Identification of Type VI Collagen Synthesizing Cells in Human Diabetic Glomerulosclerosis Using Renal Biopsy Sections

Although the role of extracellular matrices in the development of glomerulosclerosis has been discussed widely, the cellular origin of type VI collagen in diabetic nephropathy (DN) has remained relatively unexplored. This study reports the distribution and cellular origin of type VI collagen in DN. Type VI collagen‐specific oligonucleotide probes and monoclonal antibody were used to assess the relative expression of mRNA for \alpha1 (VI) chain and its translated protein in paraffin‐embedded renal biopsy sections of DN. By immunohistochemistry, compared to the control, increased deposition of type VI collagen was noted in the diffuse and nodular lesions of diabetic glomeruli. For cellular localization of type VI collagen mRNA, paraffin‐embedded renal sections of the control and DN were hybridized in situ with digoxigenin (Dig)‐labeled antisense oligo‐DNA probe complementary to a part of \alpha1 (VI) mRNA. In comparison to the control kidney sections, increased numbers of intraglomerular cells (both mesangial and epithelial cells) were positive for α1 (VI) mRNA in renal biopsy sections of DN. From the results, we conclude that overexpression of type VI collagen by intraglomerular cells with its increased deposition might significantly contribute to the glomerulosclerosis found in DN.


Introduction
Diabetes mellitus (DM) is associated with multiple connective tissue changes, including generalized thickening of basement membranes. About 40% of DM patients develop nephropathy [3]. In diabetic nephropathy (DN), increased accumulation of extracellular matrix (ECM) results in diffuse and nodular glomerulosclerosis. As the origin and the source of all the components of the ECM in DN has not yet been clearly understood, the identification of ECM producing cells is critical for a better understanding of the sclerotic process in DN. Several components of ECM including type III and type IV collagens have been intensively studied in DN and other renal diseases [7,14,15,17]. We have also reported increased deposition of type III and type IV collagens and their enhanced synthesis in diabetic glomerulosclerosis [16]. A few reports demonstrating distribution of type VI collagen in DN are found [12]. However, no report concerning the identification of cells synthesizing this collagen in human renal tissue has been found hitherto. In an attempt to further elucidate the composition of sclerotic matrix in diabetic glomeruli, we investigated the distribution of type VI collagen and its cellular origin in renal tissues biopsied from the patients with DN.

Kidney tissue
Renal biopsies from 15 patients with DM, with typical histologic changes of DN as described in the WHO classification of glomerular diseases [6], and 5 samples of renal tissue obtained in conjunction with renal surgery without any noticeable histological abnormality were used in this study as control. The renal biopsy tissue was processed for routine light and electron microscopic examinations. For the light microscopy, the tissue was fixed in 10% formalin and embedded in paraffin and were routinely processed by various staining methods, including hematoxylin-eosin (HE), periodic acid schiff (PAS), periodic acid schiff methenamine silver (PAM) and Masson's trichrome to examine the histological alterations. For the electron microscopy, the tissue was fixed in 2.5% glutaraldehyde, osmicated and embedded in Epon. The antigenic markers for IgG, IgA, IgM, C3 and C1q were also examined on the other portions of renal tissue section by immunofluorescence microscopy.

Immunohistochemistry
Paraffin sections (4 µm) were deparaffinized and endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol (30 min). After mild treatment with trypsin, the sections were reacted with goat serum to block nonspecific binding of monoclonal antibody (20 min). As immunohistochemical control, monoclonal antibody was substituted by either 0.01 M PBS or mouse IgG diluted with PBS (similar concentration as that of primary antibody). Monoclonal antibody against type VI collagen was purchased from Fuji Chemical and was diluted to 1 : 50 with PBS (pH 7.2). The sections were reacted with the primary antibody (1 h) and processed further by streptavidin-biotin method using Histofine SAB kit (Nichirei, Tokyo). The staining intensity of type VI collagen was graded semiquantitatively according to the following scale: (0) = no staining, (+) = weak staining, (++) = moderate staining, (+++) = strong staining.

In situ hybridization (ISH)
Oligo-DNA probes used: For ISH, digoxigenin (Dig)-labeled synthetic oligo-DNAs were used as probes. A sequence complementary to mRNA of human pro α1 (VI) chain (382-426) [5] was chosen to locate type VI collagen mRNA-positive cells. A computer-assisted search (GenBank) of the above antisense sequence as well as that of sense sequence revealed no significant homology with any of the known sequences other than that of pro α1 (VI) chain. The oligo-DNAs were synthesized by an automatic DNA synthesizer.

Labeling of oligo-DNAs by digoxigenin:
The oligo-DNAs were labeled at their 3 -end with Dig using digoxigenin tail labeling kit as recommended by Boehringer Mannheim. The digoxigenin labeling of the probes were verified immunochemically on nitrocellulose filter using anti-Dig antibody (Boehringer Mannheim).

Dot blot hybridization:
The hybridizabilities of the Dig-labeled oligo-DNA probes were confirmed by dot blot hybridization as reported [8,15]. Briefly, various amounts (1 pg-10 ng/spot) of sense α1 (VI) oligo-DNA was fixed into two nitrocellulose filters and were hybridized with Dig-labeled sense and antisense α1 (VI) oligo-DNAs. The hybrids were detected immunochemically using anti-Dig antibody.

Localization of type VI collagen by immunohistochemistry
Type VI collagen was predominantly present in the mesangium (+) and glomerular basement membrane (GBM) (+) of the control glomeruli (Fig. 1A). Compared to the control glomeruli, increased immunostaining (+++) for type VI collagen was recorded in nodular and diffuse lesions of the glomeruli in DN (Fig. 1B). In the late stage of glomerulosclerosis, i.e., in globally sclerotic glomeruli, immunostaining for type VI collagen was slightly lower than earlier stages of glomerulosclerosis, although the collagen distributed diffusely in the sclerotic matrix (Fig. 1C).

Detection of Dig-labeled pro α1 (VI) oligo-DNA hybridized on nitrocellulose filter by immunochemistry
When Dig-labeled antisense pro α1 (VI) oligo-DNA was hybridized with unlabeled sense pro α1 (VI) oligo-DNA on nitrocellulose filter, at least 10 pg of the sense oligo-DNA was detected on nitrocellulose filter. No hybridization signals were found when Dig-labeled sense pro α1 (VI) oligo-DNA was hybridized with unlabeled sense pro α1 (VI) oligo-DNA on nitrocellulose filter (data not shown).

Localization of α1 (VI) mRNA by ISH
It was found that occasionally glomerular epithelial cells and mesangial cells hybridized with Diglabeled antisense α1 (VI) oligo-DNA in normal kidney. In diabetic glomeruli, proportionally increased number of intraglomerular cells (mainly epithelial cells and infrequently mesangial cells) hybridized with Dig-labeled antisense pro α1 (VI) oligo-DNA ( Fig. 2B and C). However, the numbers of α1 (VI) mRNA positive cells were decreased in advanced sclerotic glomeruli. Control experiments have established the specificity of the positive hybridization signals (Fig. 2A).

Discussion
Type VI collagen is an ubiquitous protein and its monomer consists of three distinct polypeptide chains, α1 (VI), α2 (VI) and α3 (VI) chains. In this study, utilizing non-radioactive ISH technique, we localized α1 (VI) mRNA in control and diseased glomeruli. The cells containing pro α1 (VI) chain mRNAs were identified in formalin-fixed paraffin-embedded renal biopsy specimens obtained from the patients of DN. ISH was done using a synthetic oligo-DNA complementary to mRNA of human pro α1 (VI) chain. Since it is rather difficult to distinguish mRNA-positive cell types in the glomeruli, which consist of mesangial, epithelial and endothelial cells, the specific intraglomerular type VI collagen mRNA-positive cell types was identified in renal sections by counterstaining with periodic acid schiff (PAS) after ISH [16]. In the renal biopsy sections of DN, the positive signals were predominantly found in the glomerular epithelial cells and occasionally in the mesangial cells.
Increased α1 (VI) mRNA positive cells and its protein in diabetic glomeruli reflected the overproduction of the type VI collagen in DN. Our results correlated with the findings of Mohan et al. [10], who using radioimmunoassay, reported 2.8-fold higher level of type VI collagen in human diabetic glomerular basement membrane preparations. Similarly, Nerlich et al. [12] showed increased immunostaining for type VI collagen in human diabetic glomerulosclerosis. Oomura et al. [13] also reported increased accumulation for various types of collagens including type VI collagen in human glomerular diseases.
In the current study, we were able to specify the type VI collagen synthesizing cells in DN, but the triggering factors for the increased synthesis of type VI collagen in diabetic glomeruli is not clear from this study. Experimental studies revealed that glomerular cells had the potential to produce different types of collagens in altered environment. Rat mesangial cells were able to produce interstitial collagens including type III collagen [1] and when cells were grown in a high glucose medium accumulated more fibronectin, laminin and type IV collagen than the cells grown in a low glucose medium [4]. The hyperglycemia elevates the levels of glomerular mRNAs that encode type IV collagen in the experimental diabetic kidneys [9]. When rat sciatic nerves were exposed to high glucose concentrations, it up-regulated the mRNA levels of α1 (VI), α2 (VI) and α3 (VI) chains of type VI collagen and caused accumulation of type VI collagen in the perineurial cells, Schwann cells, and fibroblasts [11]. In human DN, increased in situ expression of α1 chain mRNAs for type III and type IV collagens were noted in the mesangial and glomerular epithelial cells [16]. By comprising the above findings, it is likely that the hyperglycemic environment of DM might induced the excessive synthesis of type VI collagen similar to that of other components of ECM and might subsequently contribute to the sclerotic process in DN.
In conclusion, excessive synthesis of type VI collagen by intraglomerular cells (glomerular epithelial and mesangial cells) might contribute significantly to the glomerulosclerosis in diabetic nephropathy.