Tenascin Is Synthesized and Secreted by Rat Mesangial Cells in ...

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OH), which is known to be toxic to gbomerular epithe- hal cells, was added. The ..... Erickson. HP, Bourdon. MA: Tenascin: An extra- cellular matrix protein.

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


W. Majesky,




angial L.D. Truong, Pathology,

MW. Baylor

Majesky, College

J. Pindur, of Medicine.

L.D. Truong, Department Hospital, Houston, TX Soc.

(J. Am.

Department Houston,

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Key Words:


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enascin (TN), a recently described component of the extracellular matrix, is a large oligomeric protein composed of six similar subunits joined together at the amino terminus by disulfide bonds (1). Although there are minor differences among species, each subunit with molecular weight ranging from 1 90 to 280 kd is composed of a cysteine-rich amino acid terminal domain followed by epidermab growth factor-bike homologous repeats, fibronectin Type III homologous repeats, and a fibrinogen (3 and #{244} chain homologous domain at the carboxyl terminal end ( 1 5). There are only a few original studies in which the presence of TN in kidney tissue is mentioned (1-9). Some of those studies deal with the developmental aspect of TN expression and have suggested that the epithelium/mesenchyme interface of the S-shaped tubules in embryonic kidneys contains a barge amount of TN, which is known to play an important robe in nephrogenesis (6,7). In contrast, the expression of TN in mature kidney tissue in normal or pathologic conditions has not been adequately evabuated (5). The mesangium is a highly specialized precapibbary tissue of the renal gbomerulus, which is composed of intrinsic mesangial cells, surrounded by mesangial matrix (8). The normal mesangiab matrix is known to be composed mainly of collagen type IV, baminin, 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 mesangial matrix, also called mesangial sclerosis, is a common feature of several gbomerular diseases, regardless of etiology (8). The expanded mesangial matrix is traditionally thought to be in part rebated to -


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data have suggested that TN is variably seen in rare mesangial areas and in stroma surrounding some tubules. The enlargement of the mesangial matrix (mesangial sclerosis) is a common feature of many renal diseases and is thought to be partially related to


in glomerular

ings suggest that mesangial cells in culture synthesize 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


as a component

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oversynthesis of the normal components of the mesangial matrix (8-12); however, the possibility that mesangial cells are also the source of other mesangial matrix proteins that participate in the process of mesangial sclerosis has not been explored. In this study, we present data to suggest that rat mesangial cells in culture express various species of mRNA for TN, that TN is secreted by mesangial cells 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 process of mesangiab sclerosis and gbomerular sclerosis.

film (Eastman Kodak Co. Rochester, NY) at -70#{176}C. The following probes were used for the Northern hybridization: for TN, a 2.3-kb EcoRI, mouse cDNA (1 5) subcloned into pBluescript II; for fibronectin, a 1 .2-kb EcoRI, rat cDNA fragment released from pRLF-l (16); for smooth muscle actin, an obigonucleotide probe, 5’-AGT GCT GTC CTC TTC TTC ACA CATA-3’, specific for smooth muscle a-actin (17,18). ,


Rat mesangiab cells were cultured according to established techniques ( 1 2). Briefly, fresh kidneys were obtained from anesthetized male Sprague-Dawley rats (Harlan Sprague-Dawley, Houston, TX) weighing between 1 50 and 200 g. Individual gbomeruli (with less than 2% contaminating tubular fragments) were isolated by compression of the renal cortical tissue through nylon sieves of graded pore sizes (1 3). Gbmerubar explants from three rats were cultured in RPMI medium (Sigma Chemical Company, St. Louis, MO) supplemented with fetal calf serum (20%), dexamethasone (0.002 zg/mL), transferrin (5 zg/mL), penicillin-streptomycin (0.01 tL/mL), and HEPES buffer (1 0 oL/mL) (all of these reagents were obtained from Sigma Chemical Company). Initially, only epithelloid cells grew out of the gbomerular explants, but between 20 and 30 days, these cells were completely replaced by spindle cells. These spindle cells were subcultured every 3 to 5 days and maintained their characteristics up to 80 passages; cells between passages 4 to 1 0 were used for all studies.

The antibody used for the immunoblotting study was a gift from Dr. Eleanor J. Mackie (Basel University, Basel, Switzerland). This rabbit pobycbonal antibody is directed against TN extracted from cultured rat embryo fibroblasts (19). The antibody used for immunohistochemistry is a mouse monocbonal antibody against TN extracted from a glioma cell line commercially available from Dako (Dakopatts, Carpinteria, CA 120]). Although these two antibodies were previously characterized ( 1 9,20) and found to be specific for TN, we have proceeded to confirm their specificity by both Western blotting and absorption studies. For the monocbonal antibody, the Westem blotting study included protein extracted from whole human kidney and human aortic wall, baminin (Gibco BRL, Gaithersburg, MD), and human plasma fibronectin (Sigma Chemical Company). The procedures for Western blotting study and protein extraction are described below. For the polycbonal antibody, the Western blotting study included protein extracted from isolated rat gbomeruli ( 1 3), laminin, and human plasma fibronectin. For the absorption study, the antibodies at optimal dilutions for immunohistochemistry were mixed with human plasma fibronectin (15 and 50 g/mL) for 2 h at room temperature and were then used for immunostaining of tissue sections.






of Rat Mesangial




Total RNA was isolated from cultured mesangial cells grown to confluence by acid guanidine extraction. The details of the method were previously described (14). A similar technique was used to extract total RNA from snap-frozen renal cortical and medublary tissue from normal Sprague-Dawbey rats. Repeated efforts to obtain total RNA from gbomerular isolates failed to produce intact RNA suitable for Northern blotting study. The RNA was subjected to agarose gel electrophoresis and transferred to a nylon membrane (Zeta Probe; Bio-Rad Laboratories, Richmond, CA). Blots were hybridized with a cDNA probe labeled with 132P] dCTP by random primer extension (Amersham Corp., Arlington Heights, IL) and exposed to Kodak X-AR5

I 772


The immunobbotting studies were done for protein extracted from cultured rat mesangial cells and from conditioned medium. Rat mesangial cells grown to confluence were washed twice with phosphate-buffered saline and maintained in serum-free medium for 36 h. The conditioned medium was collected and subjected to protein precipitation with 1 0% trichloroacetic acid (TCA) at 4#{176}C for 1 h, followed by centrifugation at 1 2000g for 1 5 mm and repeated washing with ethanol/ether (1 : 1 vol/vol) to remove TCA. After being washed, the TCA-precipitated protein was dissolved directly in sodium dodecyl sulfate (SDS) sample buffer (0.0625 M Tris-HC1 [pH 6.8], 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.002% bromophenol blue). The cell monolayer was directly dis-







solved in SDS sample buffer. Protein samples obtamed from the conditioned medium and the cell monolayer, respectively, were boiled for 5 mm and subjected to ebectrophoresis, with polyacrylamide gel. The acrybamide concentrations of the stacking and running gels were 4 and 6% respectively. The procedure of Towbin et at. was used with slight modification to transfer proteins from the gel to the Zeta Probe membrane by electrobbotting (2 1). The membrane was immunobbotted for TN with rabbit-polycbonal antibody at a 1 :4,000 dilution. Biotinybated goat antirabbit immunogbobubin G (IgG) was used as a secondary antibody by the avidin-biotin-peroxidase complex technique (22).




Immunohistochemical staining for TN was performed for cultured rat mesangial cells and for kidney tissue from five normal Sprague-Dawbey rats. To assess whether there is human relevance of the staining reaction, a similar study of 1 8 human renal biopsies showing expansion of mesangial matrix was also performed (three IgA nephropathy, three focal segmental sclerosis, three mesangial proliferative lupus nephritis, three focal proliferative lupus nephritis, three membranoprobiferative gbomerubonephritis. and three diabetic gbomerubosclerosis). Three normal kidney samples obtained from nephrectomy specimen for renal cell carcinoma were also included in this study. Mesangial cells grown to confluence were gently trypsinized and subjected to cytocentrifuge preparation. Mesangial cells were also grown to monolayer on pobybysine-coated histology glass slides. Both the monolayer and the cytocentrifuge preparation were fixed with acetone at 4#{176}C for 6 mm and air dried. Kidney tissues from rats and humans were fixed and paraffin embedded or snap frozen and cut at 4 m. The immunostamning was done by a standard avidin-biotin-peroxidase complex technique (22) with the mouse monocbonab antibody described above (20). Briefly, the following steps were Involved: blocking with normal horse serum ( 1 / 100 dilution) to prevent nonspecific binding; primary antibody (1 /50 dilution); secondary antibody (polycbonal horse anti-mouse immunogbobulins, 1 / 1 00 dilution); avidin-biotin-peroxidase complex ( 1 / 1 00 dilution); color development with diaminobenzidine (0.5 mg%, Sigma Chemical Company); counterstain with hematoxybin or methyl green. All of the reagents for this procedure, unless otherwise indicated, were from Vector Laboratory (Burlingame, CA). The negative controls included replacement of the primary antibody, the secondary antibody, or the avidin-blotin peroxidase complex, in different combinations, with buffer. For positive control, tissue from a decubitus ulcer was used.


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of the Gene

Expression Mesangial Medulla


for TN by Cultured Rat Renal Cortex,


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The cultured cells displayed several features of mesangial cells including absence of epithebial and endothebial differentiation by electron microscopy, positive immunohistochemicab staining for a-smooth muscle actin, negative staining for keratin and Factor VIII-related antigen, and the ability of these cells to grow normally when the amino nucleoside of puromycmn (1 tg/mL; 1CM Pharmaceutical, Cleveland, OH), which is known to be toxic to gbomerular epithehal cells, was added. The mesangiab nature of these cells was also supported by the expression of mRNA for a-smooth muscle actin and fibronectin. The Northern hybridization study showed that rat mesangiab cells in culture expressed two distinct TN mRNA species (approximately 6.8 and 8.0 kb [Figure 1 J). The renal cortex and medulla, respectively, showed two bands similar to that of mesangial cells. Additionally, a third band of bow density was also noted for the renal medulla (Figure 1 ). The Northern blotting findings for renal cortex and medulla seemed to correspond with the immunohistochemical staining of the normal rat kidney tissue, where TN staining in the cortex was limited to mesangium and arteriobar wall, but there was diffuse interstitial staining in the deep medulla (see also the results of immunohistochemical staining below).

Monospecificity clonal

of the Polyclonal


The Western blotting antibody showed two

Against study distinct



TN using bands

the monocbonal of TN for the

123 8.OkB_ 6.8kB.

Figure 1. Expression of the gene for TN by cultured rat mesangial cells (Lane 1), normal rat renal cortex (Lane 2), and normal rat renal medulla (Lane 3). Total cellular RNA is extracted from cultured rat mesangial cells, normal rat renal cortex, and normal rat renal medulla. A total of 10 g of RNA from each specimen is electrophoresed, transferred to a nylon membrane, and hybridized with a 32P-labeled cDNA probe for TN. Two distinct species of mRNA for TN (approximately 6.8 and 8.0 kb) are seen for the cultured rat mesangial cells. The renal cortex and medulla, respectively, show two bands similar to those of mesangial cells. Additionally, a third band of low density is noted for the renal medulla.







human whole kidney and human aortic wall, respectiveby. No band was seen in the baminin or fibronectin lane (Figure 2). The Western blotting study using the pobycbonab antibody showed at beast three distinct bands of TN for the gbomerubar isolates but no bands for the baminin or fibronectin bane (Figure 3). The absorption by fibronectin did not induce any changes in the immunohistochemicab staining pattern by either antibody. These results indicate that both antibodies are specific for TN and that neither of them cross-react with fibronectin or baminin.

TN Isoforms





of the


in the

the Condition Cultured








Four distinct bands of TN isoforms were identified in the Western blotting study of the total proteins extracted for the conditioned medium (Figure 4); their molecular weights were approximately 320, 280, 220, and 200 kd, respectively. The blotting study of the corresponding mesangial cell bysates also reveabed four distinct bands of TN isoforms. Although 1






2. Western blotting studies with the monoclonal anagainst TN. Lane I represents the control in which the primary antibody is replaced by buffer. Lane 2 represents the molecular weight markers. Protein samples extracted from whole human kidney, human aortic wall, lammm, and fibronectin are in Lanes 3, 4, 5 and 6, respectively. Distinct bands of TN isoforms are seen for the kidney and aorta tissue, whereas no bands are present in the laminin and fibronectin lanes. tibody

rN 200


Figure 3. Western blotting studies with the polyclonal antibody against TN. Lane I represents the molecular weight markers. Protein extracted from rat glomerular isolates, lammm, and fibronectin are in Lanes 2, 3, and 4, respectively. Three bands of TN isoforms were noted in Lane 1, whereas Lanes 2 and 3 displayed no bands.










c kD 320 280

>TN 200 kD


220 200



Figure 4. Western blotting study shows TN isoforms in both cultured mesangial cell lysates and the corresponding conditioned medium. Lane A represents the control in which the polyclonal antibody against TN is replaced by buffer. MW represents molecular weight markers. Proteins extracted from mesangial cell lysates and from conditioned medium are in Lanes B and C, respectively. Both Lanes B and C show four distinct bands of similar molecular weight but of different density. Lane B also shows a faint band of low molecular weight. This band most probably represents the proteolytic product related to cell protease and indeed is not seen in the Lane C for the conditioned medium.

the molecular weights of these four bands were similar to those of the four bands seen in the conditioned medium, the density of each of the corresponding pair of bands was markedly different. Additionally, another faint band of low molecular weight was also observed for the mesangial cell lysates. Although the remote possibility of this band representing a TN isoform cannot be completely ruled out, most probably this band represents a proteolytic product rebated to cell protease; the repeated presence of this band in the cell-associated protein extract and its consistent absence in the protein extracted from the supernatant are consistent with this hypothesis. These observations indicate that isoforms of TN exist and that the nature of the soluble and mesangial cell-associated TN is probably different.,

Immunohistochemistry The mesangial cells in the cytocentrifuge preparations displayed strong, diffuse cytoplasmic staining for TN (Figure 5a). In the monolayer obtained at Day 7 after plating in primary culture, there was staining of the extracellular matrix and less cytoplasmic staining (Figure 5c). There was global, diffuse stainIng of the mesangial matrix of gbomeruli from normal rats (Figure 5e). The controls for these staining reactions, including the ones in which the primary antibody was replaced by buffer, showed negative results (Figure 5b, d, and f). All three normal human kidney samples showed global, diffuse staining of the mesangial matrix, whereas the gbomerular capillaries were negative (Figure 5g); this pattern of staining was similar to that of normal rat gbomeruli (Figure

























et al





Figure 5. Immunohistochemical staining for TN. (a) Cultured mesangial cells are mildly trypsinized and cytocentrifuged; staining for TN is noted in the cytoplasm of these cells. (b) In the control study, in which the primary antibody is replaced by nonimmune serum, staining is not noted in these cells. (c) Staining of mesangial cells cultured to monolayer on glass slides shows a positive reaction for TN in the extracellular matrix. (d) Extracellular matrix is not stained in the control. (e) Kidney tissue from normal rat displays global and diffuse staining of the mesangial matrix for TN; the wall of an arteriole also shows positive staining. (f) The mesangium shows a negative reaction in the control. (g) A normal glomerulus from a human specimen shows global mesongial staining. (h) A human glomerulus with diabetic glomerulosclerosis shows strong staining of a mesangial nodule (arrowheads). Other mesangial areas with milder mesangial enlargement also display a weak staining. Avidin-biotin-peroxidase method with slight counterstain, x I 600 for all pictures. 5e). Staining of the human renal biopsies showing IgA nephropathy, focal segmental sclerosis, mesangial proliferative lupus nephritis, focal proliferative lupus nephritis, membranoproliferative gbomerubonephritis, and diabetic gbomerubosclerosis, respectively, showed that, in each of these lesions, whenever there was an expansion of the mesangial matrix, this matrix was stained strongly for TN. This observation was most pronounced in the cases of diabetic


of the



of Nephrology

Studies addressing the expression of TN in mature kidney tissue in either normal or pathologic conditions are scanty and preliminary (5,6,20,23-26). These studies have alternatively suggested that, in normal mature kidney tissue, TN is either absent (6) or is focally present in rare vascular walls, some mesangial areas, stroma immediately outside Bowman’s capsule of rare gbomerubi, and stroma outside some tubules, especially the ones in the medulla (5,20,24). In pathologic conditions, increased TN expression in the mesangium and interstitium, associated with mesangiab sclerosis and interstitial fibrosis, has been briefly mentioned (5,24). Nevertheless, the cell types responsible for TN synthesis, factors controlling this synthesis, and the significance of TN expression in kidney tissue have not been addressed. In this study, we have demonstrated that TN is synthesized by rat mesangial cells in culture and is secreted into culture medium. Supporting evidence includes the observations that ( 1 ) rat mesangial cells express mRNA for TN; (2) TN is detected in the conditioned medium by immunoblotting; (3) TN is detected in mesangial cell lysates (which contain both mesangial cells and the surrounding matrix); and (4) TN is immunohistochemicably detected in the extraceblular matrix surrounding the cultured mesangial cells and in the cytoplasm of these cells as well. Moreover, TN may be a component of normal mesangial matrix, as evidenced by the global, diffuse mesangial staining for TN. Although repeated efforts to extract total RNA from gbomerular isolates yield degraded products unsuitable for Northern blotting study, TN mRNA were clearly identified from both the cortex and the medulla of normal rat kidney. suggesting that TN is indeed synthesized in vivo. It is interesting to note that different species of TN mRNA were found both for cultured rat mesangial cells and for rat kidney tissue, indicating that isoforms of TN are synthesized. Isoforms of TN, identified in several species including chicken, human, mouse, and rat (1 ,2, 1 5,23,27), are due to alternative splicing, whereby different insertional fibronectinlike homologous repeats are added or deleted from the subunit of the TN molecule (23,25-27). The functional consequences of TN variants containing one or more alternate fibronectin Type III domains are not well understood. Chiquet-Ehrismann et at. (28) showed that the smallest form of TN bound more strongly to fibronectin and was preferentially incorporated into a fibronectin-rich extracellular matrix than larger TN forms that contained extra fibronec-






tin Type III repeats. Murphy-Ullrich et at. showed that the ability of TN to reduce the number of focal contacts in cultured endotheliab cells was dependent on the inclusion of an extra fibronectin Type III repeat defined by a monocbonal antibody (29). It is also noted that the immunoblotting study of proteins extracted from the conditioned medium, the mesangial cell lysates, and the glomerular isolates, respectively, has revealed bands of TN protein with different molecular weights. Several studies, using Western blotting, have reported up to two TN isoforms in whole-kidney tissue in several species including human, mouse, and chicken (23,26,30). The differential expression of TN isoforms in the cortex and medulla is not addressed in any of these studies. TN isoforms are also reported in other organs including rat vascular wall, chicken gizzard, chicken lung, and mouse brain ( 1 ,23,25,26). It is interesting to note from our study that, although only two species of TN mRNA were found in both mesangiab cells and kidney tissue, up to four corresponding TN isoforms were observed. The explanation for this discrepancy is not clear but may be rebated to several observations. First, although the two apparently distinct TN mRNA bands were identified in the Northern blot, each of them may be in fact composed of multiple mRNA, which because of their similar molecular weight, are detected as one band. Second, significant glycosybation of TN has been demonstrated in viva and in vitro (1 1 5) and may alter the molecular weight of the TN subunits, accounting for additional bands in the immunobbotting study (7). Third, the observation that TN may bind to chondroitin sulfate proteoglycans, also a synthetic product of mesangial cells in culture. may have produced the results seen in our immunoblotting study ( 1 ); however, the batter possibility seems remote because the reducing condition used in the ebectrophoresis tends to dissociate such binding interactions. Mesanglab cell proliferation and mesangial sclerosis are seen in several pathologic processes invobving the kidney and may play an important robe in gbomeruboscberosis (8). It has been documented that, despite minor quantitative differences seen in varbus types of gbomerubar diseases, all components of normal mesangiab matrix participate in the process of mesangial sclerosis (9- 1 1 ). Furthermore, mesangial cells in culture are known to synthesize these components (9- 1 1 ). Our study, documenting the synthesis of TN by rat mesangial cells in culture, suggests that TN may also play a significant role in the process of mesangial sclerosis and gbomerubosclerosis. The human relevance of this observation is also supported by our finding that human renal biopsies show strong staining for TN in areas with expansion of the mesangiab matrix, especially in diabetic gbmerubosclerosis. ,


In conclusion, our study documents that rat mesangial cells in culture synthesize TN and that TN may be a component of the mesangial matrix in both normal and pathologic conditions. Further studies addressing the mechanisms controlling the expression of TN by mesangial cells, and possibly by other cell types in the gbomerulus, may provide additional insight into the process of gbomerubosclerosis.

ACKNOWLEDGMENTS This study was supported in part by funds from the Moran Foundation (Project #2900047) and by generous start-up funds from the Department of Pathology. Baylor College of Medicine. We thank Drs. Juan Lechago. and Mary Ostrowski for their review of the manuscript. Ms. Melinda Sanchez’s secretarial help is appreciated. We also thank Drs. E. Mackie and P. Ekblom for providing tenascin polyclonal antibody and cDNA clone. respectively.

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