Different activation of mitogen-activated protein kinases inexperimental proliferative glomerulonephritis

DIRK BOKEMEYER, KELLY E. GUGLIELMI, ANN MCGINTY, ANDREY SOROKIN, ELIAS A. LIANOS,and MICHAEL J. DUNN

Department of Medicine, Division of Nephrology, Medical College of Wisconsin, Milwaukee, WI, USA, and Medizinische Poliklinik,University of Bonn, Bonn, Germany

Different activation of mitogen-activated protein kinases in ex-perimental proliferative glomerulonephritis. Mitogen-activatedprotein (MAP) kinases are critical for cell signaling goals such ascellular proliferation and induction of apoptosis. We examinedwhether MAP kinases, as a point of convergence for multipleextracellular stimuli, are activated in proliferative glomerulone-phritis (GN) in vivo. Accelerated crescentic anti-glomerular base-ment membrane (GBM) GN was induced in rats preimmunizedwith rabbit IgG by administration of rabbit anti-rat GBM serum.Whole cortical tissue and isolated glomeruli were then subjectedto kinase activity assays and Western blot analysis. Corticalactivity of the archetypal MAP kinase, extracellular signal-regu-lated kinase (ERK), was increased significantly one, three, andseven days after induction of GN. In contrast, activation of MAPkinases with antiproliferative actions, stress-activated proteinkinase, and p38 MAP kinase was detectable only in the earlystages of proliferative GN (days one and three), implying thatdifferent MAP kinases serve distinct roles in the pathogenesis of GN.

A major focus for research into the pathogenesis ofglomerulonephritis (GN) has been to define extracellulargrowth-promoting factors controlling cell growth duringproliferative forms of GN. Several cytokines are implicatedin the pathogenesis of proliferative GN and may accountfor hypercellularity [1]. Mitogen-activated protein (MAP)kinases are important mediators in the intracellular net-work of interacting proteins transducing extracellular cuesto intracellular responses [2]. Because in vitro studies haveshown MAP kinases to be a key point in the control ofcellular growth, these might be crucial mediators of cellularproliferation in renal diseases mediated by extracellularstimuli. Extracellular signal-regulated kinase (ERK), thebest described mammalian MAP kinase, regulates theexpression of many genes by phosphorylating several tran-scription factors [2]. ERK’s activity is regulated by MAP

kinase/ERK kinase (MEK), which phosphorylates boththreonine and tyrosine regulatory sites in ERK [2]. In vitrostudies have established the pivotal role of the ERKsignaling pathway in the control of cellular proliferation [2].In contrast to ERK, the recently described MAP kinasesstress-activated protein kinase (SAPK) and p38 MAP ki-nase are thought to inhibit cell growth and to induceapoptosis [2, 3]. Although much is known about the func-tion of MAP kinases in vitro, little is known about their rolein physiological or pathophysiological conditions or theiractivation in vivo.

METHODS

Proliferative GN was induced as described previously [4].Briefly, male Sprague-Dawley rats (180 to 200 g) wereimmunized with 1-mg i.p. rabbit IgG emulsified in completeFreund’s adjuvant. On days 5 and 6 thereafter, animalswere given rabbit immune serum raised against rat partic-ulate glomerular basement membrane (GBM) or controlnonimmune rabbit serum, via the tail vein. Studies wereperformed on days 1, 3, and 7 following the secondinjection. Renal cortical sections and isolated glomeruliwere lysed in Triton X-100 buffer.

Western blot analysis was performed as described previ-ously [5]. Briefly, soluble lysate (30 mg) of whole corticaltissue or isolated glomeruli were mixed with Laemmlibuffer and were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Cellulo-ses were probed with polyclonal antibodies against p42ERK and phosphorylated p38 MAP kinase (New EnglandBiolabs, Beverly, MA, USA) or with monoclonal antibodiesagainst MEK-1 (Transduction Laboratories, Lexington,KY, USA).

Kinase activity assays were performed as described pre-viously [5]. Briefly, 200-mg soluble lysate was incubated for90 minutes with 2-ml polyclonal antibody recognizing p42ERK. Immunocomplexes were adsorbed on protein A-sepharose and were washed and resuspended in a 60-mlkinase buffer containing 50 mM adenosine triphosphate

Key words: inflammation, cell proliferation, anti-glomerular basementmembrane, extracellular signal-regulated kinase, MAP kinase/ERK ki-nase, stress-activated protein kinase.

© 1998 by the International Society of Nephrology

Kidney International, Vol. 54, Suppl. 67 (1998), pp. S-189–S-191

S-189

(ATP), 5-mCi [g-32P]ATP, and 15-mg myelin basic protein.The reaction was initiated by incubation at 30°C for 15minutes. Laemmli buffer was added to terminate thereaction, and samples were subjected to SDS-PAGE.

RESULTS AND DISCUSSION

We evaluated the in vivo activity of MAP kinases in thekidney of rats with crescentic GN. Sprague-Dawley ratsdeveloped accelerated GN with heavy proteinuria as earlyas one day following the anti-GBM antibody injections [4].Prominent lesions of proliferation and crescent formationwere apparent as early as day 3 [4], and most animals diedby days 9 to 11. We examined the activation of ERK ondays 1, 3, and 7 in whole cortical tissue. Anti-ERK Westernblot analysis and immunocomplex ERK activity assaysshowed a slight increase in activation of ERK on day 1 andsignificant activation on days 3 and 7 (Fig. 1). Quantifica-tion of myelin basic protein phosphorylation at day 3revealed a fourfold increase in ERK activity in rats injectedwith anti-GBM immune serum. Cellular proliferation isthought to be pivotal for the development of crescentformation and progression of proliferative GN to end-stagerenal disease. The proliferative response to injury in GN

may be augmented by convergence of multiple cytokines onERK, inducing its activation.

Previously, up-regulation of MEK protein levels, thekinase upstream from ERK, has been thought to beimportant in the long-term regulation of ERK in culturedglomerular mesangial cells [6]. Here, we show up-regula-tion of MEK protein levels in rats with experimentalproliferative GN. As shown in Figure 2, the changes ofMEK protein in whole cortex samples were not significant.However, in isolated glomeruli, there was a dramaticincrease in MEK protein at day 3 and day 7. Thus, inagreement with the location of the prominent cellularproliferation, MEK protein levels were found elevatedprincipally in glomeruli. It is therefore likely that theelevated MEK protein levels contribute to ERK activationin vivo.

Previously, we have shown SAPK to be activated onlyearly in the development of proliferative GN [4]. SAPK andp38 MAP kinase have recently been suggested to inducecellular apoptosis and to inhibit cellular proliferation [2, 3].Figure 3 shows the activation of p38 MAP kinase in isolatedglomeruli on days 1 and 3, but not on day 7, implyinginvolvement of p38 MAP kinase only in the early stage in

Fig. 1. Activation of extracellular signal-regulated kinase (ERK) in crescentic glomerulonephritis (GN). Upper panel: Western blot analysis showsERK2 [p42 mitogen-activated protein (MAP) kinase] in whole cortical tissue lysates. At each time point, one control and one experimental animal werestudied. Rats were sacrificed at the indicated times following the second injection of anti-glomerular basement membrane (GBM) serum. Activationof ERK is identifiable by an increase of phosphorylated protein forms, detected by bands with delayed mobility (indicated by stars), compared with theunshifted unphosphorylated ERK forms. Lower panels: ERK activity assayed by the ability of immunoprecipitated ERK to phosphorylate myelin basicprotein (MBP).

Fig. 2. Western blot analysis of MAP kinase/ERK kinase (MEK) protein expression incortical and glomerular lysates.

Bokemeyer et al: Activation of MAP kinases in glomerulonephritisS-190

the pathogenesis of this disease model. No significantchanges of p38 MAP kinase activity were seen in wholecortical tissue, pointing to the glomeruli as the major site ofp38 MAP kinase activation during early stages of prolifer-ative GN. Mesangial cell apoptosis occurs during anti-Thy1GN and is thought to account for the resolution of glomer-ular hypercellularity in this disease model [7]. It is, there-fore, tempting to speculate that the observed p38 MAPkinase activation might account for a similar induction ofapoptosis in proliferative GN. However, further studies areneeded for a better understanding of the role of p38 MAPkinase and SAPK in crescentic GN.

Activation of ERK and p38 MAP kinase in proliferativeGN, as shown in this study, is a novel finding and points toMAP kinases as a putative regulator of the proliferativeresponse to immune injury in vivo. Activation of p38 MAPkinase follows a different time course to that of ERK,suggesting distinct roles for these two MAP kinases roles atdifferent stages of proliferative GN.

ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health GrantsHL 22563 and DK 41684 (to M.J.D.), by a grant of the DeutscheForschungsgemeinschaft BO 1288/3-1 (to D.B.), and a Veterans Affarsmerit review grant (to E.A.L.).

APPENDIXAbbreviations used in this article are: ATP, adenosine triphosphate;

ERK, extracellular signal-regulated kinase; GBM, glomerular basementmembrane; GN, glomerulonephritis; MAP, mitogen-activated protein;MEK, MAP kinase/ERK kinase; SAPK, stress-activated protein kinase.

Reprint requests to Dirk Bokemeyer, M.D., Medizinische Poliklinik, Uni-versity of Bonn, Wilhelmstr. 35-37, D-53111 Bonn, Germany.E-mail: bokemeyer@uni-bonn.de

REFERENCES1. ABBOUD HE: Growth factors in glomerulonephritis. Kidney Int 43:

252–267, 19932. BOKEMEYER D, SOROKIN A, DUNN MJ: Multiple intracellular MAP

kinase signaling cascades. Kidney Int 49:1187–1198, 19963. XIA Z, DICKENS M, RAINGEAUD J, DAVIS RJ, GREENBERG ME:

Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis.Science 270:1326–1331, 1995

4. BOKEMEYER D, GUGLIELMI KE, MCGINTY A, SOROKIN A, LIANOS EA,DUNN MJ: Activation of extracellular signal-regulated kinase inproliferative glomerulonephritis in rats. J Clin Invest 100:582–588,1997

5. BOKEMEYER D, SOROKIN A, DUNN MJ: Differential regulation of thedual-specificity protein-tyrosine phosphatases CL100, B23 and PAC1in mesangial cells. J Am Soc Nephrol 8:40–50, 1997

6. SCHRAMEK H, SOROKIN A, WATSON RD, DUNN MJ: Differentiallong-term regulation of MEK and of p42 MAPK in rat glomerularmesangial cells. Am J Physiol 270:C40–C48, 1996

7. BAKER AJ, MOONEY A, HUGHES J, LOMBARDI D, JOHNSON RJ, SAVILLJ: Mesangial cell apoptosis: The major mechanism for resolution ofglomerular hypercellularity in experimental proliferative nephritis.J Clin Invest 94:2105–2116, 1994

Fig. 3. Activation of p38 mitogen-activated protein (MAP) kinase in crescentic glomerulonephritis (GN). At each time point, one control and twoexperimental animal were studied. Whole lysates of isolated glomeruli were analyzed by Western blot analysis employing an antiserum against thephosphorylated and activated form of p38 MAP kinase.

Bokemeyer et al: Activation of MAP kinases in glomerulonephritis S-191