Absence of α6(IV) collagen in kidney and skin of X-linked Alport syndrome patients

Ab stract. To identify the abnormalities of the type IV collagen α6 chain, α6(IV), in Alport syndrome, we examined renal and skin tissue using rat monoclonal antibodies against non-consensus amino acid sequences of α6(IV). Immunofluorescence of normal human kidney and skin tissue revealed linear α6(IV) staining in the basement membrane (BM) of Bowman’s capsule, in some tubules, and also in the epidermal BM. Renal specimens from five male patients of four families with X-linked Alport syndrome showed no reactivity for α6(IV) in Bowman’s capsules and tubules. In these patients, α1(IV) and α2(IV) were normal, whereas α3(IV), α4(IV), and α5(IV) were absent from the BMs of the kidney. In skin tissue of male patients, neither α5(IV) nor α6(IV) were detected. The epidermal BM of female heterozygotes with X-linked Alport syndrome showed a mosaic staining for α5(IV) and α6(IV). These findings indicate that, in addition to a disturbed α3(IV)-α4(IV)-α5(IV) network, patients with X-linked Alport syndrome have abnormalities in α6(IV) of the renal and epidermal BMs at the protein level. Received October 6, 1995; received in revised form April 25, 1996; accepted April 29, 1996     

Urinary excretion of glomerular basement membrane-related peptides in children with renal disorders

Urinary excretion of glomerular basement membrane (GBM)-related peptides was analysed in 72 patients with a variety of renal diseases by immunoblotting using polyclonal antibodies against either collagenase or pepsin digests of human GBM. The specificity of the antibodies was verified by elution of antibodies bound to urinary GBM-related peptides on nitrocellulose blots and demonstration of reactivity of the eluted antibodies with the respective GBM digests. Furthermore, six mice immunized with urinary GBM-related peptides all developed focal linear deposits of mouse IgG along their GBM, linear and mesangial deposits of C3 in the glomeruli and serum antibodies reactive with human GBM. Monoclonal antibodies against urinary GBM-related peptides of one of the mice reacted with different peptides of the non-collagenous and collagenous domains of type IV collagen, the major structural protein of GBM. In the majority of the 75 patients' urines tested, excretion of GBM-related peptides with molecular weights of 33, 50, 80 and 150 kilodaltons (kD) was detectable. Patients with a diminished glomerular filtration rate (GFR) demonstrated excretion of the 33 kD peptide more frequently (91%) and never of the 80 kD peptide as compared with patients with normal GFR (33 kD [42%] 80 kD [87%]). The pattern of urinary GBM-related peptides was not specific for the underlying renal disease as in Alport's syndrome.     

Glomerular basement membrane type IV collagen antigens in Goodpasture's and Alport's syndromes

Type IV collagen, the main constituent of the renal glomerular basement membrane, is involved in Goodpasture's syndrome, and autoimmune disease, and in Alport's syndrome, a genetic disease. There are 6 alpha chains, α1(IV) through α6(IV), in type IV collagen in mammals. Immunohistochemical studies, using α-chain-specific monoclonal antibodies on tissue specimens from healthy people and patients with Alport's syndrome, have shown that there are 3 forms of type IV collagen molecules in mammalian basement membranes, namely α1/α1/α2, α3/α4/α5, and α5/α5/α6. Antibody specificity analysis of sera from patients with Goodpasture's syndrome show that all sera have autoantibody, with the highest titer against α3(IV)NC1, although they also have titers against the other 5α chains. This indicates that α3(IV)NC1 is the major target antigen of the disease, although the glomerular basement membrane contains the α1 through α5 chains. Experimental glomerulonephritis in rats, induced by the injection of 6 recombinant α(IV)NC1s with an adjuvant, has shown that α3(IV)NC1 and α4(IV)NC1 are nephritogenic. The lack of, or very poor, nephritogenicity of the other 4 α(IV)NC1s can be explained by the high immunologic tolerance against these chains, which are distributed widely in basement membranes of the whole body. This paper was presented at the 2nd International Forum “The Frontiers of Nephrology,” Tokyo, May 10, 1998.     

Alport syndrome with neurofibromatosis type-I: a case report

We report a 9-year-old boy with repeated fractures of the tibia from age 6 months and microscopic hematuria from age 2 years. His maternal family has a history of nephritis and his paternal family has neurofibromatosis type-I (NF-I). The boy’s renal biopsy revealed an irregular attenuation and splitting of the glomerular basement membrane. The skin biopsy was stained with monoclonal antibody against the α5 chain of type IV collagen; the epidermal basement membrane was negative in the boy and segmentally positive in the boy’s mother. We conclude that the patient inherited Alport syndrome from his mother and NF-I from his father. We postulate this was a chance association and that this case does not suggest any relationship between the two diseases. Received August 21, 1996; received in revised form and accepted March 20, 1997     

肝硬化病人血清TNF-α.Ⅳ型胶原蛋白水平的研究

目的:为探讨肝硬化病人Ⅳ型胶原(CⅣ)和肿瘤坏死因子(TNF-a)在肝硬化病中的临床意义。方法:对30例肝硬化病人应用双抗体夹心法检测血清CⅣ.TNF-a水平。结果:肝硬化病人血清CⅣ·TNF-a含量较对照组升高(P<0.05),血清CⅣ与TNF含量两者变化相一致。结论:血清CⅣ含量升高能反映肝纤维化的程度,TNF-a参与肝纤维化的形成。     

Sera from patients with anti-GBM nephritis including Goodpasture syndrome show heterogenous reactivity to recombinant NC1 domain of type IV collagen {alpha} chains

BACKGROUND.: Goodpasture (GP) syndrome is defined by the clinical associationof pulmonary haemorrhage with rapidly progressive glomerulonephritis.The disease is caused by pathogenic autoantibodies directedagainst type IV collagen, which is a major structural componentof glomerular basement membranes (GBM). METHODS.: The non-collagenous domains (NC1) of all six human type IV collagenchains was produced in E. coli as recombinant fusion proteinswith glutathione-S transferase. Sera from 10 patients with differenttypes of anti-GBM nephritis, including GP syndrome, were testedfor reactivity with the six proteins using immunoblotting ofdenatured and reduced proteins and ELISA without reduction. RESULTS.: All 10 sera reacted with the 3(IV) collagen chain by immunoblottingand ELISA. One serum also recognized the 2(IV) 4(IV), 5(IV)and 6(IV) chains by immunoblotting. ELISA measurements revealedreactivity of several other sera with 2(IV), 4(IV) or 6(IV)but not with 5(IV) collagen chains. No reactivity was observedwith the 1(IV) chain. CONCLUSION.: Autoantibodies in anti-GBM nephritis may not be directed onlyagainst the 3(IV) collagen chain and they frequently recognizeconformational epitopes.     

Nine novel <Emphasis Type="Italic">COL4A3</Emphasis> and <Emphasis Type="Italic">COL4A4</Emphasis> mutations and polymorphisms identified in inherited membrane diseases

Both thin basement membrane nephropathy (TBMN) and autosomal recessive Alport syndrome result from mutations in the COL4A3 and COL4A4 genes, and this study documents further mutations and polymorphisms in these genes. Thirteen unrelated children with TBMN and five individuals with autosomal recessive Alport syndrome were examined for mutations in the 52 exons of COL4A3 and the 47 coding exons of COL4A4 using single-stranded conformation polymorphism (SSCP) analysis. Amplicons producing different electrophoretic patterns were sequenced, and mutations were defined as variants that changed an amino acid but were not present in 50 non-hematuric normals. Three further novel mutations were identified. These were IVS 22-5 T>A in the COL4A3 gene in a consanguineous family with autosomal recessive Alport syndrome, and R1677C and R1682Q in the COL4A4 gene. In addition, six novel polymorphisms (G455G, I462I, G736G and IVS 38-8 G>A in COL4A3, and L658L and A1577A in COL4A4) were demonstrated. Many different COL4A3 and COL4A4 mutations cause TBMN and autosomal recessive Alport syndrome. The identification of polymorphisms in these genes is particularly important to enable diagnostic laboratories to distinguish mutations from uncommon normal variants.     

Laminin and type IV collagen in the human testis

Specimens of normal human testis and biopsies from testes with Sertoli-cell-only syndrome in which the seminiferous tubules had a remarkably thickened lamina propria, were investigated immunohistochemically using specific antibodies against human laminin and human type IV collagen. In the normal testis, both laminin and type IV collagen were localized to the epithelial basement membranes and the peritubular cell layers. In addition, laminin was found in the Sertoli cells. In the pathological testis, structures representing invaginations of the tubular basement membrane were positive for both laminin and type IV collagen. The presence of laminin and type IV collagen in the myoid cell layers, and laminin in the Sertoli cells from both normal and pathological testis and its indication for the secretion of these substances by the myoid and Sertoli cells is discussed.     

膀胱癌组织Ⅳ型胶原的表达意义

目的 研究Ⅳ型胶原在膀胱癌中的表达及其临床意义。方法 应用免疫组化方法检测Ⅳ型胶原在65例膀胱癌及19例非肿瘤性膀胱组织中的表达,分析其与膀胱癌的病理分级、分期、复发、多发的关系。结果 Ⅳ型胶原表达与膀胱癌的病理分级、分期相关(P<0.01),而与肿瘤的复发、多发无明显相关(P>0.05),非肿瘤性膀胱组织未见Ⅳ型胶原染色。结论 Ⅳ型胶原的表达与膀胱癌浸润和转移相关,对膀胱癌的预后判断及治疗具有一定的意义。     

Nitric oxide in the developing kidney

Although nitric oxide (NO) has a well-established role in regulating renal function in the adult, recent studies point to perhaps an even more critical role for NO in maintaining basal renal blood flow (RBF) and glomerular filtration rate (GFR) in the developing kidney. The immature kidney has enhanced renal hemodynamic and functional responses to stimulation and inhibition of NO synthesis when compared with the adult, and these increased responses are not mediated by prostaglandins. Increased intrarenal activity of NO in the developing kidney counter-regulates the highly activated renin angiotensin system by modulating the angiotensin II-mediated vasoconstriction of the developing renal vasculature, the angiotensin II effects on GFR, as well as renin release. Localization studies demonstrate that NO acts on neonatal RBF and stabilization of GFR through an intrarenal distribution of the synthesizing enzyme, nitric oxide synthase, that is different from that of the adult. The developing kidney is dependent on NO to maintain RBF and GFR during periods of hypoxemia, protecting against renal injury, such as acute renal failure. In summary, NO is vital in the developing kidney to maintain normal physiological function and to protect the immature kidney during pathophysiological stress. Received February 12, 1996; received in revised form and accepted February 28, 1996     

成年人肾小球基底膜厚度及基底膜变薄标准研究

目的 了解成年人肾小球基底膜（GBM）厚度及拟建议薄基底膜肾病（TBMN）的GBM弥漫变薄的标准。 方法 选取肾癌根治性切除患者29例,分析性别、年龄、尿常规、Scr以及既往史、家族史等临床资料。选取远离病灶的肾皮质组织,进行光镜、免疫荧光及透射电镜检查,并进行GBM厚度测量和Ⅳ型胶原α3、α5链免疫荧光检查。 结果 29例中,男15例、女14例,年龄（55.9±14.9）岁（20~80岁）,所有病例均无肾脏病家族史。肾组织GBM厚度为（363.6±46.8） nm。GBM厚度与性别相关,男性为（384.0±41.7） nm,女性为（335.0±39.2） nm,差异有统计学意义（P = 0.008）。建议以均数减去两倍标准差作为GBM变薄的标准,即GBM厚度＜270 nm。 结论 成年人肾组织的GBM厚度为（363.6±46.8） nm。GBM厚度和性别相关,男性GBM厚度大于女性,差异有统计学意义。TBMN的GBM弥漫变薄的诊断标准建议为GBM厚度＜270 nm,也建议今后制定TBMN标准中应考虑男女的差异。     

Oxygen reduces accumulation of type IV collagen in endothelial cell subcellular matrix via oxidative stress

Anchorage-dependent cells in culture attach initially to proteins adsorbed to the culture substrate from the medium, and produce and deposit a subcellular matrix during the course of the cultivation. The aim of this study was to determine whether the concentration of O(2) in the culture atmosphere affects the accumulation of type IV collagen and laminin under human endothelial-cell monolayers. Enzyme-linked immunoassays on decellularized polystyrene substrates showed less type IV collagen, but not less laminin, under cells incubated in the standard atmosphere (5% CO(2) in air, i.e., approximately 20% O(2)) compared to an atmosphere of 5% O(2) and 5% CO(2) in N(2). Type IV collagen accumulation was inhibited via oxidative stress, because the inhibitory effect of 20% O(2) was antagonized by antioxidant ascorbic acid, and mimicked by prooxidant pyrogallol and exogenous H(2)O(2). Measurements of endogenous H(2)O(2) accumulation demonstrated that endothelial cells partially adapt to the high O(2) concentration. These results may have implications in endothelium modeling in vitro and in engineering of endothelial cell sheets and endothelialized vascular grafts.     

Persistent familial hematuria in children and the locus for thin basement membrane nephropathy

This study examined how often children with persistent familial hematuria were from families where hematuria segregated with the known genetic locus for the condition known as benign familial hematuria or thin basement membrane nephropathy (TBMN) at COL4A3/COL4A4. Twenty-one unrelated children with persistent familial hematuria as well as their families were studied for segregation of hematuria with haplotypes at the COL4A3/COL4A4 locus for benign familial hematuria and at the COL4A5 locus for X-linked Alport syndrome. Eight families (38%) had hematuria that segregated with COL4A3/COL4A4, and four (19%) had hematuria that segregated with COL4A5. At most, eight of the other nine families could be explained by disease at the COL4A3/COL4A4 locus if de novo mutations, non-penetrant hematuria or coincidental hematuria in unaffected family members was present individually or in combination. This study confirms that persistent familial hematuria is not always linked to COL4A3/COL4A4 (or COL4A5) and suggests the possibility of a further genetic locus for benign familial hematuria. This study also highlights the risk of excluding X-linked Alport syndrome on the basis of the absence of a family history or of kidney failure.     

Laminin, type IV collagen, and fibronectin in normal and cryptorchid human testes. An immunohistochemical study

An immunohistochemical study of laminin, type IV collagen, and fibronectin was carried out in the testes of normal men and in the cryptorchid and contralateral scrotal testes of cryptorchid men from 2 to 40 years of age. The integrated optical density (IOD) per unit area of the lamina propria was measured in the immunostained sections. Fibronectin was found throughout the thickness of the lamina propria of the seminiferous tubules and in the interstitial connective tissue. No differences between normal and cryptorchid testes were found. Laminin was observed in the innermost part of the lamina propria of the seminiferous tubules and surrounding the endothelium of blood capillaries from infancy. No differences were found between normal and cryptorchid testes in the prepubertal period. In adult cryptorchid testes, laminin formed more numerous and deeper invaginations towards the seminiferous epithelium than in normal adult testes. Type IV collagen appeared throughout the thickness of the lamina propria of normal testes as well as in the wall of interstitial blood vessels. From infancy, the lamina propria of seminiferous tubules, but not blood vessel walls, showed lesser immunostaining for type IV collagen and a lower IOD of this component than did control tests from men of the same age. No differences between unilateral and bilateral cryptorchidism were found. The contralateral scrotal testes of cryptorchid males showed intermediate immunostaining for type IV collagen between that of normal control testes and that of cryptorchid testes. These findings suggest that the lamina propria of seminiferous tubules is lesioned at an early age in both cryptorchid and contralateral scrotal testes of cryptorchid men.     

Expression of complement regulatory proteins in the developing human kidney

Complement homologous restriction factor CD59 and complement receptor CD35 are typically involved in the regulation of the host defense system. Recent observations in the human fetal kidney suggest a further role for complement cell surface regulators CD35 and CD59 in kidney development and maturation. We investigated this possible role by localizing CD35 and CD59 protein and mRNA in the developing and adult kidney. Adult tissue and fetal tissue ontogeny were analyzed using immunohistochemistry and in situ hybridization. CD35 protein and mRNA were localized to the podocyte of the glomerulus in the human fetal and adult kidney. Expression was initiated after vascularization of the early developing glomerulus. CD59 protein and mRNA were observed as early as 8 weeks’ gestation and were localized primarily to the ureteric duct epithelium in the fetal kidney and predominantly to the collecting duct in the adult. Interestingly, CD59 expression was translocated from the basolateral surface in the fetal kidney to the apical surface in the adult kidney. The specific spatial and temporal expression of CD35 and CD59 suggests a possible role for these complement regulatory proteins in renal cell differentiation. Received: 29 October 1999 / Revised: 21 March 2000 / Accepted: 22 March 2000     

Glomerular basement membrane outpockets and glomerular growth in the postnatal development of the rat kidney

Distribution of glomerular basement membrane (GBM) outpockets and dimensional growth of glomeruli were studied in the maturing stage of rat glomeruli after completion of nephrogenesis. We observed the postnatal rat glomeruli from 5 to 60 days of age by transmission electron microscopy and estimated the structural development of glomeruli by computerized morphometry. On day 5, the GBM was double in structure, possessing an epithelial and endothelial lamina densa. After day 10, the lamina densa of the GBM was single and sent branches toward the epithelial side making outpockets. There is no change in the distributional pattern of the outpockets, at least from day 10 to day 60, although they decreased considerably in number between days 20 and 40. They were found almost exclusively on the peripheral surface of the glomerulus. The rat glomeruli increased in volume constantly in this period, and the capillary volume increased more significantly than the mesangial volume. The GBM surface area increased in parallel with the glomerular tuft volume. The growing mode of capillaries was different before and after day 40; namely before day 40 elongation was predominant, whereas after day 40 widening was more pronounced. These results indicate that if the outpockets are the other site of GBM assembly after fusion of double basement membranes, the GBM must be redistributed from the peripheral to the paramesangial site to enable elongation and branch formation of capillaries during the growth of glomeruli. Received April 27, 1995; received in revised form and accepted October 19, 1995     

Procollagen type III N-peptide and type IV collagen 7S-domain in the sera of breast cancer patients

We investigated the usefulness of serum levels of procollagen type III N-peptide (P III P) and the type IV collagen, 7S-domain, (IV-C) as markers of recurrent and metastatic breast cancer. Serum P III P and IV-C levels were found to be significantly higher in patients who showed postoperative recurrence, with 76.9% of P III P-positive cancer bearing patients and 68.0% of IV-C-positive cancer bearing patients having metastases in the bone and/or liver. Furthermore, 66.7% of all patients with metastases in the bone and/or liver were P III P-positive and 75.6% were IV-C-positive. Patients with liver metastases were either P III P- or IV-C-positive, and the levels were higher than those in patients with bone metastases. Thus, a positive correlation of serum P III P and IV-C levels was observed. These findings suggest that both serum P III P and IV-C levels are useful markers of recurrent and metastatic breast cancer, especially of disease in the bone and/or liver.     

Two novel alternatively spliced 9-bp exons in the COL4A5 gene

The existence of an alternatively used 18-bp sequence has been described in type IV collagen α5 chain mRNA from human kidney (Guo et al., Kidney Int 44, 1993). In this study we have shown that this sequence is encoded by two exons, termed 41A and 41B, that are located in a 9-kb intron 41 that was sequenced in its entirety. The sequences of exon 41A and 41B were shown to be present in mRNAs from a variety of tissues. Received: 13 April 2000 / Revised: 20 June 2000 / Accepted: 20 June 2000