Puromycin aminonucleoside inhibits mesangial cell-induced contraction of collagen gels by stimulating production of reactive oxygen species

Kidney International, VoL 47 (1995), pp. 811—81 7

Puromycin aminonucleoside inhibits mesangial cell-induced

contraction of collagen gels by stimulating production of reactive oxygen species Ro ZEni', MENACHEM AILENBERG, THOMAS K. WADDELL, GREGORY P. DOWNEY, and MELVIN SILVERMAN MRC Membrane Biology Group, Department of Medicine, and Division of Respiratoiy Diseases, University of Toronto, Toronto, Ontario, Canada

Puromycin aminonucleoside inhibits mesangial cell-induced contraction of collagen gels by stimulating production of reactive oxygen species. Glomerular epithelial cell injuly is thought to be the primary reason for the development of proteinuria in puromycin aminonucleoside nephrosis (PAN), the rat model of nephrotic syndrome. By comparison mesangial cells are considered resistant to the effects of puromycin. The purpose of the present study was to investigate whether puromycin in non cytotoxic concentrations caused mesangial cell dysfunction, with particular reference to cell-extracellular matrix interactions. Mesangial cells, when em-

ulonephritis. The glomerular epithelial cell appears to be the primasy cellular target in PAN. Within 24 hours of parenteral administration of puromycin, albuminuria occurs and proteinuria

ensues by 48 hours [11. There are detectable alterations of the width of the podocyte foot processes within 24 hours, and after 48

hours the loss of foot process structure is marked. Podocyte cytoskeletal disaggregation and detachment of the glomerular

bedded in collagen gels, contract after exposure to minimal essential

epithelial cell from the basement membrane is most severe on day

medium (MEM) containing fetal bovine serum (FBS). This contractility, measured by determining changes in area of the collagen gel, is inhibited by puromycin in a dose dependant manner from 2.5 g/ml to 160 .tg/ml. At these concentrations there is no alteration of cell viability as measured by the tetrazolium salt (Mfl') method and trypan blue exclusion. Immunocytochemistry with rhodamine phalloidin reveals that actin filaments are not disrupted. The antioxidants, superoxide dismutase (SOD) and catalase as well as diphenylene iodonium (DPI), a flavoprotein inhibitor, not only counteracted the effect of puromycin on gel contraction, but also enhanced gel contraction when added to mesangial cells on their own. Aminotriazole, an inhibitor of endogenous catalase, inhibited mesangial cell-induced gel contraction in a dose dependent manner (5 mM to 40 mM), and this effect was completely reversed by addition of catalase. Mesangial cells preloaded with dihydrorhodamine and exposed to puro-

5 [1]. These morphological changes are accompanied by a decrease in proteoglycan and increased collagen I, laminin and fibronectin mRNA level expression in the glomerular basement membrane within the first 48 hours [2]. Cultured glomerular epithelial cells are also affected by puromycin in both a time and

mycin (5 gJml to 160 Wml) exhibited a dose dependent increase in

ROS have been measured in whole glomeruli in adriamycin-

rhodamine 123 fluorescence, indicating production of reactive oxygen

induced nephrotic syndrome which may be analogous to PAN [5], and ROS are also produced by cultured kidney slices exposed to

species (ROS). This effect was blocked by the addition of DPI. We conclude that: (i) puromycin, in concentrations similar to those that cause glomerular epithelial cell changes in vitro, also inhibits mesangial cellinduced contractility in collagen gels; (ii) endogenous ROS production by mesangial cells inhibits their ability to contract collagen gels; and (iii) mesangial cells stimulated by puromycin produce increased amounts of ROS which causes inhibition of mesangial cell-collagen gel contraction. Thus mesangial cells may play a previously unrecognized role in the pathogenesis of PAN, either directly through ROS-induced mesangial cell

dose dependant manner, with increasingly severe cell injury noted at concentrations between 25 .tg/ml and 400 .tWml [3]. Reactive oxygen species (ROS), which include H202 and 02,

are highly reactive oxidizing agents that can damage cellular components of cells. They have been implicated in the pathogenesis of PAN both in vitro and in vivo [4]. Increased amounts of

puromycin [6]. Cultured glomerular epithelial cells exposed to puromycin produce H2O2, and toxicity produced by this oxidizing

agent can be reversed by the antioxidants catalase and desferrioxamine [3]. In the rat puromycin model, administration of the antioxidants, SOD and allopurinol, reduced proteinuria and prevented glomerular injury [7]. Taken together there is substantial dysfunction or indirectly through increased production of ROS and evidence implicating ROS production in the pathogenesis of PAN secondary damage to adjacent glomerular epithelial cells. with the glomerular epithelial cell as the main target. Mesangial cells on the other hand are generally considered to be resistant to puromycin nephrotoxicity. Despite the fact that mesangial cells Puromycin aminonucleoside nephrosis in rats has been exten- are well known to produce relatively large amounts of ROS under sively used as a model to understand the pathogenesis of ne- a variety of experimental disease conditions, comparatively little phrotic syndrome in humans, particularly minimal change glomer- information is available on the potential role of mesangial cells in the pathogenesis of PAN. Mesangial cells, similar to other cells of mesenchymal origin, when embedded in a three dimensional type I collagen gel can be Received for publication July 28, 1994 and in revised form October 3, 1994 stimulated by serum to cause a reduction in area of the collagen Accepted for publication October 3, 1994 gel [8—li]. This serum-induced contraction of the collagen gel © 1995 by the International Society of Nephrology requires the cells to have an intact cytoskeleton, the ability to 811

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Zent et al: Mesangial cells and PAN

spread and migrate, to form cell substratum attachments mediated by integrins, a flexible substratum and signaling pathways linking these various components [12]. In the present study we investigated the effect of puromycin on mesangial cells and their

MiT colorimetric assay. Cells were cultured on 24-well plastic plates in a concentration of iO cells/well and allowed to grow

overnight in MEM and 15% FBS. They were washed three times in HBSS and then incubated with the various substances tested for interactions with extracellular matrix using an in vitro collagen gel 24 hours. Cells were washed again in HBSS and incubated with contraction assay. Our results indicate that puromycin, in a dose MIT in a concentration of 1 mg/ml for two hours at 37°C. The dependent manner, inhibits mesangial cell gel contraction. More- cells were treated with dimethyl-sulfoxide for 30 minutes. The over, this effect is mediated by a puromycin-dependent increase in plates were read on a Du Pont multiwell spectrophotometer at a mesangial cell production of ROS. These findings imply that the wavelength of 560 nm. mesangial cell may play a more significant role in the pathogenesis Actin filament staining. Mesangial cell F-actin was visualized, as of PAN than has heretofore been recognized. previously described, using rhodamine-conjugated phalloidin [8, Methods Materials

Minimal essential medium (MEM), Hanks balanced salt solu-

14].

Detection of ROS produced by mesangial cells Production of ROS from puromycin-treated mesangial cells was examined using dihydrorhodamine which can be oxidised to the fluorescent product rhodamine 123 [16, 17]. A stock solution of

tion (HBSS) and fetal bovine serum were supplied by Gibco (Grande Island, NY, USA). Type I collagen, puromycin amino- 2 m of dihydrorhodamine was prepared in dimethyl sulfoxide. nucleoside, MIT [3,(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazo- Mesangial cells cultured at a density of 105/ml in 24 well plates hum bromidel, bovine liver catalase, superoxide dismutase were incubated for 24 hours in MEM with 30 U/ml horseradish (SOD), aminotriazole, colchicine, desferrioxamine B mesylate peroxidase and 2 m of dihydrorhodamine at 37°C. Increasing and allopurinol were obtained from Sigma Laboratories (St. concentrations of puromycin from 20 jLg/ml to 160 g/ml were Louis, MO, USA). Diphenylene iodonium (DPI) was a gift from Dr. Sergio Grinstein (Hospital for Sick Children, Toronto, Ontario, Canada). Rhodamine-conjugated phalloidin and dihydrorhodamine 123 were obtained from Molecular Probes (Eugene, OR, USA). Tissue culture

added to the cells. The inhibitory effect of DPI at a concentration of 0.625 n on cells treated with puromycin was also assessed. The

cells were trypsinized and filtered through a 35 j.m filter. The generation of rhodamine 123 was analyzed on a Becton Dickinson

FACscan with excitation at 488 nm and the fluorescence measured at 525 nm.

Statistics Rat glomeruli were isolated from kidneys of three-week-old male Sprague-Dawley rats (approximately 15 to 50 g) by the All experiments were performed in duplicate and on at least graded sieve technique. Mesangial cells were trypsinized and three separate occasions. The results of the contraction assays plated onto 100 mm plates, 6 well or 24 well plates in MEM were normalized as shown in the Methods section. The means and containing 15% FBS as previously described [13]. For rhodamine- standard errors of the pooled data of the contraction assays, MIT phalloidin staining cells were plated on 12 mm glass slides assays and dihydrorhodamine assays were calculated and shown positioned in wells. Experiments were performed on cells between graphically. The regression coefficients of these curves was deterpassages 5 and 15. mined and the significance of these regression coefficients was

compared by the Student's t-test. In Figure 4 the mean and Gel contraction assay

The gel contraction assay was based on previously described procedures [8, 14]. Mesangial cells were placed in a solution of MEM and type I collagen which was allowed to gelate for three hours. The substances tested were dissolved in MEM and added for 24 hours, after which contraction of the gels was induced by the addition 3% FBS in MEM. The degree of contraction was

standard error of the assays were calculated and their significance was compared using the Student's t-test.

Results Effect of puromycin on mesangial cell-induced collagen gel contraction

Mesangial cells plated within hydrated collagen I gels attached

measured after an additional 24 hours and quantitated. The and assumed a bipolar elongated shape. Incubation of the gel for longest and shortest axes of the gel was measured using a 24 hours after the addition of MEM resulted in a small amount of dissecting microscope (Wild-Leitz) at 16 times magnification, and

the percentage of maximal contraction was calculated using the following formula.

% gel contraction 100 times (area MEM treated gel — area PBS and test substance treated gel)

(area MEM treated gel — area FBS treated gel)

Cell viability studies

Cell viability was assessed by the tetrazohium salt (MIT) method [151 as well as by trypan blue dye exclusion [8, 14, 15].

gel contraction. Addition of MEM containing FBS (final concentration 3% PBS) resulted in maximal gel contraction. As demonstrated in Figure 1, when puromycin was added to MEM in the

presence of FBS there was a dose dependant inhibition of the FBS-induced gel contraction. In the concentration range of 5 g/ml to 160 Wml no cytotoxicity was noted, suggesting that the inhibition of gel contraction was not simply due to impairment of cell viability. As an intact cytoskeleton is necessary for gel contraction [8] we

determined whether puromycin treatment of mesangial cells disrupted the actin filaments. Cells were plated on cover slips, treated with puromycin in concentrations up to 160 j.Lg/ml and

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Zent et al: Mesangial cells and PAN

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Fig. 1. The dose-dependent inhibitoiy effect of puromycin on FBS-induced

mesangial cell-collagen gel contraction. Mesangial cells, embedded in collagen gels, were treated with increasing concentrations of puromycin for 24 hours and then exposed to 3% FBS for a further 24 hours. Percent gel contractility was calculated using the formula outlined in the Methods

section. The data points represent the mean the standard error of three experiments performed in duplicate.

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Aminotriazole, mtc Fig. 2. The inhibitoiy effect of aminotriazole •

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mesangial cell-collagen gel contraction and its complete reversal by the addition of 0.4 mg/mI catalase (• •). Mesangial cells were embedded in collagen gels and % contractility calculated as described in the Methods section. The data points represent the mean the standard error of three experiments performed in duplicate.

stained with rhodamine phalloidin. The actin filaments of mesangial cells were intact under these conditions (data not shown). In other experiments, coichicine, in concentrations up to 8 nM, did not inhibit gel contraction (data not shown), indicating that this assay is not dependent on microtubular function.

and SOD each caused significant gel contraction in the absence of FBS (Fig. 3). Taken together the results of sections (a) and (b) above confirm not only that ROS inhibit the ability of mesangial cells to contract collagen gels, but also that endogenous ROS produced by mes-

Effect of ROS on MC-induced gel contraction

angial cells under our present culture conditions are acting to oppose collagen gel contraction.

(a) The effect of aminotriazole. Our first objective was to (c) The effect of ROS scavengers on puromycin toxicity. Although determine what effect, if any, ROS had on mesangial cell- all the gel contraction assays were performed in the noncytotoxic mediated gel contraction. For this purpose we studied the effect of range of puromycin, we wished to determine whether ROS aminotriazole, an inhibitor of endogenous catalase, on mesangial cells embedded in collagen gels. Treatment of cells with aminotriazole [5, 18] results in increased H202 release into the medium

scavengers prevent cytotoxic injury to mesangial cells in a manner

duced H202. As shown in Figure 2, when aminotriazole (5 mM to 40 mM) was added to MEM in the presence of FBS, there was a dose dependant inhibition of the FBS-induced gel contraction in the absence of cytotoxicity (confirmed by MTTassay and trypan blue exclusion, data not shown). This inhibitory effect of aminotriazole on gel contraction was completely reversed by concomitant addition of catalase 0.4 mg/ml, confirming that inhibition of gel contraction was due to an increase in endogenously produced H202.

puromycin was accompanied by addition of ROS scavengers, was assessed. To ensure that the antioxidants tested did not interfere with the optical density reading of the assay, controls using these substances in the absence of puromycin were performed. Mesangi-

similar to that demonstrated in cultured glomerular epithelial cells [3]. Cell viability, using MIT assays (see Methods) on presumably due to impaired degradation of constitutively pro- mesangial cells in the presence of puromycin alone or when

al cells treated with the antioxidants alone gave optical density readings similar to the control mesangial cells (data not shown). The data shown in Figure 4 indicates that up to 160 gig/mI there was no observable toxicity of puromycin. For concentrations greater than 160 j.g/ml there was progressive puromycin cytotox(b) Evidence that ROS produced by mesangial cells under normal icity which was largely reversed by ROS scavengers. The data of conditions inhibit gel contraction. To further confirm that ROS the MIT assay correlated with the mesangial cell cyotoxicity played a role in mesangial cell collagen gel contraction we observed with Trypan blue exclusion staining (data not shown). demonstrated that the addition of antioxidants to the system These data further suggest that puromycin treatment of mesangial induced gel contraction in the absence of FBS. The fiavoprotein cells in vitro causes increased ROS production which in turn inhibitor DPI [19, 201, catalase and the combination of catalase affects cellular function and viability.

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Zent et al: Mesangial cells and PAN

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DPI CAT CAT/SOD Fig. 3. Induction of gel contraction by ROS scavengers SOD, catalase or DPI in the absence of FBS. Mesangial cells were embedded in collagen as described in the legend for Figure 1. However, no FBS was added after 24 hours. Contraction is expressed as a % relative to a gel with the addition of MEM alone. The data points represent the mean the standard error of three experiments performed in duplicate. *A significant (P < 0.02) change in contraction induced by catalase, DPI and catalase/SOD when compared to MEM alone.

(d) The effect of ROS scavengers on puromycin inhibition of gel

contraction. We next wished to determine whether or not the inhibitory effect of puromycin on collagen gel contraction was actually due to puromycin-induced ROS production. As a first

o 200 400 600 800 1000 1200 1400 Puromycin, pg/mi Fig. 4. Mesangial cell viability measured by MIT absorbance assay (Methods). Puromycin alone (A— —A), puromycin and SOD (• — — •), puromycm and catalase ('V — — V), puromycin and catalase and SOD (• and

puromycin and DPI (•

•)

•) are compared to control mesangial

cells. Mesangial cells were plated in 24-well plates and then treated with various concentrations of puromycin with or without ROS scavengers for a further 24 hours, after which a cell viability assay was performed. The data points represent the mean the standard error of three experiments performed in duplicate. *A significant (P < 0.02) difference in cell viability between cells treated with puromycin alone and cells treated with the various ROS inhibitors.

step the effect of ROS scavengers on puromycin treated collagen gels was assessed. Results shown in Figure 5 demonstrate that addition of catalase (0.4 mg/mI) and SOD (1000 ji/ml) to puro- cells treated with increasing concentrations of puromycin there mycin-treated mesangial cell-collagen gels, alone or in combina- was a progressive increase of rhodamine 123 fluorescence. Moretion, decreased the inhibitory effect of puromycin. As demon- over, also shown in Figure 7, DPI inhibited the ROS production strated in Figure 6, DPI which inhibited flavoproteins in the stimulated by the addition of puromycin to mesangial cells. These concentration 0.625 tiM, also reversed the inhibitory effect of results provide strong evidence that puromycin increases producpuromycin. In contrast, neither desferrioxamine (0.5 m to 8 mM) tion of ROS by mesangial cells. nor allopurinol (0.5 n to 2 nM) reversed the inhibitory effect of Discussion puromycin on the collagen gels (data not shown). Previous studies on the pathogenesis of PAN have tended to (e) ROS production by mesangial cells treated with puromycin. The reversal of puromycin inhibition of gel contraction by ROS focus almost exclusively on puromycin-induced changes in gbscavengers (Figs. 5 and 6) provides indirect evidence that the merular epithelial cells or the glomerular epithelial cell-glomerueffect of puromycin was mediated by increased mesangial cell bar basement membrane interaction, as being primarily responsiROS production. To directly examine whether puromycin stimu- ble for proteinuria. In contrast, the mesangial cell is considered to lated mesangial cells to produce increased ROS, the fluorescent be "resistant" to these concentrations of puromycin. In fact, probe dihydrorhodamine 123 [16, 17] was used to measure puromycin sensitivity is widely used to distinguish between medirectly ROS production over 24 hours, a time frame similar to sangial cells and glomerular epithelial cells under primary culture the gel contraction assay. To ensure that the increase in fluores- conditions [21]. Notwithstanding these considerations, in the cence of rhodamine 123 was due to ROS production by mesangial present study we have demonstrated that puromycin, in concen-

cells rather than due to the puromycin or DPI, similar experi-

trations similar to those that cause pathological changes to

ments were carried out in a cell-free system initially, with mesangi-

glomerular epithelial cells in culture and in vivo, also result in mesangial cell dysfunction. In particular the results of the present study show that puromycin inhibits FBS-induced mesangial cellcollagen gel contraction, an assay which reflects the integrity of

al cells being placed in the medium for only half an hour prior to flow cytometry. The dihydrorhodamine fluorescence changes in the initially cell-free system with DPI (0.625 nM) and puromycin (5

.tg/ml to 160 gJml) were similar to nonstimulated mesangial cells. However, inspection of Figure 7 revealed that in mesangial

the interactions between the cell cytoskeleton and cell-substratum attachments. Moreover, these effects of puromycin on mesangial

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Zent et a!: Mesangial cells and PAN

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Fig. 5. Reversal of the inhibito,y effect of puromycin (—•) on FBSinduced MC collagen gel contraction by the addition of antioxidants SOD (U—U), catalase (A— — A) and SOD and catalase (V — — Y). Mesangial cells were embedded in collagen gels and % contractility calculated as described in the Methods section. SOD (1000 pJml), catalase (0.4 mg/mI) and the combination of the two reverse the inhibitory effect of puromycin

in a dose dependent manner. The data points represent the mean the

Fig. 6. Reversal of the inhibitory effect of puromycin (—•) on FBSinduced MC collagen gel contraction by the addition of DPI (0.625 pM) (U—U). MC were embedded in collagen gels and % contractility calcu-

lated as described in the Methods section. The data points represent the mean the standard error of three experiments performed in duplicate. *A significant (P < 0.02) difference in inhibition of gel contraction by puromycin when treated with DPI.

standard error of three experiments performed in duplicate. *A significant (P < 0.02) difference in inhibition of gel contraction by puromycin when treated with ROS inhibitors SOD and catalase.

cells are caused by a puromycin-mediated induction of increased ROS production.

In the present study no morphologic evidence of puromycin induced changes in actin filaments was found. In addition, microtubules do not appear to be involved in the gel contraction assay.

Instead, the most likely cause of puromycin inhibition of FBSinduced contraction is due to alterations at the level of mesangial cell-extracellular matrix attachment or alterations in the cell signaling processes that govern the mesangial cell-extracellular matrix interaction. The inhibitory effect of aminotriazole on FBS-induced mesangial cell-collagen gel contraction and its reversal by catalase, as well

as the induction of gel contraction by DPI and catalase in the absence of FBS, demonstrate that endogenous mesangial cell ROS play a modulatory role in mesangial cell-collagen gel interactions under normal steady-state resting culture conditions. ROS have been shown to act as modulators of cell signaling in other cell

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have not determined the mechanism whereby ROS affect the mesangial cell-collagen gel assay; however, it is plausible that Puromycin, pg/mi ROS is similarly a regulator of cell signaling under these condi- Fig. 7. Mesangial cell induced dihydrorhodamine fluorescence changes tions.

Although H202 would appear to be implicated, we have not identified unequivocally which ROS are actually responsible for the inhibitory effect on collagen gel contraction. The finding that DPI reverses the puromycin changes implicates ROS formed by

(Methods) following puromycin (5 pg/mI to 160 pg/mI) treatment (—•) and after the addition of DPI (0.625 nM) (U U). The data points represent the mean the standard error of three experiments performed

in duplicate. *A significant (P < 0.02) difference in fluorescence by puromycin-treated mesangial cells, when compared to cells treated with puromycin and DPI.

Zent et al. Mesangial cells and PAN

816

Fig. 8. Migratoiy cultured cells plated on plastic substratum (S) spread and generate vectorial forces (VA, VA') which originate from the elastic nature of actin filaments. These forces are transmitted to the substratum via attachment loci (Bi, B2). The rigidity of the plastic in turn generates vectorial forces (VC,VC') that oppose the forces generated by the actin filaments [21]. These two forces are opposite in

Actin filaments A

I

B'

P

VA

VC

* *

VA'

VC'

Bi

direction and equal in magnitude (VA + VA' = VC + VC'). Thus, any reduction in the forces of VC and VC' brought about by plating cells on a flexible substrate (collagen I in this study) results in VA + VA' > VC + VC', leading to gel contraction. This contraction is achieved by a newly created attachment point (B') following the migratory movement (arrow M) of the cell. The forces generated by actin filaments pull the newly established attachment (B') to its original position (Bi), while the cell remains attached to the substratum. Hence, in order to achieve gel contraction, all the following requirements must be fulfilled: (1) cell spreading by elaborate actin filaments that produce contracting forces; (2) cell migration that permits formation of new cell-substratum contacts; (3) cell-substratum attachment that generate the necessary binding site for transmission of forces to substratum; (4) fierible substratum that allows shrinkage of the substratum. Perturbation of any of the above four requirements will inhibit gel contraction. Abbreviations are: F, plasma membrane; N, nucleus.

fiavoproteins as being at least in part responsible for the inhibitory and tumor necrosis factor in cultured mesangial cells [25]; howeffect of puromycin on gel contraction. In contrast to the findings ever, little else is known about the role of these cells in PAN. We

of Diamond, Bonventre and Karnovsky [7], allopurinol did not reverse the effect produced by puromycin. Consequently, it is unlikely that production of ROS by the xanthine oxidase pathway plays a significant role in our mesangial cell studies.

have shown that puromycin affects mesangial cell-interactions with collagen gels. For gel contraction to occur mesangial cells

must be able to produce a force. The origin of this force is complex and somewhat controversial, however, actin filaments

Although all effects of puromycin in the gel assays were somehow play an important role [26] because their disruption, for performed in the non-cytotoxic range, our study confirms that example by cytochalasin D, inhibits gel contraction [8]. Cell mesangial cells in culture are more resistant to the cytotoxic migration is also a significant parameter and there must be an affects of puromycin than glomerular epithelial cells. Cytotoxicity intact cell-extracellular matrix interface in the form of a cell

was noted at concentrations greater than 160 tg/ml in mesangial adhesion molecule in order that the generated force be transmitcells compared to 25 ig/ml in glomerular epithelial cells [3]. At ted to the gel [9]. Signaling pathways are necessary to regulate all concentrations greater than 160 g/ml the puromycin-induced these the mesangial cell-ECM interactions. Collagen gel contraction by mesangial cells can be induced by cytotoxicity of mesangial cells was reversed by the addition of ROS inhibitors. This observation is similar to the earlier reported various growth factors, hormones, vasoactive mediators and exeffects of puromycin on glomerular epithelial cells in culture, tracellular matrix components found in FBS [10, 11]. When suggesting that the mechanism of injury in mesangial cell is similar mesangial cells are embedded in extracellular matrix their arto that of glomerular epithelial cells, albeit at higher concentra- rangement is unlike muscle tissue, and their immediate interaction is with extracellular matrix rather than directly with adjacent tions of puromycin. Resting mesangial cells produce basal rates of H202 of approx- mesangial cells [26]. Mesangial cells in culture respond to agonists imately 0.4 nM per iO cells [241, which is significantly higher than which are known to induce smooth muscle contraction in vivo by glomerular epithelial cells which produce almost no detectable a reduction in their cell volume [14]. But this rounding up and H202 levels in the resting state [3, 24]. Since mesangial cells have reduction in cell volume that is observed in culture systems and a much greater potential to produce ROS than glomerular often referred to as "contractility" is actually associated with a epithelial cells, they may in fact be a more important source of reduction in contractile forces, hence inhibition of the gel conROS than the glomerular epithelial cells in PAN nephrosis. traction assay (Fig. 8). For example, cells treated with TGF-13 Consequently, the effects of enhanced mesangial cell ROS pro- become flattened, exhibit enhanced expression of actin filaments

duction may not just be limited to changing mesangial cell- and induce contractility when embedded in collagen gels. In

extracellular matrix interactions, but could instead cause second- contrast, dibuteryl cAMP and ROS causes the same cells to round up and ultimately inhibits contractility in the collagen gel model ary damage to adjacent glomerular epithelial cells. Puromycin has been shown to increase platelet activating factor [14].

Zent et al: Mesangial cells and PAN

The collagen gel assay is a useful model for elucidating the physiological and pathological interactions between the mesangial cell and extracellular matrix; however, the physiologic relevancy of

the assay is not established. In order to do so it is necessary to reconcile the fact that agonists that act as vasoconstrictors in vivo cause reduction in cell volume and decrease contractility in the gel model. We propose the following mechanism: forces arising from mesangial cell extracellular matrix interactions (Fig. 8) are transmitted to the microfibrils [27] that connect the glomerular basement membrane to the mesangial cell, and together they form a

biomechanical unit. Under resting conditions the resultant of these forces maintains the capillaries in the open state. When cells are exposed to "constrictor" agents, the actin filaments undergo complex changes causing reduction in cell volume (rounding up) leading to relaxation of the forces acting in the adjacent capillaries

with a consequent reduction of capillary diameter (that is, constriction). Within this model framework, mesangial cell-extracellular matrix interactions could exert considerable influence on glomerular filtration dynamics which in turn would influence the ultrafiltration conditions in the glomerulus. Following the same reasoning, the proteinuria of puromycin nephrotoxicity may not only be caused by the cytotoxic effects of puromycin on the glomerular epithelial cell (leading to detachment), but also may be directly related to altered mesangial cell-extracellular matrix interactions.

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4. Si-iii-i SV: Role of reactive oxygen metabolites in experimental glomerular disease. Kidney mt 35:1093—1106, 1989 5. UEDA N, GUIDET B, Si-jAil SV: Measurement of intracellular genera-

tion of hydrogen peroxide by rat glomeruli in vitro. Kidney mt 45:788—793, 1994

6. RICARDO SD, BERTRAM JF, RYAN GB: Reactive oxygen species in aminonucleoside nephrosis: In vitro studies. Kidney mt 45:1057—1069, 1994 7. DIAMOND JR, BONVENTRE JF, KARNOVSKY MJ: A role for oxygen free

radicals in aminonucleoside nephrosis. Kidney mt 29:478—483, 1986 8. AILENBERG M, WEINSTEIN T, LI I, SILVERMAN M: Activation of procollagenase IV by cytochalasin D and concanavlin A in cultured rat mesangial cells: Linkage to cytoskeletal reorganisation. JASN 4:1760— 1770, 1994 9. KUPPER TS, FERGUSON TA: A potential pathophysiological role for a2131 integrins in human eye disease involving vitreoretinal traction. FASEB 7:1401—1406, 1994 10. KJTAMURA M, MARUYAMA N, MITARA! T, NAGASAWA R, YOSHIDA H,

SAII 0: Extracellular matrix contraction by cultured mesangial cells: Modulation by transforming growth factor-J3 and matrix components. Exp Mol Pathol 56:132—143, 1992 11. KITAMURA M, MITARAI T, MARUYAMA N, NAGASAWA R, YOSHIDA H,

SAicI 0: Mesangial cell behaviour in a three-dimensional extracellular matrix. Kidney mt 40:653—661, 1991 12. KORNBERG L, JULIANO RL: Signal transduction from the extracellular matrix: The integrin-tyrosine kinase connection. TiPS 13:93—95, 1992 13. HARPER PA, ROBINSON JM, HOOVER RL, WRIGHT TC, KARNOVSKY

MJ: Improved methods for culturing rat glomerular cells. Kidney Int 26:875—880, 1984

In conclusion, we have demonstrated that puromycin affects 14. AILENBERG M, TUNG PS, FRITZ IB: Transforming growth factor j3 elicits shape changes and increases contractility of testicular peritumesangial cell-collagen gel contractility in concentrations similar bular cells. Biol Reprod 42:499—509, 1990 to those that cause morphological and cytotoxic changes of

glomerular epithelial cells in culture and that this effect is mediated by ROS production. Based on our in vitro studies we believe ROS act as modulators of mesangial cell signaling. The effect of ROS production by the mesangial cells after exposure to puromycin may not just be limited to the mesangial cell itself, but may also influence the adjacent glomerular epithelial cells causing glomerular epithelial cell toxicity and possibly glomerular epithe-

hal cell detachment. Thus, mesangial cells may play a more important role in the pathogenesis of PAN nephrosis than has heretofore been recognized. Acknowledgements

This work was supported by a group grant from the Medical Research Council of Canada and The Kidney Foundation of Canada. Preliminary aspects of this work were presented at the Clinical Research Meeting, Baltimore in May 1994. Roy Zent is the recipient of a fellowship from Miles Canada. We thank Brian La and David Naimark for their help with the statistical analysis

as well as Cheryl Smith and Andrea Sue-A-Quan for their technical assistance using the FACScanner. Reprint requests to M. Silverman, Room 7207 Medical Sciences Building, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.

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