowed the analysis of antibody specificities in 77 sera from well-documented patients., Results: One construct that harbors the aminoterminal third of the alpha3(IV)NC1 was recognized by all sera, indicating that it represents the dominant target of the B-cell response in Goodpasture disease. Seventy percent of the samples recognized other parts of the molecule as well. However, only reactivity to the N-terminus of the alpha3(IV)NC1 correlated with prognosis, that is, kidney survival after six months of follow-up., Conclusion: The results indicate the crucial importance of antibody recognition of this particular domain for the pathogenesis of Goodpasture disease, thereby opening new avenues for the development of better diagnostic and therapeutic procedures.

Background: Tissue injury in Goodpasture (GP) syndrome (rapidly progressive glomerular nephritis and pulmonary hemorrhage) is mediated by antibasement membrane antibodies that are targeted to the alpha3(IV) chain of type IV collagen, one of five alpha(IV) chains that occur in the glomerular basement membrane. GP antibodies are known to bind epitopes within the carboxyl terminal noncollagenous domain (NC1) of the alpha3(IV) chain, termed the GP autoantigen. Whether epitopes also exist in the 1400-residue collagenous domain is unknown because studies to date have focused solely on the NC1 domain. A knowledge of GP epitopes is important for the understanding of the etiology and pathogenesis of the disease and for the development of therapeutic strategies., Methods: A cDNA construct was prepared for the full-length human alpha3(IV) chain. The construct was stably transfected into human embryonic kidney 293 cells. The purified full-length r-alpha3(IV) chain was characterized by electrophoresis and electron microscopy. The capacity of this chain for binding of GP antibodies from five patients was compared with that of the human r-alpha3(IV)NC1 domain by competitive enzyme-linked immunosorbent assay., Results: The r-alpha3(IV) chain was secreted from 293 cells as a single polypeptide chain that did not spontaneously undergo assembly into a triple-helical molecule. An analysis of GP-antibody binding to the full-length r-alpha3(IV) chain showed binding exclusively to the globular NC1 domain., Conclusion: The full-length human alpha3(IV) chain possesses the capacity to bind GP autoantibodies. The epitope(s) is found exclusively on the nontriple-helical NC1 domain of the alpha3(IV) chain, indicating the presence of specific immunogenic properties. The alpha3(IV) chain alone does not spontaneously undergo assembly into a triple-helical homotrimeric molecule, suggesting that coassembly with either the alpha4(IV) and/or the alpha5(IV) chain may be required for triple-helix formation.

Anti-neutrophil cytoplasmic antibodies (ANCA) in sera from patients with clinically proven vasculitis have been described as reacting with proteins present in the granules of human neutrophils. We have studied sera from 59 ANCA positive patients to further characterize the antibody response. In addition to the antigens previously identified in the vasculitic syndromes (myeloperoxidase and serine proteinase 3) the majority of these sera contained antibodies that reacted with a cytosolic extract of neutrophils on Western blots. Nearly 40% of these sera had antibodies directed against a cytosolic protein(s) of molecular mass 48 kD. This protein was purified from neutrophil cytosol by ammonium sulphate fractionation, anion exchange and reverse phase chromatography. Amino acid sequence analysis of a proteolytic fragment of this protein identified it as alpha enolase. The anti-enolase antibodies only recognized the alpha isoform and were present in sera giving either a pANCA or cANCA staining pattern by indirect immunofluorescence. Antibodies to alpha enolase were also found in sera from patients with systemic lupus erythematosus, particularly those with renal disease. We conclude that the antibody response in ANCA positive vasculitis is not restricted to neutrophil granule proteins.

Goodpasture syndrome is an autoimmune disease causing rapidly progressive glomerulonephritis and pulmonary hemorrhage. The clinical manifestations are caused by autoantibodies that bind to a constituent, termed the Goodpasture autoantigen, of alveolar and glomerular basement membranes. Searches for the identity of this constituent have recently culminated in the discovery of two new chains (alpha 3 and alpha 4) of type IV collagen and the identification of the alpha 3 chain as the Goodpasture autoantigen. The gene, COL4A3, encoding this autoantigen was recently cloned and localized to the q35-37 region of chromosome 2. The major protomeric form of the alpha 3 chain is a homotrimer. The alpha 3-protomers associate through NC1-to-NC1 interactions mainly with each other to form a suprastructure, although some associate with protomers containing the alpha 1(IV) and alpha 2(IV) chains. The alpha 3-protomers also form suprastructures involving triple helical interactions of three or more protomers. The Goodpasture epitope is localized to the carboxylterminal region of the alpha 3(IV) chain, encompassing the last 36 residues of the chain, as the primary interaction site, and its structure is discontinuous.