Cultured Rat Mesangial Cells Contain Smooth Muscle ... - Europe PMC

Amerncan Journal of Pathology, Vol, 142, No. 2, February 1993 Copyight © American Society for Investigative Pathology

Cultured Rat Mesangial Cells Contain Smooth Muscle a-Actin not Found in Vivo Marlies Elger,* Detlev Drenckhahn,t Rainer Nobiling,t Peter Mundel,* and Wilhelm Kriz* From the Department of Anatomy and Cell Biology I*

and Department of Pbysiology,t University of Heidelberg, D-6900 Heidelberg, and Department of Anatomy,* University of Wurzburg, D-8700-Wurzburg, Germany

A monoclonal antibody against smooth muscle a-actin (SM a-actin) was used to study the expression of SM a-actin in kidney sections and mesangial ceUl (MC) cultures. In the tissue sections, indirect immunofluorescence revealed intense labeling of vascular smooth muscle cells and precapilary pericytes for SM a-actin. Glomerular ceUs including MC were negative, with the exception of scattered smooth muscle ceUs in the waUl of the intraglomerular segment of the efferent arteriole. In contrast, in MC cultures 50 to 95% of the ceUs displayed bright fluorescence. Immunoreactivity for SM a-actin first appeared 3 days after explantation of glomeruli and increased until the primary culture reached subconfluence. In each subculture (I to 10) expression of SM a-actin was weak on day I and pronounced at subconfluence. Growth arrest of subconfluent cultures for I to 7 days in serumfree medium did not alter the percentage of cells positive for SM a-actin. However, exposure of MC to serum-free medium beginning on the first day of subculture curtailed expression of SM &-actin. Double-labeling with antibodies against proliferating ceU nuclear antigen and SM c-actin revealed SM a-actin-positive filaments in both replicating and resting ceUs. In summary, our results demonstrate that some process or processes associated with cel proliferation and ceUl growth of MC are accompanied by de novo expression of SM a-actin. The relevance to the contractile behavior of the difference in SM a-actin expression under in vitro and in vivo conditions is unknownL (Am J Pathol 1993, 142:497509)

Glomerular mesangial cells (MC) occupy a central axial position relative to the glomerular capillaries and possess some of the morphological characteristics of vascular smooth muscle cells (SMC), such as bundles of actin filaments that are associated with isoforms of the major components of the contractile apparatus of muscle, ie, myosin, tropomyosin, a-actinin, vinculin, and talin,1-' in conjunction with immunocytochemical evidence for a smooth muscle-like cyclic guanosine monophosphate-dependent protein kinase.10-13 On the basis of these findings, it has been proposed that MC are capable of isotonic contraction and may contribute to the regulation of glomerular blood flow and filtration rate.14 This view has been supported by observations that MC in tissue culture are able to contract and that the membrane potential is depolarized when cells are exposed to contractile stimuli.6.1015-22 However, the question of whether MC do in fact contract in vivo and whether such contractions affect glomerular microcirculation is still a subject of debate.723 26 In order to further characterize the contractile apparatus of MC, an established antibody against smooth muscle a-actin (SM a-actin)27 was applied. This actin isoform is generally considered to be the most specific biochemical marker for smooth muscle differentiation27 and, thus, its presence would be evidence for functional contractility in MC. The expression of SM a-actin was studied in normal kidney tissue and in culture.

Materials and Methods Culture of MC and SMC Kidneys were obtained from male Wistar rats (1 50-g body weight). Primary cultures of MC were obtained as outgrowths from collagenase-treated glomerular Supported by the Deutsche Forschungsgemeinschaft, "Forschergruppe Niere" grant Kr 546/5-3, and Sonderforschungsbereich 176, Projekt Dr A 14. Accepted for publication July 27,1992. Address reprint requests to Dr. Marlies Elger, Institute of Anatomy and Cell Biology I, Im Neuenheimer Feld 307, University of Heidelberg, 6900 Heidelberg, Federal Republic of Germany.

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remnants, as described previously.22'28 In brief, intact glomeruli were obtained by serial sieving of renal cortex homogenates. The glomeruli were incubated with 2.5 mg/ml collagenase type IV (Sigma, Taufkirchen, FRG) for 30 to 50 minutes at 37 C, resulting in digestion of the glomerular basement membrane and dissociation of its epithelial components. Washed glomerular remnants (the number of cells per remnant was estimated to range from 10 to 60) were plated on glass coverslips (LHD, Heidelberg, FRG) on the bottom of 24-well microtiter plates (Costar, Greiner, NOrtingen, FRG) or in plastic culture flasks (Greiner) at a density of approximately 200 remnants/cm2. The culture medium was Dulbecco's modified Eagle's medium (GIBCO, Karlsruhe, FRG) supplemented with 10 or 20% fetal calf serum (FCS). The cells were maintained in a humidified atmosphere with 5% CO2 at 37 C. Subcultures were prepared from cells grown to confluence in plastic flasks. Confluent MC were disaggregated by 5-minute treatment with 0.05%/0.02% trypsin/EDTA (Seromed, Berlin, Germany), washed, reconstituted in Dulbecco's modified Eagle's medium at a 1:4 to 1:10 dilution, and plated at a density of 1500 to 2000

cells/well. SMC were obtained from the medium of aortas of the same rats. The aortas were stripped from the adventitia, minced into pieces of about 1 mm2, incubated with collagenase, and plated as described above for the MC. In MC cultures, the first outgrowth after explantation of glomeruli was noted after 3 to 5 days. Most of the cells had an irregular stellate or multipolar spindle shape. Growth was not inhibited in medium containing D-valine, indicating the presence of D-amino acid oxidase activity, thus distinguishing this cell type from fibroblasts.29 The identity of MC was further confirmed by positive immunofluorescence with antibody against Thy-1 .128 and by the absence of reaction with antibody against von Willebrand factor, a specific marker for endothelial cells (see below).

Growth Arrest of MC Cultures In two separate MC culture lines, the effect of cell proliferation on expression of SM a-actin was studied. Proliferation in MC culture was suppressed by incubation of the cells in serum-free Dulbecco's modified Eagle's medium for 1 to 10 days. This was performed at two different stages of culture growth. 1) At day 0 of subcultures cells were plated directly into FCS-free medium (ie, before the onset of replication after subcultivation). First and second subcultures were investigated. 2) In other culture plates

derived from the same subcultures, FCS was withdrawn at subconfluence (thus in replicating cultures). The viability of the cells was not significantly affected, as assessed by trypan blue vital dye exclusion. Absence of mitotic activity was confirmed by the lack of incorporation of the thymidine analogue bromodesoxyuridine (Amersham, Braunschweig, FRG). Bromodesoxyuridine was added to the culture to a final concentration of 15 pmol/l either at the beginning of incubation in FCS-free medium or 24 h before examination; cells were then visualized by indirect immunohistochemistry using a monoclonal antibody (antibromodesoxyuridine; Amersham). Growth arrest was considered to have occured if the labeling index fell by >95%.

Processing of Kidneys for Standard Transmission Electron Microscopy (TEM) Kidneys of male Munich Wistar rats (150 to 200 g of body weight) were fixed by aortic retrograde perfusion with 1.5% glutaraldehyde and 1.5% paraformaldehyde and embedded in Epon as described previously.23 Ultrathin section series were examined in a Philips 301 electron microscope.

Processing of Kidneys for Immunohistochemistry The kidneys of male Munich Wistar rats (150 to 200 g body weight) were perfused with phosphate-buffered saline (PBS) (pH 7.4). Kidneys to be used for cryosectioning were frozen in isopentane cooled by liquid nitrogen. Frozen sections were prepared with a 2800 Frigocut E cryostat (Reichert-Jung GmbH, Nussloch, FRG) for 5-pm sections or with an Ultracut E/FC 4E (Reichert-Jung) for 1-pm sections. For semithin resin sections (1 pm), flushing of the kidney with PBS was followed by perfusion fixation with 0. 1% glutaraldehyde and 2% paraformaldehyde in PBS for 3 minutes at room temperature.7 Tissue was embedded in Lowicryl (London Resin Corporation, London, UK), and series of semithin sections were cut on an Ultracut E microtome (Reichert-Jung).

Indirect Immunofluorescence of Cell Culture and Tissue Sections Immunohistochemistry was performed as described

recently.7'30 Cells grown on glass slides were fixed, permeabilized with ice-cold acetone for 5 to 10 min, and washed with PBS at room temperature. Several specimens were further treated with 0.1% Triton X-100 (Sigma, Munich, Germany) in PBS for 5 to 10

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min. Fixed cells, frozen sections, or resin sections immersed in PBS with 10% horse serum and 1% bovine serum albumin for 15 min. Incubation was performed at room temperature with one of the following antibodies: a mouse monoclonal IgG-antibody raised against the amino-terminal decapeptide of SM a-actin (Progen, Heidelberg, FRG),27 a murine monoclonal IgG antibody against the thymocyte antigen Thy-1.1 (clone MRC, OX-7; Camon, Wiesbaden, FRG) (in this case, the fixation time for cultured cells was reduced to 30 to 60 seconds), or a murine antiserum to factor VIII-related antigen (von Willebrand factor) (Boehringer, Mannheim, FRG). Antibody binding was visualized with fluorescein isothiocyanateconjugated goat anti-mouse IgG (Dianova, Hamburg, FRG). In resin sections, antibody against SM a-actin was visualized with biotinylated anti-mouse IgG from sheep (Amersham, Braunschweig, FRG) followed by Texas Red-conjugated streptavidin (Amersham). For double-labeling of cell cultures with anti-SM a-actin either a rabbit antiserum against thymocyte antigen Thy-1 .1, followed by tetramethylrhodamine B isothiocyanate-conjugated anti-rabbit IgG Fab fragment, or tetramethylrhodamine B isothiocyanate-labeled phalloidin (rhodamine-phalloidin) was used. Antithymocyte serum was kindly provided by Prof. Dr. R. Stahl, Universitatsklinik Frankfurt, and rhodamine-phalloidin by Prof. Dr. H. Faulstich, Max Planck Institute for Medical Research (Heidelberg, FRG). The specimens were mounted in 2.5% diazabicyclooctane (Sigma) dissolved in 40% glycerol in distilled water and were observed with a Polyvar fluorescent microscope (Reichert-Jung).

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Quantification of Immunoreactive Cells in Culture The percentage of cells immunoreactive with antibodies against SM a-actin and Thy-1 was determined in cultures maintained in FCS-containing or serum-free medium. In order to facilitate cell counting, the nuclei were stained with fluorochrome 4,6diamidino-2-phenylindole dihydrochloride (Sigma) after immunolabeling with antibodies. Staining was performed in 0.001% 4,6-diamidino-2-phenylindole dihydrochloride in distilled water for 10 minutes at room temperature. Epifluorescence for 4,6-diamidino-2-phenylindole dihydrochloride was observed at a wavelength of 360 or 430 nm. The cells were photographed systematically from at least two glass slides and printed at a final magnification of x1000. In subconfluent cultures, 300 cells treated with one of

the antibodies were evaluated. In 1-day-old subcultures and 3-day-old primary cultures, approximately 100 cells were analyzed. The following cultures were used for quantification. 1) Cultures in FCS-containing medium were used. Primary cultures and subcultures of four separate culture lines of MC and several passages of SMC were evaluated for immunoreactivity with SM a-actin. The cultures were investigated 1 day after plating (or, in the case of primary cultures, 3 to 4 days after plating) and at subconfluence. Part of these cultures were also used for counts of Thy-ipositive cells. 2) Growth-arrested cultures in FCSfree medium were used. Quiescent cells of two separate culture lines were studied for immunoreactivity with SM a-actin and Thy-1 at the stages (initial and subconfluent) described above.

Immunohistochemical Staining for Identification of Proliferating Cells in MC Culture Several replicating subconfluent MC cultures were double-labeled for proliferating cell nuclear antigen (PCNA) and SM a-actin. PCNA is expressed in the late G1, S, G2, and M phases of the cell cycle,31 ie, at all stages other than Go (resting). Cells at subconfluence were incubated first with anti-PCNA (Amersham), a murine IgM monoclonal antibody, followed by a class-specific biotinylated anti-mouse IgM antibody and streptavidin-biotin and Texas Red. The cells were then stained with anti-SM a-actin, an IgG murine antibody, followed by a fluorescein isothiocyanate-conjugated anti-mouse IgG as described above. Specificity of secondary staining was examined by omission of each of the primary antibodies.

Results The occurrence of SM a-actin in MC was studied in rat kidney sections and in cultures of MC by indirect immunofluorescence with a specific monoclonal antibody (anti-SM a-actin).27 Immunoreactivity of MC in situ and in vitro was compared.

Kidney Sections In kidney sections incubated with antibody to SM a-actin, the wall of renal arteries, arterioles, and descending vasa recta was strongly labeled. The staining could be localized to the SMC of arteries and arterioles (Figure 1) and to the precapillary pericytes of descending vasa recta (data not shown).

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There were no differences in staining patterns between sections of frozen and resin-embedded tissue. No immunoreaction was noted in other renal structures. Tissue sections also revealed that staining of afferent arterioles ended abruptly at the entry into the glomerular tuft (Figure 1). Glomerular cells, including MC, were negative for SM a-actin staining. The extraglomerular MC were also negative. Staining was evident at the origin of the efferent arteriole (EA). The labeling of EAs decreased distally and disappeared when the EAs split into the peritubular capillaries. In addition, in some glomerular profiles scattered SM a-actin-positive cells were also encountered in the middle of the tuft (Figure 2). Compared with the staining intensity in afferent arterioles and EAs, the signal obtained from these cells was weak. These cells were traced through semithin serial sections incu.1,4 "De cieariy ;,4-uaentateo witn anti-Mvi a-actin anci4 couiu

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tified as belonging to the intraglomerular segment of the EA. This segment extends up to 30 pm into the glomerulus.32 By TEM it was seen that the endothelium of the intraglomerular segment of the EA was completely surrounded by a layer of MC with several interspersed SMC (Figure 3). The SMC could be clearly distinguished from neighboring MC by the circumferential orientation of their microfilament bundles, by cytoplasmic densities associated with intermediate filaments, and by numerous caveolae arranged in characteristic rows along the plasma membrane. In contrast, microfilament bundles of the MC ran in all directions, and the cells were not endowed with caveolae. Thus, the occasional intraglomerular SM a-actin-positive cells are probably identical to the SMC in the wall of the intraglomerular segment of the EA. Glomerular MC have been found to possess specific Thy-1 immunoreactivity.28'33'34 As expected, in this study strong immunofluorescence specific for Thy-1 was revealed in the mesangial location (Figure IInn contrast, contrast, vascular vascular SMC SMC were 4).4). were negative negative for for Thy-1.

Figure 4. Sectiont throuigh the renial cortex incnibated with anti-Thy-1 A isothiocyanate-labeled second andfluiorescein is intenscly with its vascuilarpole is shown. The mesatngialanitibodv. regioni (M) glomenrluis labeled. Fluioresceini isothiocyanazefluorescence (a). The same section for ultraviolet lightofexcitation, with showing fluorescence the demonistrationz MD, nionispecific of the thick histology, (b). macuila densa ascending limb of Henle. Semithin frozen section; X 650.

Figure 1. Immunofluorescence micrograph of rat kidnzey cortex labeled u'ith anti-SM a-actitn. A glomenular profile (G) with afferenzt arteriole (AA) and EA is shoun. SMC of glomenrlar arterioles are intensely labeled, uhereas glomenilar cells, inzclnidinig MC, and extraglomenrlar MC (EGM) are niegative. Erythrocytes (E) have a faint azutoimmunofluiorescentce. Staining of the vessels differs in inztenzsity. Ini the afferent arteriole segment immediately before its entry into the glomenrlus, staining is less intense (arrou), apparently corresponding to the presence of grantular cells. The labeling of the SMC in the EA decreases distally (douible arrouw). TAL, thick ascenzdinig limb of Ilenles loop. Semithin Lowicryl sectionz, perfusion fixation with 0.10% glutaraldebyde and 2% paraformaldehyde; x 570. Figure 2. Sectionfrom a set ofsemithin serialsectionsstained uith anti-SMa-actiz. A glomenrluis uith itdividual SM a-actin -positive cellsencircling a vessel (arrow) is shoun. In the series of sections this vessel uwasfolloued to its exitfrom the glomenrluis (niot shown), where it continued into the EA. Thus, the SM a-actin-positive cells are located in the uwall of the intraglomenrlar segmetnt of the EA. AA, afferenzt arteriole; BC, Bouman is capsule. Semithin Lowicryl section, 0.1% glutaraldehyde and 2% paraformaldehyde; x 650. Figure 3. TEM micrographs ofglomerularprofiles showing the intraglomerularsegment ofan EA in cross-sectioni. The sections belong to a set ofserial uiltrathin sections and are located approximately 20 ym (a) anzd 5 lim (b) from the glomenrlar exit of the EA. a: In the intraglomenrlar segment of the EA (asterisk), MC and a SMC are seen. b: SMC are characterized by abundant caveoleac (arrou'heads) and circuimferenitial arraYs of microfilament buindles (MF). Processes of MC are also rich in microfilaments, uihich lack a preferential orienitationi. EN, enzdotheliuim; PB, primary branches of the afferetnt arteriole. a, x 920; b, X 24, 000.

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Cell Culture Cultures of glomerular MC and aortic SMC were conducted in parallel. Cell cultures maintained in serumcontaining medium were investigated from the primary culture to the tenth passage. In addition, MC from first and second subcultures were examined in arrested conditions. The successful culture of MC was verified by the presence of Thy-1 immunoreactivity. In all mesangial cultures, the majority of cells (70 to 99%) were Thy-1 positive (Figure 5). There was no evidence for a change in the percentage of Thy-1-positive cells with increasing number of subcultures. All cultured MC were negative for von Willebrand factor. As shown in Figures 5, 6, and 7 and summarized in Figure 8, the reaction of cultured MC with anti-SM a-actin was completely different from that of MC in situ. Isolated glomeruli studied 2 days after explantation were essentially negative for anti-SM a-actin staining, similar to glomeruli in vivo. Cells in the primary cultures generally began to grow out from the

glomeruli about 3 days after plating. At this time, only a few clusters of distinctly SM a-actin-positive cells per glass slide (50% of the cultured cells showed distinct SM a-actin reactivity similar to that seen in cells in proliferating cultures. Moreover, double-immunostaining of replicating cultures with antibodies against SM a-actin and PCNA showed that both proliferating and nonproliferating cells were SM a-actin positive, suggesting that once a cell has expressed SM a-actin further proliferative stimulus is not needed for the cell to remain positive for SM a-actin. This is in agreement with findings in cultured SMC, where SM a-actin synthesis was also found in both cycling and noncycling cells.44 These results suggest that the expression of SM a-actin is suppressed in glomeruli under normal physiological conditions. However, stimuli that cause proliferation of MC in situ as well as in vitro abolish this suppression and induce synthesis of SM a-actin. In addition, the presence of this protein in both cycling and noncycling MC, as well as the observation that in growtharrested cultures (maintained in serum-free medium beginning immediately after subcultivation) some immunoreactivity for SM a-actin develops despite the lack of proliferation, suggests that other factors are also involved in induction of SM a-actin expression. The results from MC cultures showed that frequently there was a higher percentage of cells that were positive for F-actin than cells that were positive for SM a-actin. Cells containing stress fibers with

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F-actin but not expressing SM a-actin, as shown by double-labeling, generally were small and/or postmitotic cells. In contrast, large cells usually expressed a large amount of SM a-actin. Thus, it appears that SM a-actin is expressed in a somewhat later stage of stress fiber formation. Moreover, cells undergoing hypertrophic growth overexpress SM a-actin. The change in SM a-actin expression between freshly passaged cultures and older cultures (days) is in part due to the greater abundance of the small SM a-actin-negative cell phenotype shortly after subcultivation. The phenotypic changes of the MC and the expression of SM a-actin may depend, in addition to stimuli associated with proliferation, on environmental conditions such as the composition of the extracellular matrix and/or the culture medium accessible to the cells. This view is supported by observations in multilayered MC cultures. Cells of the superficial layers usually express SM a-actin, whereas deep layers are nonreactive. This is especially seen in the mesangial hillocks. Studies with TEM revealed that the extracellular matrix in the superficial layers and that in the center of the hillocks are different (Nagata, personal communication).6 Labeling of sections through mesangial hillocks showed that only the superficial layers resembling the two-dimensional MC culture were reactive for SM a-actin, whereas the cells in the middle of the hillocks did not express SM a-actin (Nagata and Elger, unpublished observations). The influence of extracellular matrix and the dimensionality of the culture on expression of SM a-actin has also been demonstrated in cultured vascular SMC.45 It may be speculated that in glomerulonephritis changes in matrix components, eg, de novo synthesis of collagen types or other substances not usually present in the mesangium, may stimulate synthesis of SM a-actin. Studies on MC cultures will further elucidate the factors that lead to expression of SM a-actin in altered physiological and pathological settings.

Relevance As shown in several studies, MC in culture are able to contract in an isotonic fashion.11'171 8 This capability is best seen when MC are cultured on thin films of silicone rubber, on which contraction of cells can be demonstrated by the appearance or enhancement of wrinkles and by a reduction of the cell surface area.18 Evidence for dynamic contractility of MC has also been derived from cultures grown on glass slides19 and in three-dimensional cultures in type IV collagen gels.46 A variety of hormones such as

angiotensin 11 and arginine vasopressin,17 as well as autacoids such as leukotrienes,47 platelet-activating factor,16 and endothelin-1,46 are able to evoke contractions of cultured MC. In these experiments, generally 30 to 40% of the cells are observed to contract. Compared with our immunocytochemical results, this proportion of responsive cells seems rather low. As shown by Mene and Dunn,19 the proportion of contractile cells in MC culture can be increased to approximately 75% by inhibition of prostaglandin synthesis, suggesting that the contraction of some of the cells is normally blocked by an action of prostaglandins. Treatment of MC cultures with platelet-derived growth factor consistently initiates contraction of about 75%.11 In agreement with our study, in which we observed a decrease in the density of SM a-actin-positive microfilaments with the number of passages, other groups have found that the ability of cultured MC to contract also decreases with continued passage in vitro (in primary cultures and first passages of cells contraction was most vigorous, whereas in long term cultures contractility was decreased).17'48 These observations suggest that the capacity of MC to contract correlates to some degree with their expression of SM a-actin. It is widely assumed that MC in situ are capable of isotonic contraction. Based on this assumption, MC have often been presumed to participate in the regulation of glomerular blood flow and ultrafiltration coefficient. For instance, the decrease in ultrafiltration coefficient in response to substances such as arginine vasopressin or angiotensin 11 has been proposed to be effected by mesangial contraction.14 However, this conclusion is based solely on indirect evidence. The observation of dynamic contractions in cultured MC cannot be extrapolated directly to the in vivo situation. As shown in this study, there are substantial differences between the in vivo and in vitro compositions of the contractile cytoskeleton in MC. Observations of reductions in tuft size in isolated glomeruli in response to angiotensin 1149-52 are also not absolutely conclusive. In these preparations the opposing forces against which MC contraction normally acts in vivo, ie, the distending forces exerted on the capillary wall by the blood pressure, are absent. Therefore, a totally different balance of forces exists in the two conditions. In fact, direct observation of glomeruli under in vivo conditions has repeatedly failed to show tuft contraction even in response to high doses of vasoactive substances.53 Thus, whether MC in situ are capable of dynamic contraction remains to be established. We suggest that the role of MC in situ may instead be static in nature, i.e., serving to counteract the distending

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forces to which glomerular capillaries and mesan-

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Acknowledgments The authors thank Drs. Kevin Lemley and Rudiger Waldherr for helpful discussions and Mr. Michael Pafreiter for carrying out the cell cultures. The expert technical assistance of Ms. Bruni Hahnel, Ms. Maria Harlacher, Ms. Inge Hartmann, Ms. Hiltraud Hosser, and Ms. Christa Merte-Grebe and the photographic assistance of Ms. Ingrid Ertel are gratefully acknowledged. The authors thank Dr. Kevin Lemley for linguistic help with the manuscript.

References 1. Becker CG: Demonstration of actomyosin in mesangial cells of the renal glomerulus. Am J Pathol 1972, 66:97110 2. Scheinman JI, Fish AJ, Matas AJ, Michael AF: The immunohistopathology of glomerular antigens: the glomerular basement membrane, actomyosin, and fibroblast surface antigens in normal, diseased, and transplanted human kidneys. Am J Pathol 1978, 90:71-88 3. Andrews PM, Coffey AK: Cytoplasmic contractile elements in glomerular cells. Fed Proc 1983, 42:30463052 4. Foidart JB, Dechenne CA, Mahieu P, Creutz CE, DeMey J: Tissue culture of normal rat glomeruli: isolation and morphological characterization of two homogeneous cell lines. Invest Cell Pathol 1979, 2:15-21 5. Norgaard JO: Cellular outgrowth from isolated glomeruli. Lab Invest 1983, 48:526-542 6. Sterzel RB, Lovett DH, Foellmer HG, Perfetto M, Biemesderfer D, Kashgarian M: Mesangial cell hillocks: nodular foci of exaggerated growth of cells and matrix in prolonged culture. Am J Pathol 1986, 125:130-140 7. Drenckhahn D, Schnittler H, Nobiling R, Kriz W: Ultrastructural organization of contractile proteins in rat glomerular mesangial cells. Am J Pathol 1990,

137:1343-1351 8. Ishino T, Kobayashi R, Wakui H, Fukushima Y, Nakamoto Y, Miura AB: Biochemical characterization of contractile proteins of rat cultured mesangial cells. Kidney Int 1991, 39:1118-1124 9. Drenckhahn D, Franke RP: Ultrastructural organization of contractile and cytoskeletal proteins in glomerular podocytes of chicken, rat, and man. Lab Invest 1988, 59:673-682 10. Kreisberg JI, Venkatachalam MA, Radnik RA, Patel PY: Role of myosin light-chain phosphorylation and microtubules in stress fiber morphology in cultured mesangial cells. Am J Physiol 1985, 249:F227-F235 11. Mene P, Simonson MS, Dunn MJ: Physiology of the mesangial cell. Physiol Rev 1989, 69:1347-1424

12. Mene P, Simonson S, Dunn MJ: Phospholipids in signal transduction of mesangial cells. Am J Physiol 1989, 256:F375-F386 13. Pfeilschifter J: Cross-talk between transmembrane signalling systems: a prerequisite for the delicate regulation of glomerular haemodynamics by mesangial cells. Eur J Clin Invest 1989, 19:347-361 14. Ichikawa I, Brenner BM: Evidence for glomerular actions of ADH and dibutyryl cyclic AMP in the rat. Am J Physiol 1977, 233:F103-F1 13 15. Ausiello DA, Kreisberg JI, Roy C, Karnovsky MJ: Contraction of cultured rat glomerular mesangial cells after stimulation with angiotensin 11 and arginine vasopressin. J Clin Invest 1980, 65:754-760 16. Schlondorff D, Satriano JA, Hagege J, Perez J, Baud L: Effect of platelet activating factor and serum-treated zymosan on PGE2 synthesis, arachidonic acid release, and contraction of cultured rat mesangial cells. J Clin Invest 1984, 73:1227-1231 17. Kreisberg JI, Venkatachalam K, Troyer D: Contractile properties of cultured glomerular mesangial cells. Am J Physiol 1985, 249:F457-F463 18. Singhal PC, Scharschmidt LA, Gibbons N, Hays RM: Contraction and relaxation of cultured mesangial cells on a silicone rubber surface. Kidney Int 1986, 30:862873 19. Mene P, Dunn MJ: Eicosanoids and control of mesangial cell contraction. Circ Res 1988, 62:916-925 20. Singhal PC, Hays RM: Actin filament morphology in living and nonliving cultured mesangial cells: formation and dissolution. Nephron 1988, 50:28-33 21. Olivera A, Lamas S, Rodriguez-Puyol D, L6pez-Novoa JM: Adenosine induces mesangial cell contraction by an Al-type receptor. Kidney Int 1989, 35:1300-1305 22. Nobiling R, BOhrle CP: The mesangial cell culture: a tool for the study of the electrophysiological and pharmacological properties of the glomerular mesangial cell. Differentiation 1987, 36:47-56 23. Elger M, Sakai T, Kriz W: Role of mesangial cell contraction in adaptation of the glomerular tuft to changes in extracellular volume. Pfluegers Arch 1990, 415:598605 24. Kriz W, Elger M, Lemley KV, Sakai T: Mesangial cellglomerular basement membrane connections counteract glomerular capillary and mesangium expansion. Am J Nephrol 1990, 10:4-13 25. Kriz W, Elger M, Lemley KV, Sakai T: Structure of the glomerular mesangium: a biomechanical interpretation. Kidney Int 1990, 38(suppl. 30):S2-S9 26. Jaremko G, Larsson L, Bohman SO: Angiotensin-induced structural changes in the rat glomerulus. J Ultrastruct Mol Struct Res 1989, 100:300A-301A 27. Skalli 0, Ropraz P, Trzeciak A, Benzonana G, Gillessen D, Gabbiani G: A monoclonal antibody against a-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol 1986, 103:2787-2796 28. Lovett DH, Ryan JL, Sterzel RB: A thymocyte-activating factor derived from glomerular mesangial cells. J

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Immunol 1983, 130:1796-1801 29. Scott FG, Migeon BR: D-Valine as a selective agent for normal human and rodent epithelial cells in culture. Cell 1975, 5:11-17 30. Mundel P, Gilbert P, Kriz W: Podocytes in glomerulus of rat kidney express a characteristic 44 kD protein. J Histochem Cytochem 1991, 39:1047-1056 31. Kurki P, Vanderlaan M, Dolbeare F, Gray J, Tan EM: Expression of proliferating cell nuclear antigen (PCNA)/ cyclin during cell cycle. Exp Cell Res 1986, 166:209219 32. Elger M, Sakai T, Winkler D, Kriz W: Structure of the outflow segment of the efferent arteriole in rat superficial glomeruli. Contrib Nephrol 1991, 95:22-33 33. Paul LC, Rennke HG, Milford EL, Carpenter CB: Thy-1.1 in glomeruli of rat kidneys. Kidney Int 1984, 25:771-777 34. Holthofer H, Decandido S, Schlondorff D: Identification of specific glomerular cell types in culture by use of lectin and antibody binding. Cell Div Dev 1990, 30:181-194 35. Skalli 0, Pelte M-F, Peclet M-C, Gabbiani G, Gugliotta P, Bussolati G, Ravazzola M, Orci L: a-Smooth muscle actin, a differentation marker of smooth muscle cells, is present in microfilamentous bundles of pericytes. J Histochem Cytochem 1989, 37:315-321 36. Nehls V, Drenckhahn D: Heterogeneity of microvascular pericytes for smooth muscle type a-actin. J Cell Biol 1991, 1:147-154 37. Vandekerckhove J, Weber K: Mammalian cytoplasmic actins are the products of at least two genes and differ in primary structure in at least 25 identified positions from skeletal muscle actins. Proc Natl Acad Sci USA 1978, 75:1106-1110 38. Babai F, Musevi-Aghdam J, Schurch W, Royal A, Gabbiani G: Coexpression of a-sarcomeric actin, a-smooth muscle actin and desmin during myogenesis in rat and mouse embryos. I. Skeletal muscle. Differentiation 1990, 44:132-142 39. Skalli 0, Schurch W, Seemayer T, Lagac6 R, Montandon D, Pittet B, Gabbiani G: Myofibroblasts from diverse pathologic settings are heterogeneous in their content of actin isoforms and intermediate filament proteins. Lab Invest 1989, 60:275-285 40. Rubbia-Brandt L, Sappino AP, Gabbiani G: Locally applied GM-CSF induces the accumulation of a-smooth muscle actin containing myofibroblasts. Virchows Arch B Cell Pathol 1991, 60:73-82

41. DeNofrio D, Hoock TC, Herman IM: Functional sorting of actin isoforms in microvascular pericytes. J Cell Biol 1989, 109:191-202 42. Kocher 0, Madri JA: Modulation of actin mRNAs in cultured vascular cells by matrix components and TGF-f. In Vitro Cell Dev Biol 1990, 25:424-434 43. Johnson RJ, lida H, Alpers CE, Majesky MW, Schwartz SM, Pritzl P, Gordon K, Gown AM: Expression of smooth muscle cell phenotype by rat mesangial cells in immune complex nephritis. J Clin Invest 1991, 87:847858 44. Blank RS, Thompson MM, Owens GK: Cell cycle versus density dependence of smooth muscle a actin expression in cultured rat aortic smooth muscle cells. J Cell Biol 1988, 107:299-306 45. Strauch AR, Berman MD, Miller HR: Substrate-associated macromolecules promote cytodifferentiation of BC3H1 myogenic cells. J Cell Physiol 1991, 146:337348 46. Simonson MS, Dunn MJ: Endothelin-1 stimulates contraction of rat glomerular mesangial cells and potentiates f-adrenergic-mediated cyclic adenosine monophosphate accumulation. J Clin Invest 1990, 85:790797 47. Simonson MS, Dunn MJ: Leukotriene C4 and D4 contract rat glomerular mesangial cells. Kidney Int 1986, 30:524-531 48. Venkatachalam MA, Kreisberg JI: Agonist-induced isotonic contraction of cultured mesangial cells after multiple passage. Am J Physiol 1985, 249:C48-C55 49. Bernik MB: Contractile activity of human glomeruli in culture. Nephron 1969, 6:1-10 50. Sraer JD, Sraer J, Ardaillou R, Mimoune 0: Evidence for renal glomerular receptors for angiotensin 11. Kidney Int 1974, 6:241-246 51. Scharschmidt LA, Douglas JG, Dunn MJ: Angiotensin II and eicosanoids in the control of glomerular size in the rat and human. Am J Physiol 1986, 250:F348-F356 52. De Arriba G, Barrio V, Olivera A, Rodriguez-Puyol D, Lopez-Novoa JM: Atrial natriuretic peptide inhibits angiotensin 11-induced contraction of isolated glomeruli and cultured glomerular mesangial cells of rats: the role of calcium. J Lab Clin Med 1988, 111:466-474 53. Steinhausen M, Endlich K, Wiegman D: Glomerular blood flow. Kidney Int 1990, 38:769-784 54. Sakai T, Kriz W: The structural relationship between mesangial cells and basement membrane of the renal glomerulus. Anat Embryol 1987, 176:373-386