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Tenascin Is Synthesized and Secreted by Rat Mesangial Cells in Culture and Is Present in Extracellular Matrix in Human Glomerular Diseases1 Luan D. Truong,2 Mark W. Majesky, and Jana Pindur angial staining for TN by immunohistochemical tech- L.D. Truong, MW. Majesky, J. Pindur, Department of niques and that, in glomerular diseases character- Pathology, Baylor College of Medicine. Houston, TX ized by the expansion of the mesangial matrix such L.D. Truong, Department of Pathology, The Methodist as diabetic glomerulosclerosis, the expanded mes- Hospital, Houston, TX angial matrix was stained positive for TN. These find- ings suggest that mesangial cells in culture synthe- (J. Am. Soc. Nephrol. 1994; 4:1771-1777) size TN and that TN is a component of the mesangial matrix; moreover, increased synthesis of TN may play a significant role in the pathogenesis of mesangial ABSTRACT sclerosis and glomerulosclerosis. Tenascin (TN) is a large oligomeric protein recently Key Words: Tenascin. mesangial cell. extracellular matrix described as a component of the extracellular ma- trix. The distribution of TN in adult kidney tissue has not been adequately evaluated, but preliminary T enascin (TN), a recently described component of data have suggested that TN is variably seen in rare the extracellular matrix, is a large oligomeric mesangial areas and in stroma surrounding some protein composed of six similar subunits joined to- tubules. The enlargement of the mesangial matrix gether at the amino terminus by disulfide bonds (1). (mesangial sclerosis) is a common feature of many Although there are minor differences among species, renal diseases and is thought to be partially related each subunit with molecular weight ranging from to oversynthesis of the normal components of the 1 90 to 280 kd is composed of a cysteine-rich amino mesangial matrix (collagen type IV, laminin, fibro- acid terminal domain followed by epidermab growth nectin, and heparan sulfate proteoglycans) by mes- factor-bike homologous repeats, fibronectin Type III homologous repeats, and a fibrinogen (3 and #{244}chain angial cells. However, the possibility that mesangial homologous domain at the carboxyl terminal end ( 1 - cells are also the source of other extracellular matrix 5). There are only a few original studies in which the proteins that participate in the process of mesangial presence of TN in kidney tissue is mentioned (1-9). sclerosis has not been explored. In this study, the Some of those studies deal with the developmental synthesis of TN by cultured rat mesangial cells was aspect of TN expression and have suggested that the documented by the following observations: (1) epithelium/mesenchyme interface of the S-shaped Northern hybridization of total PNA extracted from tubules in embryonic kidneys contains a barge mesangial cells showed two distinct species of TN amount of TN, which is known to play an important mRNA; (2) immunoblotting of the protein extracted robe in nephrogenesis (6,7). In contrast, the expres- k from the conditioned medium demonstrated four TN sion of TN in mature kidney tissue in normal or protein bands; (3) immunoblotting of the protein ex- pathologic conditions has not been adequately evab- uated (5). tracted from the mesangial cell lysate demonstrated The mesangium is a highly specialized precapibbary at least four TN protein bands; and (4) immunohisto- tissue of the renal gbomerulus, which is composed of chemical techniques identified TN within the cyto- intrinsic mesangial cells, surrounded by mesangial plasm of mesangial cells and in the surrounding ex- matrix (8). The normal mesangiab matrix is known to tracellular matrix. It was also found that normal rat be composed mainly of collagen type IV, baminin, and human glomeruli showed global, diffuse mes- fibronectin, and heparan sulfate proteoglycans, all of which have been known to be synthesized by mesangial cells in culture (8- 1 1 ). Enlargement of the ‘ Received January 22, 1993. Accepted september io, 1993. 2correspondence to Dr. L.D. Truong, Department of Pathology. MS205, The mesangial matrix, also called mesangial sclerosis, is Methodist Hospital, Houston, TX 77030. a common feature of several gbomerular diseases, 1046-6673/0410-177 1$03.00/0 regardless of etiology (8). The expanded mesangial Journal of the American society of Nephroiogy Copyright © 1994 by the American 5ociety of Nephrology matrix is traditionally thought to be in part rebated to Journal of the American Society of Nephrology 1771 Tenascin and Mesangial Cells 1 ‘ oversynthesis of the normal components of the mes- film (Eastman Kodak Co. , Rochester, NY) at -70#{176}C. angial matrix (8-12); however, the possibility that The following probes were used for the Northern mesangial cells are also the source of other mesangial hybridization: for TN, a 2.3-kb EcoRI, mouse cDNA matrix proteins that participate in the process of (1 5) subcloned into pBluescript II; for fibronectin, a mesangial sclerosis has not been explored. 1 .2-kb EcoRI, rat cDNA fragment released from In this study, we present data to suggest that rat pRLF-l (16); for smooth muscle actin, an obigonucle- mesangial cells in culture express various species of otide probe, 5’-AGT GCT GTC CTC TTC TTC ACA mRNA for TN, that TN is secreted by mesangial cells CATA-3’, specific for smooth muscle a-actin (17,18). into the culture medium, and that TN is incorporated into the extracellular matrix. These observations may in turn imply a significant robe for TN in the Antibodies process of mesangiab sclerosis and gbomerular scle- rosis. The antibody used for the immunoblotting study was a gift from Dr. Eleanor J. Mackie (Basel Univer- sity, Basel, Switzerland). This rabbit pobycbonal an- MATERIALS AND METHODS tibody is directed against TN extracted from cultured rat embryo fibroblasts (19). The antibody used for Culture of Rat Mesangial Cells immunohistochemistry is a mouse monocbonal anti- Rat mesangiab cells were cultured according to es- body against TN extracted from a glioma cell line tablished techniques ( 1 2). Briefly, fresh kidneys were commercially available from Dako (Dakopatts, Car- obtained from anesthetized male Sprague-Dawley pinteria, CA 120]). Although these two antibodies rats (Harlan Sprague-Dawley, Houston, TX) weighing were previously characterized ( 1 9,20) and found to between 1 50 and 200 g. Individual gbomeruli (with be specific for TN, we have proceeded to confirm less than 2% contaminating tubular fragments) were their specificity by both Western blotting and absorp- isolated by compression of the renal cortical tissue tion studies. For the monocbonal antibody, the West- through nylon sieves of graded pore sizes (1 3). Gb- em blotting study included protein extracted from merubar explants from three rats were cultured in whole human kidney and human aortic wall, baminin RPMI medium (Sigma Chemical Company, St. Louis, (Gibco BRL, Gaithersburg, MD), and human plasma MO) supplemented with fetal calf serum (20%), dex- fibronectin (Sigma Chemical Company). The proce- amethasone (0.002 zg/mL), transferrin (5 zg/mL), dures for Western blotting study and protein extrac- penicillin-streptomycin (0.01 tL/mL), and HEPES tion are described below. For the polycbonal antibody, buffer (1 0 oL/mL) (all of these reagents were obtained the Western blotting study included protein extracted from Sigma Chemical Company). Initially, only epi- from isolated rat gbomeruli ( 1 3), laminin, and human thelloid cells grew out of the gbomerular explants, but plasma fibronectin. For the absorption study, the between 20 and 30 days, these cells were completely antibodies at optimal dilutions for immunohisto- replaced by spindle cells. These spindle cells were chemistry were mixed with human plasma fibronec- subcultured every 3 to 5 days and maintained their tin (15 and 50 g/mL) for 2 h at room temperature characteristics up to 80 passages; cells between pas- and were then used for immunostaining of tissue sages 4 to 1 0 were used for all studies. sections. Northern Hybridization Study Immunoblotting Study Total RNA was isolated from cultured mesangial The immunobbotting studies were done for protein cells grown to confluence by acid guanidine extrac- extracted from cultured rat mesangial cells and from tion. The details of the method were previously de- conditioned medium. Rat mesangial cells grown to scribed (14). A similar technique was used to extract confluence were washed twice with phosphate-buff- total RNA from snap-frozen renal cortical and med- ered saline and maintained in serum-free medium ublary tissue from normal Sprague-Dawbey rats. Re- for 36 h. The conditioned medium was collected and peated efforts to obtain total RNA from gbomerular subjected to protein precipitation with 1 0% trichlo- isolates failed to produce intact RNA suitable for roacetic acid (TCA) at 4#{176}Cfor 1 h, followed by cen- Northern blotting study. trifugation at 1 2000g for 1 5 mm and repeated wash- The RNA was subjected to agarose gel electropho- ing with ethanol/ether (1 : 1 vol/vol) to remove TCA. resis and transferred to a nylon membrane (Zeta After being washed, the TCA-precipitated protein Probe; Bio-Rad Laboratories, Richmond, CA). Blots was dissolved directly in sodium dodecyl sulfate (SDS) were hybridized with a cDNA probe labeled with 132P] sample buffer (0.0625 M Tris-HC1 [pH 6.8], 2% SDS, dCTP by random primer extension (Amersham Corp., 5% 2-mercaptoethanol, 10% glycerol, 0.002% bro- Arlington Heights, IL) and exposed to Kodak X-AR5 mophenol blue). The cell monolayer was directly dis- I 772 Volume 4 ‘ Number 10 ‘ 1994 Truong et al solved in SDS sample buffer. Protein samples ob- RESULTS tamed from the conditioned medium and the cell Expression of the Gene for TN by Cultured Rat monolayer, respectively, were boiled for 5 mm and Mesangial Cells, Normal Rat Renal Cortex, and subjected to ebectrophoresis, with polyacrylamide gel. Medulla The acrybamide concentrations of the stacking and running gels were 4 and 6% respectively.

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