Differential expression of two basement membrane collagen genes, COL4A6 and COL4A5, demonstrated by immunofluorescence staining using peptide-specific monoclonal antibodies.

Genes for the human alpha 5(IV) and alpha 6(IV) collagen chains have a unique arrangement in that they are colocalized on chromosome Xq22 in a head-to-head fashion and appear to share a common bidirectional promoter. In addition we reported a novel observation that the COL4A6 gene is transcribed from two alternative promoters in a tissue-specific manner (Sugimoto, M., T. Oohashi, and Y. Ninomiya. 1994. Proc. Natl. Acad. Sci. USA. 91:11679-11683). To know whether the translation products of both genes are colocalized in various tissues, we raised alpha 5(IV) and alpha 6(IV) chain-specific rat monoclonal antibodies against synthetic peptides reflecting sequences near the carboxy terminus of each noncollagenous (NC)1 domain. By Western blotting alpha 6(IV) chain-specific antibody recognized 27-kD monomers and associated dimers of the human type IV collagen NC1 domain, which is the first demonstration of the presence in tissues of the alpha 6(IV) polypeptide as predicted from its cDNA sequence. Immunofluorescence studies using anti-alpha 6(IV) antibody demonstrated that in human adult kidney the alpha 6(IV) chain was never detected in the glomerular basement membrane, whereas the basement membranes of the Bowman's capsules and distal tubules were positive. The staining pattern of the glomerular basement membrane was quite different from that obtained with the anti-alpha 5(IV) peptide antibody. The alpha 5(IV) and alpha 6(IV) chains were colocalized in the basement membrane in the skin, smooth muscle cells, and adipocytes; however, little if any reaction was seen in basement membranes of cardiac muscles and hepatic sinusoidal endothelial cells. Thus, both genes are expressed in a tissue-specific manner, perhaps due to the unique function of the bidirectional promoter for both genes, which is presumably different from that for COL4A1 and COL4A2.

Glycosphingolipid patterns of rat kidney. Dependence on age and sex.

The composition and quantity of glycosphingolipids in kidney from 3-50-week-old male and female rats have been investigated. Glycosphingolipids were separated by high-performance liquid chromatography on a diethylaminoethyl-porous glass and porous glass column followed by high-performance thin-layer chromatography. The quantity of neutral glycolipids in male rat kidney decreased significantly between 3 and 25 weeks; the quantity in 25-week-old kidney became half the quantity found in 3-week-old kidney and remained constant thereafter. The quantity of neutral glycolipids in female rat kidney did not show age-dependent changes. The composition of neutral glycolipids revealed by high-performance thin-layer chromatography was constant, regardless of age and sex. Monoglycosylceramide and tetraglycosylceramide were the major compoments at all ages in both sexes. In contrast to the patterns of neutral glycolipids, ganglioside content was higher in female rats than males, and total ganglioside content increased in both sexes with increasing age. The most remarkable change was an increase of disialoganglioside in female rat kidney between 5 and 25 weeks. The ratio of disialogangliosides to monosialogangliosides (D/M value) in female rat kidney increased 4-fold during this period. The ganglioside pattern in female rat kidney was affected by spaying. The quantity of disialogangliosides decreased rapidly on the 7th day after spaying, but the level went back to normal on the 14th day after spaying. The data strongly suggest that sex hormones influence glycosphingolipid synthesis in rat kidney.

Properties of a coenzyme, pyrroloquinoline quinone: generation of an active oxygen species during a reduction-oxidation cycle in the presence of NAD(P)H and O2.

The oxidation of NAD(P)H by pyrroloquinoline quinone (PQQ) was non-enzymatically carried out at physiological pH in the presence of O2. The PQQ-NAD(P)H system requires about 1 mol of O2 for the oxidation of 1 mol of NAD(P)H. The oxidation of NAD(P)H occurred at a pseudo-first-order rate with respect to NAD(P)H and was of zero order with respect to PQQ concentration in in the presence of O2: k0[PQQ] [NAD(P)H] = k1 [NAD(P)H], where k0[PQQ] = k1, in which [PQQ] represents the initial concentration of PQQ. k0 values for NADH and NADPH were 3.4.10(2) M-1.min-1 and 2.0.10(2) M-1.min-1, respectively, at 25 degrees C and at 258 microM O2 (initial concentration). The system produced O-2, probably by the interaction of PQQ.H and/or NAD(P).with O2, during the oxidation of NAD(P)H. PQQH2 and PQQ.H were easily oxidized to PQQ in the presence of O2, yielding H2O2.

Actual invasive potential of human hepatocellular carcinoma revealed by in situ gelatin zymography.

BACKGROUND: The matrix-degrading proteinases are believed to play an important role in the invasion and metastasis of hepatocellular carcinoma (HCC), but no one has ever seen the in situ matrix-degrading activity in HCCs. PURPOSE: To demonstrate the cellular localization of actual gelatinolytic activity and to investigate the invasive potential of human HCC. EXPERIMENTAL DESIGN: HCC cases (30) were subjected to in situ gelatin zymography and SDS-gelatin gel zymogram. RESULTS: In situ gelatin zymography revealed a heterogeneous gelatinolytic activity in HCC cells, as well as stromal cells of noncancerous livers. The gelatinolytic intensity was stronger in 15 HCC nodules than in the corresponding noncancerous livers and was significantly associated with the cancer invasion to the capsule of the HCCs and to the portal veins. An intense gelatinolytic activity was detected in HCC cells in the front of tumor invasion. SDS-gelatin gel zymogram revealed gelatinases A and B that were mostly in latent forms. CONCLUSIONS: The present study demonstrates high gelatinolytic activity at the invasive front of HCCs at a cellular level and that HCC has an invasive potential with the gelatin (matrix)-degrading metalloproteinases. Furthermore, it suggests the importance of the activation mechanism of gelatinolytic enzymes in the invasion and metastasis of HCCs.

Quantitative immunoelectron-microscopic analysis of the type IV collagen alpha1-6 chains in the glomerular basement membrane in childhood thin basement membrane disease.

AIM: Thin basement membrane disease (TBMD) is characterized histologically by diffuse thinning of glomerular basement membrane (GBM). Although recent genetic analysis has shown that TBMD might be included within type IV collagen disorders, conventional immunohistochemical studies demonstrated normal labeling of type IV collagen alpha chains in the GBM. We have, however, successfully used confocal laser scanning microscopy to demonstrate a significantly reduced signal of type IV collagen alpha5 chain (alpha5(IV)) along capillary walls in TBMD. In order to further understand the association of type IV collagen with TBMD, we used immunoelectron microscopy to examine renal biopsies from 6 children with TBMD and six control children with minimal change nephrotic syndrome. METHODS: Ultrathin sections of LR gold resin were incubated with a rat monoclonal antibody against human alpha1(IV), alpha2(IV), alpha3(IV), alpha4(IV) alpha5(IV) or alpha6(IV) followed by colloidal gold conjugated goat anti-rat IgG. After taking electron micrographs, the labeling was quantitatively evaluated in the area occupied by the segments of basement membrane. The basement membrane was divided into three equal segments viz. subepithelial side, central portion and subendothelial side. RESULTS: In control subjects, the number of gold particles for alpha1(IV) or alpha2(IV) was significantly greater in the subendothelial side and central portion than in the subepithelial side of the GBM, whilst alpha3(IV), alpha4(IV) or alpha5(IV) labeling was significantly more prominent in the central portion compared to the subepithelial and subendothelial side of the GBM. TBMD samples showed a similar distribution pattern except that the subepithelial side and central portion of the GBM had a significantly reduced amount of alpha5(IV) antigen compared to control subjects. CONCLUSION: This is the first report demonstrating a diminished labeling intensity of alpha5(IV) in the central portion and subepithelial side of the GBM in renal biopsy specimens from patients with TBMD. These findings suggest that an abnormality of alpha5(IV) might possibly be associated with the pathogenesis of TBMD.

Localization of type IV collagen a 1 to a 6 chains in basement membrane during mouse molar germ development.

The dental basement membrane (BM) putatively mediates epithelial-mesenchymal interactions during tooth morphogenesis and cytodifferentiation. Type IV collagen alpha chains, a major network-forming protein of the dental BM, was studied and results disclosed distinct expression patterns at different stages of mouse molar germ development. At the dental placode and bud stage, the BM of the oral epithelium expressed alpha 1, alpha 2, alpha 5 and alpha 6 chains while the gubernaculum dentis, in addition to the above four chains, also expressed a 4 chain. An asymmetrical expression for alpha 4, alpha 5 and alpha 6 chains was observed at the bud stage. At the early bell stage, the BM associated with the inner enamel epithelium (IEE) of molar germ expressed alpha 1, alpha 2 and alpha 4 chains while the BM of the outer enamel epithelium (OEE) expressed only alpha 1 and a 2 chains. With the onset of dentinogenesis, the collagen a chain profile of the IEE BM gradually disappeared. Howeverfrom the early to late bell stage, the gubernaculum dentis consistently expressed alpha 1, alpha 2, alpha 5 and a 6 chains resembling fetal oral mucosa. These findings suggest that stage- and position-specific distribution of type IV collagen alpha subunits occur during molar germ development and that these changes are essential for molar morphogenesis and cytodifferentiation.

Identification of a 450-kDa human epidermal autoantigen as a new member of the plectin family.

The serum from an individual with a subepidermal blistering disease was previously shown to recognized a 450-kDa epidermal autoantigen. The molecular structure of this antigen was investigated by screening a human keratinocyte cDNA library with the patient's serum. One clone, with a 276-bp cDNA insert, that encoded an epitope recognized by the serum was isolated. Rabbit polyclonal antibodies that were prepared against the corresponding fusion protein recognized the 450-kDa epidermal antigen and stained the basal keratinocytes in human epidermis. This clone was used for further screening of the original keratinocyte and HeLa cell cDNA libraries. Two different, but closely related, 0.8- and 2.0-kb cDNAs were isolated, and their deduced amino acid sequences indicated that the encoded proteins belonged to the plectin family. Northern blot analysis of total RNA from human keratinocytes with these cDNA inserts as probes detected RNAs of approximately 12-13 kb. The 0.8-kb cDNA hybridized to polyadenylated RNA species from human skeletal muscle, heart, lung, and kidney, whereas the 2.0-kb cDNA hybridized to transcripts present only in kidney and lung. Southern blot analysis of genomic DNA from the human placenta revealed similar, but not identical, patterns of hybridization with the 0.8- and 2.0-kb cDNAs. Data suggest that the 0.8- and 2.0-kb cDNAs encode two different proteins but are derived from the same gene.

Molecular genetic and immunohistochemical study of autosomal recessive Alport's syndrome.

A DNA analysis of autosomal type IV collagen alpha3 and alpha4 chain genes (COL4A3 and COL4A4) and an immunohistochemical study of type IV collagen alpha1 to alpha6 chains were performed in an inbred family with autosomal recessive Alport's syndrome (AS). A linkage study using polymorphic markers around the COL4A3/COL4A4 genes clearly differentiated the affected patients from healthy individuals. These patients were homozygous for all markers analyzed, whereas their parents were heterozygotes. Because of the large size of both the genes and the heterogeneous range of the mutations of these genes, linkage analysis by using highly polymorphic markers is still the method of choice in genetic counseling for autosomal recessive AS, as well as for the X-linked form. Although the distribution of alpha1 and alpha2 chains in the index patient and her affected sister were normal, the alpha3 and alpha4 chains were completely defective in the renal basement membrane (BM). The alpha5 chain could be found in Bowman's capsular basement membrane (BCBM) but not in the glomerular basement membrane (GBM). In addition, our study showed, for the first time, that the alpha6 chain in BCBM is spared in this form of AS. This abnormal pattern of type IV collagen could be a useful tool for differentiation of the autosomal recessive type from the X-linked type of AS.

Ultrastructural appearance of renal and other basement membranes in the Bull terrier model of autosomal dominant hereditary nephritis.

Bull terrier hereditary nephritis may represent a model for autosomal dominant Alport's syndrome because affected dogs have the typically lamellated glomerular basement membrane (GBM) and father-to-son disease transmission occurs. This study examined the ultrastructural appearance of the renal and extrarenal basement membranes and their composition in affected Bull terriers. Affected stillborn animals and puppies had subepithelial frilling and vacuolation of the GBM. In adult dogs, lamellation was common, and subepithelial frilling and vacuolation were less prominent. Foot-process effacement and mesangial matrix expansion occurred frequently. Basement membranes in the glomeruli, tubules, and Bowman's capsule were significantly thickened and often mineralized. Immunohistochemical examination showed alpha 1(IV) and alpha 2(IV) collagen chains in all renal basement membranes; alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains in the GBM, distal tubular basement membrane, and Bowman's capsule; and the alpha 6(IV) chain in Bowman's capsule. Conversely, the basement membranes from the affected Bull terrier cornea, lens capsule, retina, skin, lung, and muscle had a normal ultrastructural appearance and were not thickened compared with membranes in normal age-matched dogs. The distribution of basement membrane abnormalities in Bull terrier hereditary nephritis may occur because the defective protein is present exclusively or more abundantly in the kidney and is structurally more important in the kidney or because of local intrarenal stresses.

Organization and expression of basement membrane collagen IV genes and their roles in human disorders.

Six distinct genes have been identified as belonging to the type IV collagen gene family. They can be organized into three sets, i.e., COL4A1/COL4A2, COL4A3/COL4A4, and COL4A5/COL4A6, which are localized on three different chromosomes in humans, 13, 2, and X, respectively. Within each set the genes are aligned head-to-head and their expression is regulated by bidirectional promoters between the genes. Transcriptional regulation of the COL4A1/COL4A2 set has been well characterized. The transcription of COL4A6 seems to be controlled by two alternative promoters. While collagen IV molecules composed of alpha1 and alpha2 chains are broadly distributed, molecules comprising combinations of the other four chains, alpha3-alpha6, are important components of specialized basement membranes. The precise chain composition of triple-helical molecules assembled from the alpha3-alpha6 chains is not entirely clear, but it is hypothesized that alpha3-alpha5 chains and alpha5 and alpha6 chains form heterotrimeric molecules. Several pieces of evidence indicate that alpha3/alpha4/alpha5 molecules and alpha5/alpha6 molecules are components of the basement membrane network. This helps explain the observation that the kidney and skin basement membranes from patients with Alport syndrome caused by mutations in the alpha5 coding gene, COL4A5, are defective in the alpha3, alpha4, and alpha6 chains together with the alpha5 chain. Large deletions involving the COL4A5 and COL4A6 genes have been found in rare cases of diffuse leiomyomatosis associated with Alport syndrome.

Differential expression of mouse alpha5(IV) and alpha6(IV) collagen genes in epithelial basement membranes.

We first completed the primary structure of the mouse alpha5(IV) and alpha6(IV) chains, from which synthetic peptides were produced and a chain-specific monoclonal antibodies were raised. Expression of collagen IV genes in various basement membranes underlying specific organ epithelia was analyzed by immunohistochemical staining using these monoclonal antibodies and other antibodies from human and bovine sequences. It was possible to predict the presence of the three collagen IV molecules: [alpha1(IV)](2) alpha2(IV), alpha3(IV)alpha4(IV)alpha5(IV), and [alpha5(IV)](2)alpha6(IV). In skin basement membrane two of the three forms, [alpha1(IV)](2)alpha2(IV) and [alpha5(IV)](2)alpha6(IV), were detected. The alpha3(IV)alpha4(IV)alpha5(IV) molecule was observed as the major form in glomerulus, alveolus, and choroid plexus, where basement membranes function as filtering units. The molecular form [alpha5(IV)](2)alpha6(IV) was present in basement membranes in tubular organs such as the epididymis, where the tubes need to expand in diameter. Thus, the distribution of the basement membranes with different molecular composition is consistent with tissue-specific function.

Diffuse mesangial sclerosis associated with Kawasaki disease: an analysis of alpha chains (alpha 1-alpha 6) of human type IV collagen in the renal basement membrane.

A case of diffuse mesangial sclerosis (DMS) associated with Kawasaki disease is reported. A previously healthy Japanese girl, aged 4 months, presented with clinical features of Kawasaki disease. At week 10 of the illness, she developed the nephrotic syndrome, which was refractory to steroid therapy. Renal biopsy demonstrated a diffuse mesangial proliferative glomerulonephritis with microcystic tubular dilatation and, ultrastructurally, marked thinning of the lamina densa in the glomerular basement membrane (GBM) and the tubular basement membrane (TBM) of the proximal tubule. She went into chronic renal failure and died at the age of 11 months. At autopsy, the kidney revealed DMS. Histologically, we found Finnish microcystic disease in its early stages in the biopsy. Using a newly developed monoclonal antibody, we analysed the alpha chains (alpha 1-alpha 6) of type IV collagen in the GBM and TBM. There was no defective constitution of alpha chains on the thin GBM, but the thin TBM of the microcystic proximal tubule showed a weak or discontinuous reactivity for alpha 1 and alpha 2 chains, suggesting faulty formation of the basement membrane. The sclerosing glomeruli of the DMS did not depend on collapse of the GBM, which was positive for alpha 3-alpha 5 chains, but mainly on the proliferation of mesangial matrix, which was positive for alpha 1 and alpha 2 chains.

Nephritogenicity and alpha-chain composition of NC1 fractions of type IV collagen from bovine renal basement membrane.

Nephritogenicity (anti-GBM-nephritis-inducing activity) and alpha-chain composition of globular-do-main (NCI) fractions of type IV collagen from bovine renal, pulmonary, and placental basement membranes (BMs) was examined by injecting these fractions with adjuvant into WKY/NCrj rats and by Western blotting using epitope-defined monoclonal antibodies to the six different alpha chains of type IV collagen. A purified nephritogenic fraction from renal BM contained alpha 1-alpha 6(IV)NCI, whereas a non-nephritogenic fraction contained only alpha 1-alpha 2(IV)NCI. Renal and pulmonary NCI had strong nephritogenic activity: placental NCI had weak activity. The renal and pulmonary fractions contained alpha 1-alpha 6(IV)NCI, and the placental fraction had a large amount of alpha 1-alpha 2(IV)NCI and a very small amount of alpha 3-alpha 6(IV)NCI. Immunohistochemical study of bovine renal BM with the monoclonal antibodies revealed that bovine glomerular BM contained alpha 1-alpha 5(IV) chains, but not the alpha 6(IV) chain. The absence of alpha 6(IV) chain in glomerular BM in bovine and in humans indicates that alpha 6(IV) chain is not a target antigen of anti-GBM nephritis. Nephritogenicity is apparently a property of alpha 3-alpha 5(IV)NCI.

Normal distribution of collagen IV in renal basement membranes in Epstein's syndrome.

BACKGROUND: Epstein's syndrome is defined as a subtype of Alport's syndrome, and is distinguished from the other subtypes by accompanying macrothrombocytopenia. Mutations in collagen IV genes are known to be involved in the pathogenesis of typical Alport's syndrome. However, the presence of an underlying genetic defect has not been demonstrated in Epstein's syndrome. AIM: To clarify the involvement of collagen IV in Epstein's syndrome. METHODS: The distribution of the alpha(IV) chain was studied in renal specimens obtained from three patients with Epstein's syndrome using chain specific monoclonal antibodies and an antigen retrieval procedure. RESULTS: The patients showed a normal distribution of alpha(IV) chains: alpha 1(IV) and alpha 2(IV) were expressed ubiquitously, whereas expression of alpha 3(IV) through to alpha 6(IV) chains was limited to the glomerular basement membrane, Bowman's capsular basement membrane, and/or a portion of the tubular basement membrane. CONCLUSIONS: These results suggest that genes other than those encoding alpha(IV) chains are responsible for the pathogenesis of Epstein's syndrome.

Clinicohistological study of oligodendroglioma and oligoastrocytoma.

The clinical and histological characteristics of oligodendroglioma and oligoastrocytoma were investigated in patients, mainly adults with supratentorial tumors, who were treated with surgery and radiotherapy, and with chemotherapy for recurrent, anaplastic tumors, or both. The median survival time was 13.2 years for oligodendroglioma (four patients), 12.7 years for anaplastic oligodendroglioma (five patients), 13.5 years for oligoastrocytoma (seven patients), and 4.8 years for anaplastic oligoastrocytoma (four patients). Two of three recurrent oligodendrogliomas and two of two recurrent oligoastrocytomas showed malignant transformation. Minigemistocytes were sometimes recognized in recurrent tumors and had a sinister prognosis. Oligodendroglioma and oligoastrocytoma may transform into each other at recurrence.

Diffuse thin glomerular basement membrane in association with idiopathic membranous glomerulonephritis.

Here we present a rare report of diffuse thin glomerular basement membrane (dTGBM) with idiopathic membranous glomerulonephritis (IMGN). dTGBM was found in 11 (2.04%) of 539 adult patients with IMGN. The male:female ratio was 1:1.2. Sixty patients with IMGN alone (matched for age and sex) were adjusted and analyzed as control patients. There was no significant difference in the clinical and laboratory data between the two groups except for the microhematuria, which was more frequently found in the dTGBM associated IMGN group (dTGBM + IMGN) (10/11, 91%) than in the IMGN group (19/60, 31.7%), and the difference was significant (p <0.001). However, no statistical difference was found in morphological parameters between the two groups except in the thickness of the glomerular basement membrane (GBM). In this study, using specific monoclonal antibodies against the chains of type IV collagen, no change in quality or location (in aberrant or no expression) was found in the collagenous composition of the dTGBM compared to the normal GBM. No prominent thickening or transformation of the GBM, which is characteristic for MGN, developed in the dTGBM + IMGN cases or IMGN. As a result, we could not detect any difference in the prognosis between these two groups. These findings support the view that IMGN can occur in patients with dTGBM, and the dTGBM patients may recover completely. The possible coexistence of these diseases was thus hypothesized due to the persisting microhematuria.

Time-dependent changes of decorin in the infarct zone after experimentally induced myocardial infarction in rats: comparison with biglycan.

Decorin, a small dermatan sulphate proteoglycan, has been postulated to interact with other components of the extracellular matrix. We examined time-dependent changes of decorin in the infarct zone after experimentally induced myocardial infarction in rats by Northern blotting, in situ hybridization, and immunohistochemistry. The expression of decorin mRNA was compared to that of biglycan mRNA. Northern blotting demonstrated that the decorin mRNA expression was not increased in the infarct zone on day 2, while increased biglycan mRNA was observed at that time (average 3.1-fold increase). Decorin mRNA expression was increased on day 7, and reached a peak (average 2.2-fold increase) around day 14. Biglycan mRNA expression also reached a peak level around day 14 (average 13.3-fold increase). In situ hybridization revealed that mRNA signals for decorin did not appear in the infarct zone on day 2, while biglycan mRNA signals were observed. Decorin mRNA signals were observed in spindle-shaped mesenchymal cells in the infarct peripheral zone on day 7. The decorin mRNA signals appeared later than those of biglycan. Immunopositive staining for decorin was observed in the infarct zone on day 7. The present results demonstrated a time-dependent increase in decorin mRNA expression in mesenchymal cells in the infarct zone in rats. Decorin mRNA appeared later and was increased to a lower extent in the infarct zone than biglycan mRNA.

Sequential changes in the localization of the type IV collagen alpha chain in the infarct zone: immunohistochemical study of experimental myocardial infarction in the rat.

Collagen, as a component of the extracellular matrix, have a role in the healing process after myocardial infarction (MI). For type IV collagen, a major structural protein present in the basal membrane of myocytes, six alpha chains [alpha 1 (IV)-alpha 6(IV)] have been identified. We examined the sequential changes in the appearance and localization of the alpha 1 (IV)-alpha 5(IV) after experimental MI in rats. Hearts were excised from 1 day to 8 weeks after permanent left coronary artery ligation. Immunohistochemical staining with monoclonal antibodies was performed. On day 3, staining for both alpha 1(IV) and alpha 2(IV) first appeared, forming a wavy pattern in the infarct peripheral zone, and the staining was not restricted to the cell membrane. The staining intensity and distribution for both alpha 1(IV) and alpha 2(IV) in the peripheral zone then gradually increased, reaching a maximum around day 7. The distribution progressed from the peripheral to the central zone of the infarct for 1-2 days, reaching the center point after 2 weeks. The staining distribution gradually decreased after reaching the maximum, but the staining had not completely disappeared at 8 weeks. In contrast, no positive staining for alpha 3(IV), alpha 4(IV) or alpha 5(IV) was observed at any time during the 8-week observation period. Thus, the present results demonstrated that in rats, type IV collagen consisting of alpha 1 and alpha 2 chains appears in the infarct zone at a relatively early phase after MI, indicating that type IV collagen composed of alpha 1 and alpha 2 chains contributes to infarct healing.

Expression of the alpha6 chain of type IV collagen in glomerular basement membranes of healthy adult dogs.

OBJECTIVE: To evaluate expression of the alpha6 chain of type IV collagen in the glomerular basement membranes (GBM) of healthy dogs. SAMPLE POPULATION: Kidney specimens from 12 healthy dogs. For comparison, kidney specimens from 8 human subjects between 25 and 83 years old also were evaluated. PROCEDURE: Sections were immunolabeled with a monospecific antibody that cross-reacts with human and canine alpha6(IV) chains and examined by means of fluorescence microscopy. RESULTS: Immunolabeling of the alpha6(IV) chain was not observed in GBM of 6 dogs < or = 30 months old but was observed in GBM of the remaining 6 dogs, all of which were > or = 45 months old. Expression of the alpha6(IV) chain was not observed in GBM of the human subjects, regardless of the age of the subject. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that the alpha6(IV) chain is expressed in GBM of healthy dogs, but the expression is age-dependent. Composition and structural organization of type IV collagen in the GBM of healthy adult dogs is different from that described for other species.

New form of X-linked dominant hereditary nephritis in dogs.

OBJECTIVE: To determine features of a new form of hereditary nephritis (HN) in dogs. ANIMALS: Parents and 16 first-generation offspring (8 males, 8 females). PROCEDURE: Adolescent dogs that developed renal failure were euthanatized and necropsied. Unaffected dogs were monitored until they were at least 2 years old. Studies included light and electron microscopy of kidneys obtained from affected and unaffected dogs and immunolabeling for collagen-IV chains in renal and epidermal basement membranes (BM). The nucleotide sequence of a portion of exon 35 of the COL4A5 gene was determined in genomic DNA isolated from affected and unaffected males. RESULTS: 7 of 8 male and 2 of 8 female offspring had proteinuria and juvenile-onset chronic renal failure, which progressed more rapidly in the males. Labeling for alpha3-alpha6(IV) chains was completely absent in renal BM of affected males and segmentally absent in affected females. Expression of alpha1-alpha2(IV) chains in glomerular BM (GBM) of affected dogs was increased. Labeling for alpha5-alpha6(IV) chains in epidermal BM was absent in affected males and segmental in affected females. Ultrastructural changes characteristic of HN were observed in GBM of affected dogs. The sequence of exon 35 of COL4A5 was normal in affected dogs. CONCLUSIONS: This renal disease is an example of X-linked dominant HN, with typical abnormalities of GBM ultrastructure and alpha(IV) chain expression. CLINICAL RELEVANCE AND IMPLICATIONS FOR HUMAN MEDICINE: Dogs with this naturally acquired progressive renal disease can be used to investigate the pathogenesis and treatment of similar disorders in human beings and dogs.