Kidney International, Vol. 61 (2002), pp. 1020–1029

Angiotensin II infusion ameliorates the early phaseof a mesangioproliferative glomerulonephritis1

ULRICH O. WENZEL, FRIEDRICH THAISS, UDO HELMCHEN, ROLF A.K. STAHL, and GUNTER WOLF

Division of Nephrology, Department of Medicine, and Department of Pathology, University of Hamburg, Hamburg, Germany

Angiotensin II infusion ameliorates the early phase of a mesan- experimental and clinical studies have documented agioproliferative glomerulonephritis. strong nephroprotective effect of antihypertensive ther-

Background. Inhibition of the renin-angiotensin system slows apy using angiotensin-converting enzyme (ACE) inhibi-the progression of chronic renal disease.tors [1, 2]. Furthermore, there is clear evidence that ACEMethods. To test whether angiotensin II (Ang II) infusion

aggravates or ameliorates an acute glomerulonephritis, the inhibitors show beneficial effects that go beyond theirpeptide was infused (200 ng/min by osmotic minipump) in rats blood pressure lowering effect. However, the mecha-with an anti-thymocyte antibody-induced glomerulonephritis nisms of action of ACE inhibitors remain incompletely(ATS).

understood. Both experimental and clinical studies re-Results. Ang II significantly increased blood pressure. Fol-lowing injection of the antibody, similar glomerular binding of vealed that angiotensin II (Ang II) plays a central rolerabbit IgG and rat complement C3 was detected in ATS and in the progression of renal disease [3]. The mechanismsAng II�ATS rats, indicating no differences in delivery and of Ang II-induced kidney damage are generally attrib-binding of the antibody. Ang II infusion, however, induced a

uted to direct vasoconstriction, growth promoting andsignificant reduction in glomerular monocyte infiltration, cellproliferation and matrix expansion in nephritic rats compared profibrotic effects. We and others have characterized theto rats with nephritis without Ang II. The antiproliferative model of anti-Thy-1 induced glomerulonephritis [4–6].effect of Ang II was inhibited by the Ang II type 1 (AT1) re- The injection of a rabbit anti-rat thymocyte serum in-ceptor blocker irbesartan, but not by the AT2 receptor blocker

duces mesangiolysis, followed by monocyte infiltrationPD 123319, indicating that this effect was likely transduced byAT1 receptors. Norepinephrine infusion (600 ng/min) pro- as well as proliferation and matrix expansion. It has beenduced a similar degree of hypertension, but did not affect demonstrated that blockade of the renin-angiotensin sys-glomerular proliferation in nephritic rats. Ang II induced the tem with ACE inhibitors or Ang II type 1 (AT1) receptorglomerular expression of the cell cycle inhibitor p27KIP1 and of

blockers ameliorates the pathologic changes of the earlytransforming growth factor-� (TGF-�) and inhibited expres-sion of monocyte chemotactic protein 1 (MCP-1). phase of the anti-thymocyte antibody-induced glomeru-

Conclusion. Ang II surprisingly ameliorates glomerular mono- lonephritis (ATS) [7, 8]. In addition, we recently foundcyte infiltration, proliferation and matrix expansion in ATS that induction of ATS in rats with renovascular hyperten-nephritis. Ang II-mediated induction of cyclin kinase inhibitors

sion aggravated the glomerular damage of ATS in theand TGF-� may contribute to the protection of the glomerulusfrom inflammatory injury by inducing cell cycle arrest and nonclipped kidney [9]. Therefore, we were interested inattenuating activation of local and recruited cells. Alterna- further dissecting the obviously deleterious effects oftively, Ang II might protect the kidney at least in part by less Ang II on the course of ATS. Surprisingly, we discoveredinflow of disease activators due to reduction of renal blood

that infusion of Ang II led to a marked suppressionflow. Therefore, activation of the renin-angiotensin system mayhave protective effects in certain pathophysiological situations. of glomerular monocyte infiltration, proliferation and

matrix expansion. This suppression was specific for AngII and was transduced by the AT1 receptor. Ang II has

Systemic hypertension is a major risk factor that deter- multiple functional and structural actions, all of whichmines the rate of progression of renal disease. Numerous tend to increase blood pressure and worsen renal func-

tion. In this context, our findings are surprising and novel1 See Editorial by de Zeeuw, p. 1176. that Ang II can ameliorate some pathophysiologic changes

in a model of acute glomerulonephritis.Key words: transforming growth factor beta, p27Kip1, monocytes/macro-phages, matrix expansion, monocyte chemotactic protein-1, cell prolif-eration, renin-angiotensin system.

METHODSReceived for publication December 18, 2000 Angiotensin II infusionand in revised form October 1, 2001Accepted for publication October 2, 2001 Studies were performed in male Sprague-Dawley rats

(Charles River, Kisslegg, Germany). The American Phy- 2002 by the International Society of Nephrology

1020

Wenzel et al: Ang II and glomerulonephritis 1021

siological Society’s guidelines of experimental animal the protein DC-assay (Biorad, Munich, Germany). Toequal amounts of protein (100 �g) Laemmli buffer 2research were followed and approval was obtained from

the local goverment. The animals had free access to (50% beta-mercaptoethanol, 50% glycerol) and 1/5staining solution (42.5% glycerol, 0.5% bromphenoltap water and standard rat chow. Rats weighing 200 g

received continuous Ang II or norepinephrine infusion blue) were added. Samples were electrophoresed on12% SDS/polyacrylamide gels. Proteins were electro-via subcutaneous osmotic minipumps (Alzet model 2002;

Alza Corp. Palo Alto, CA, USA) containing angiotensin blotted onto nitrocellulose (Hybond-ECL; Amersham,Braunschweig, Germany). The membrane was blockedII (200 ng/min) or norepinephrine (600 ng/min; Sigma,

Deisenhofen, Germany). For the experiments with im- with 5% nonfat dry milk in washing buffer [1 � phos-phate-buffered saline (PBS) 0.1% Tween 20]. The fol-mediate delivery of Ang II (implantation after injection

of the antiserum or 12 hours before injection of the lowing antibodies were used in a 1:1000 dilution: for IgG,goat antirabbit antibody (Dakopatts, Hamburg, Ger-antiserum) the minipumps were preincubated in vitro in

0.9% saline by 37�C for 12 hours according to manufac- many); for transforming growth factor-� (TGF-�), mouseantiporcine (R&D Systems, Minneapolis, MN, USA);turer’s recommondations. Rats were evaluated 6 to 10

days after implantation of minipumps. Only those rats for p27Kip1, mouse monoclonal antihuman (TransductionLaboratories Lexington, MA, USA); and for anti-prolif-with systolic blood pressure �160 mm Hg were included

for further study. erating cell nuclear antigen (PCNA), M879 (Dakopatts,Hamburg, Germany). The second antibody was a rabbit

Induction of glomerulonephritis horseradish peroxidase-conjugated antimouse or anti-goat IgG in a 1:1000 dilution (Transduction Labora-Immune-mediated mesangial cell injury was induced

five days after implantation of the minipumps by the i.v. tories). Peroxidase labeling was detected with lumines-cence immunodetection (ECL; Amersham) according toinjection of 0.5 mL/100 g body weight of an anti-rat-

thymocyte antiserum. ATS was induced in rabbits by the manufacturer’s recomendations. To control for smallvariations in protein, loading and transfer membranesrepeated immunization with thymocytes from Lewis rats

as described [6, 9, 10]. were washed and reincubated with a mouse mono-clonal anti-beta-actin antibody (Sigma).The following groups of animals were studied: (1) nor-

motensive control animals (controls); (2) normotensiveIsolation of RNA for Northern blot hybridizationcontrol animals � nephritis (ATS); (3) angiotensin II

infusion (Ang II); (4) Ang II infusion � nephritis (Ang RNA from glomeruli was isolated by the guanidiumisothiocyanate method as described by us [6, 10]. RNAII�ATS); (5) Ang II infusion � irbesartan (20 mg/kg

twice daily by gavage; Bristol Myers Squibb, Munchen, was electrophoresed through a 1.2% agarose gel con-taining 2.2 mol/L formaldehyde. Equal loading of theGermany) � nephritis; (6) Ang II infusion � PD 123319

(1.1 �g/min osmotic minipump; Sigma) � nephritis; (7) lanes was evaluated with ethidium bromide staining ofthe 18 and 28 S RNA. The RNA was transferred toAng II infusion, explantation of the pump (Ang II ex) �

nephritis; (8) Ang II infusion, implantation of the pump nylon membranes by vacuum blotting and UV cross-linked. Membranes were hybridized with a JE/MCP-112 hours before injection of the antiserum; (9) Ang II

infusion, implantation of the pump after injection of probe (a 577 bp EcoR1 fragment) after (32P) dCTP-label-ing by random oligonucleotide priming of the cDNAthe antiserum; (10) norepinephrine infusion; and (11)

norepinephrine infusion � nephritis. inserts as described earlier [8, 9]. Filters were hybridizedwith 106 cpm/probe per milliliter of hybridization buffer.Kidneys were removed 24 hours after induction of the

nephritis for examination of monocyte infiltration and The membranes were washed and subsequently autora-diography was performed at �80�C with intensifyingexpression of monocyte chemotactic protein-1 (MCP-1)

and after five days for glomerular proliferation. screens. Nylon membranes were stripped and rehybrid-ized with a 0.58 kb EcoRI1 cDNA probe of 18 S RNA

Systolic blood pressure to account for small loading and transfer variability.Systolic blood pressure was measured by tail cuff ple-

Renal morphologythysmography in awake rats as previously described [9].Kidney slices were fixed in 4% buffered formalin or

Western blotting Carnoy solution, paraffin embedded, sectioned to 3- to4-�m thick sections and stained with periodic acid SchiffWestern blotting was performed as recently described

by us [10–12]. Glomeruli were isolated by differential (PAS). Tissues were also stained with antibodies againstPCNA (M879; Dakopatts), rabbit IgG (Dakopatts), ratsieving and resuspended in Laemmli buffer 1 [33% (vol/

vol) 0.5 mol/L Tris/Cl, pH 6.8, 66% (vol/vol), and 10% C3c (Nordic, Tilborg, Netherlands) and ED-1 (Chemi-kon International, Temecula, CA, USA). The tissue sec-sodium dodecyl sulfide (SDS)]. Samples were boiled and

centrifuged. Protein concentration was determined with tions were developed with the APAAP complex as de-

Wenzel et al: Ang II and glomerulonephritis1022

Ang II does not prevent glomerularIgG/C3c deposition

Following the injection of anti-thymocyte-serum, se-lective binding of rabbit IgG was detected in the mesan-gium by immunohistology. As judged by quantitativescoring this deposition was not significantly different be-tween normotensive rats and rats receiving Ang II infu-sion (Fig. 2A). The immunohistochemical data were con-firmed by Western blotting. Figure 2B shows a Westernblot of glomerular proteins using an anti-rabbit IgG anti-body. No bands were detected in controls and Ang IIrats whereas bands of similar intensity were found inATS and Ang II � ATS animals. Similarly, glomerulardeposition of complement C3c was indistinguishableFig. 1. Systolic blood pressure measured in conscious rats after induc-

tion of the nephritis is shown. Abbreviations are: Control, normotensive among nephritic rats with and without Ang II (Fig. 2C),controls; Ang II, angiotensin II-infused animals; ATS, nephritis. *P � indicating no differences in delivery and binding of ATS0.001 versus normotensive controls. N 6 in each group.

as well as subsequent complement activation in normo-tensive and hypertensive rats.

Ang II reduces the glomerular accumulationscribed earlier [6, 9, 10, 12]. Histological studies wereof monocytes/macrophagesperformed with the investigators in a blinded fashion.

A hallmark of glomerulonephritis is glomerular infil-To evaluate the appearance of proliferating glomerulartration by monocytes/macrophages [13]. To determinecells and monocyte infiltration, at least 25 glomeruli wereglomerular infiltration by monocytes, tissues were stainedstudied from each kidney. The typical glomerular lesionswith a specific antibody against monocytes/macrophagesof the ATS nephritis with edema and expansion of matrix(ED-1). There was considerable influx of ED-1 positiveon day 5 were quantitated by scoring (grade 0 to 2) incells at 24 hours in nephritic animals. However, Ang IIPAS stained sections: grade 0 no lesion; grade 1 significantly inhibited this glomerular monocyte/macro-segmental lesions that do not cover the complete glomer-phage accumulation. (Fig. 3A). We have recently demon-ular cross-sectional area; grade 2 involvement of thestrated that MCP-1 causes monocyte/macrophage infil-complete glomerular area. Thirty glomeruli were evalu-tration in the ATS nephritis [6]. It is noteworthy thatated from each kidney. Glomerular staining for IgG andAng II inhibited MCP-1 induction as shown by NorthernC3c was assessed by a semiquantitative score from 0 toblotting in Figure 3B. No more significant difference was3, where: grade 0 no staining; grade 1 weak andfound for glomerular mononocyte/macrophage infiltra-focal staining; grade 2 continuous staining; and gradetion on day 5 of the disease (controls 0.12 0.03, ATS3 heavy staining of the mesangial area.0.12 0.03, Ang II 0.12 0.61, Ang II � ATS 0.12 0.01 ED-1 positive cells/glomerular cross section). ThisStatistical analysisfinding is very similar to our previous study, in whichResults are expressed as means � SEM unless statedan antibody against MCP-1 decreased the number of

otherwise. Statistical significance was defined as P �glomerular monocytes/macrophages significantly on day

0.05. The Mann-Whitney test was applied to compare1 but not on day 5 of the nephritis [10].

two distinct treatment groups. For comparison of allgroups one-way analysis of variance was used as a screen- Ang II inhibits glomerular cell proliferationing test, if appropriate. Glomerular proliferation is another hallmark of ATS

nephritis. To further quantify proliferative changes, glo-merular proliferation was studied by immunostaining theRESULTStissue sections for PCNA, a nuclear protein that is in-

Blood pressure creased from late G1 to the M phase of the cell cycle.Systolic blood pressures are depicted in Figure 1. Glomerular proliferation peaks in the ATS nephritis at

Blood pressure was not significantly different between day 5. As shown in Figure 4A, there was strong inductioncontrols and normotensive nephritic rats. Angiotensin of proliferation in nephritic rats on day 5. This prolifera-infusion caused an increase in systolic blood pressure. tion was, however, significantly inhibited by Ang II infu-No significant difference was found to nephritic rats re- sion. The immunohistochemical data were confirmed by

Western blotting. There was induction of PCNA in ne-ceiving Ang II infusion.

Wenzel et al: Ang II and glomerulonephritis 1023

Fig. 2. IgG and C3c binding semiquantitative scoring of glomerularIgG binding in immunohistochemistry reveals that Ang II does notinfluence binding of the anti thymocyte serum to the mesangium (A).Abbreviations are: Control; normotensive controls (N 17); ATS,nephritis (N 17); Ang II, angiotensin II (N 14); Ang II � ATS(N 17). Western blotting of glomerular proteins confirms the immuno-histochemical data (B). This Western blot is representative of six blotsfrom six independent experimental series with similar results. Densitom-etry of these blots showed 1.35 0.23 (N 6) more arbitrary unitsfor rabbit IgG in Ang II � ATS rats in comparison to ATS, which wasnot statistically significant. (C) Semiquantitative scoring of glomerularC3c binding in immunohistochemistry. Ang II does not influence bind-ing of the anti-thymocyte serum to the mesangium. Abbreviations are:Control, normotensive controls (N 13); Ang II, angiotensin II-infusedanimals (N 14); ATS, nephritis (N 13), Ang II�ATS (N 14).

Fig. 3. Ang II inhibits monocyte/macrophage infiltration ED-1 positive cells per glomerular cross section (30 glomeruli/kidney) 24 hours afterinduction of the nephritis. Monocyte/macrophage infiltration in glomeruli of ATS nephritic rats was significantly higher as in controls. Ang IIsignificantly decreased the glomerular infiltration. Abbreviations are: Control, normotensive controls (N 8 animals); Ang II, angiotensin II-infused animals (N 10); ATS, nephritis (N 9); Ang II � ATS (N 10). *P � 0.001 versus normotensive controls. #P � 0.001 versus ATS.(B) Glomerular expression of MCP-1 mRNA 24 hours after induction of the nephritis. MCP-1 was induced in nephritic animals and this inductionwas inhibited by Ang II. This blot is representative of two independent experiments. Densitometry showed a 3.2-fold reduction of MCP-1 in AngII � ATS rats compared to ATS rats. Equal loading and transfer of each lane was confirmed by rehybridization against 18S rRNA.

Wenzel et al: Ang II and glomerulonephritis1024

Fig. 4. Ang II inhibits glomerular proliferation. (A) PCNA positive cells per glomerular cross section (30 glomeruli/kidney) 5 days after inductionof the nephritis. The number of PCNA positive glomerular cells of ATS nephritic rats was significantly higher as in controls. Ang II significantlydecreased this number. Abbreviations are: Control, normotensive controls (N 9); Ang II, angiotensin II-infused animals (N 10); ATS, nephritis(N 9); Ang II � ATS (N 10). *P � 0.001 versus normotensive controls, #P � 0.001 versus ATS. (B) Western blotting for PCNA confirmsthis pattern. This Western blot is representative of five blots from five independent experimental series. Densitometry showed a 7.2 2.7-fold (N 5) reduction of PCNA in Ang II � ATS rats compared to ATS rats.

onist irbesartan (20 mg/kg twice daily by gavage) orthe AT2 receptor antagonist PD 123319 (1.1 �g/min byosmotic minipump, the pumps were implanted s.c. at thesame time as the Ang II containing pumps). We alsowere interested in whether the antiproliferative effect ofAng II is reversible. To study this, the osmotic mini-pumps containing Ang II were explanted 12 hours beforeinduction of the nephritis. Figure 6A shows that irbesar-tan but not PD 123319 inhibited the increase of blood

Fig. 5. Norepinephrine infusion does not influence proliferation. West-pressure induced by Ang II. Systolic blood pressure 12ern blotting of glomerular proteins for PCNA reveals no difference

between nephritic rats and nephritic rats that received norepinephrine. hours after removal of the pumps was no longer differentfrom normotensive controls (Fig. 6A). The antiprolifera-tive effect of Ang II, as judged by Western blotting for

phritic rats that was significantly inhibited by Ang II as PCNA, was almost completely inhibited by irbesartanshown in Figure 4B. Densitometry showed a 7.2 2.7- but not by the AT2 receptor antagonist PD 123319 (Fig.fold (N 5) reduction of PCNA in Ang II�ATS rats 6B), suggesting that this effect is transduced by AT1compared to ATS rats. receptors. After removing the pumps, the antiprolifera-

tive effect of Ang II was still evident by Western blottingNorepinephrine infusion does not inhibit proliferationfor PCNA, although to a lesser extent than in animals

To examine whether the effects of Ang II are due to receiving Ang II continuously (Fig. 6B). In order to fur-hypertension or specific for angiotensin II norepineph- ther evaluate the time dependency of the antiprolifera-rine (600 ng/min) was infused. Norepinephrine infusion tive effect of Ang II, minipumps were implanted 12 hoursproduced hypertension (controls 103 5, norepineph-

before or immediately after injection of the antiserum.rine 176 9, norepinephrine � ATS 170 4 mm Hg)Blood pressure measurement showed that rats were al-but did not affect glomerular proliferation as demon-ready mildly hypertensive after an overnight delivery ofstrated by Western blotting for PCNA (Fig. 5), indicatingAng II (144 5 vs. 100 3 mm Hg in controls, P � 0.01).that the effect of Ang II is not due to hypertension butIn both settings Ang II showed a small antiproliferativeis specific for Ang II.effect by Western blotting of glomerular PCNA. How-

The antiproliferative effect of Ang II is transduced ever, as shown in Figure 6C, implantation of the mini-by the AT1 but not by the AT2 receptor pumps five days before injection of the antiserum showed

a stronger antiproliferative effect than short-term deliv-To test whether the AT1 receptor mediates the effectsof Ang II, rats were treated with the AT1 receptor antag- ery of Ang II.

Wenzel et al: Ang II and glomerulonephritis 1025

Fig. 6. The AT1 receptor blocker irbesartan,but not the AT2 receptor blocker PD 123319,blocks the antiproliferative effect of Ang II.Systolic blood pressure was measured in con-scious rats. (A) Twice daily application of irbe-sartan by gavage but not PD 123319 inhibitedthe increase of blood pressure in Ang II in-fused rats. Explantations of the osmotic mini-pumps decreased blood pressure to normalwithin 12 hours. *P � 0.001 versus controls.Ang II (ex) is osmotic minipump explanted.(N 4 to 6 rats in each group). (B) Adminis-tration of irbesartan blocked the antiprolifera-tive effect of Ang II as judged by Westernblotting for PCNA 5 days after induction ofthe nephritis. The antiproliferative effect ofAng II persisted even if the pumps were ex-planted 12 hours before induction of the ne-phritis. However, the effect was weaker com-pared to rats receiving continuos infusion ofAng II. (C) Ang II also had a weaker antipro-liferative effect if the minipumps were inserted12 hours before injection of the antiserum oreven immediately after administration of theantiserum. The glomerular lysate used in thisWestern blot was pooled from 6 kidneys incontrols and ATS respectively and 8 kidneysin each Ang II � ATS group. The Westernblot was repeated twice with similar result.

Extracellular matrix day 5. However, Figure 7D shows the reduction of theselesions in a nephritic rat receiving Ang II infusion. Scor-The third hallmark of nephritis besides infiltration bying in the PAS stained sections showed a small but sig-monocytes/macrophages and glomerular proliferation isnificant reduction in Ang II infused nephritic animalsglomerular accumulation of extracellular matrix. Over-(Fig. 7E).production of matrix proteins is another consequence of

mesangial cell activation in the ATS [5]. Figure 7 A andTGF-� and p27KIP1

B shows the normal-appearing glomeruli of controls andTransforming growth factor-� is a pleiotropic cytokineAng II-infused rats. In contrast, a glomerulus in Figure

7C shows the typical lesions with matrix expansion on involved in a wide range of pathophysiologic processes.

Wenzel et al: Ang II and glomerulonephritis1026

Fig. 7. Extracellular matrix. Glomeruli of control (A) and Ang II (B) infused rats had a normal appearance by lightmicroscopy (PAS �400). Fivedays after induction of the nephritis, nephritic rats revealed matrix expansion with edema and diffuse endocapillary proliferation leading to acompromise of capillary loop lumens (C). The expansion was decreased in nephritic rats receiving Ang II infusion (D). Scores are shown in panelE. #P � 0.05 vs. ATS (Control, N 10; ATS, N 9; Ang II, N 10; Ang II � ATS, N 10).

Ang II infusion induced TGF-� expression as judged DISCUSSIONby Western blotting of glomerular proteins (Fig. 8A). This study shows that Ang II ameliorates rather thanDensitometry showed a 2.53 0.44-fold induction in deteriorates morphologic changes in the early phase ofAng II � ATS rats compared to controls rats. To explore mesangioproliferative nephritis. This finding is in con-the inhibition of proliferation further, we turned our trast to earlier studies that described an antiproliferativeattention to the cell cycle machinery in an attempt to and antifibrogenic effect of blockade of the renin-angio-identify the locus of the Ang II effect. Figure 8B illus-

tensin system [7, 8]. One of the most common pathologi-trates that p27KIP1 protein was clearly up-regulated in

cal features of glomerular disease is infiltration of mono-Ang II-infused rats compared to controls.cytes/macrophages. Ang II infusion reduced glomerularmonocyte infiltration. We have recently shown usingLight microscopyneutralizing antibodies that MCP-1 causes the glomeru-By light microscopy of PAS-stained tissue sections thelar monocyte infiltration in the ATS nephritis [6]. More-majority of glomeruli appeared relatively well preservedover, in the present study Ang II inhibited expressionin Ang II-infused rats. However, mild focal tubulointer-of the chemokine MCP-1. While this does not prove thestitial injury with tubular atrophy, dilation as well as inter-hypothesis, it strongly suggests that the reduced mono-stitial fibrosis with cell infiltration was observed in Ang II-cyte infiltration is caused by inhibition of induction ofinfused rats. Small renal arterial vessels of Ang II-infusedMCP-1 in Ang II infused animals. Depletion of mono-rats demonstrated focal injury with fibrinoid necrosis andcytes leads to a reduction of mesangial matrix expansionmedial hyperplasia, whereas glomerular capillaries dem-

onstrated only rare segmental hyalinosis [14]. in the ATS nephritis [15]. Moreover, inhibition of MCP-1

Wenzel et al: Ang II and glomerulonephritis 1027

Fig. 8. Glomerular expression of TGF-� andp27KIP1. TGF-� protein expression is stronglyenhanced in Ang II and Ang II � ATS infusedrats (A). This blot is representative of 4 experi-ments with qualitatively similar results. AngII induced expression of the cell cycle inhibitorp27KIP1 in the Ang II and Ang II � ATS group(B). This blot is representative of two experi-ments with qualitatively similar results.

by neutralizing antibodies reduces glomerular collagen [17]. Chevalier and coworkers recently found an antipro-liferative effect of Ang II acting via AT2 receptors inIV deposition in the ATS nephritis as shown recently

by us [10]. Therefore, the reduced matrix expansion the obstructed neonatal rat kidney [18]. However, block-ing the AT1 receptors with irbesartan clearly abolishedfound in the Ang II � ATS group might be caused

by the decreased monocyte infiltration and decreased the antiproliferative effect of Ang II, whereas an AT2receptor blocker did not show a significant effect in ourexpression of MCP-1. We recently demonstrated by dou-

ble staining that monocytes do not account for the prolif- study. These data indicate that the antiproliferative ef-fect is likely mediated by the AT1 receptor.erating cells on day 5 of the nephritis [6]. Therefore, the

reduced number of PCNA-positive glomerular cells is Increased glomerular expression of TGF-� was foundin Ang II infused rats. TGF-� is a pleiotropic cytokinenot due to a reduced number of glomerular monocytes/

macrophages. involved in a wide range of pathophysiologic processes[4]. However, there is evidence supporting the anti-The predominant biological effects of Ang II are be-

lieved to be mediated in the adult kidney through stimu- inflammatory role of TGF-� [19, 20]. It also is an inhibitorof mitogenesis in glomerular cells in vivo. Some in vitrolation of the AT1 receptor. However, we have described

that induction of the chemokine RANTES by Ang II in effects of Ang II are mediated by TGF-� [21]. Althoughthat does not prove that induction of TGF-� by Ang IIthe glomerulus is mediated by the AT2 receptor [16].

Low-dose short-term infusion of Ang II for four days mediates the found effects of Ang II, the correlation ofinduction of TGF-� and the reduced monocyte infiltra-even induced a small increase of glomerular monocyte

infiltration (from 0.25 to 0.46 monocytes/glomerular tion and decreased proliferation is intriguing.Toward resolution of inflammation, the defending ma-cross section). In contrast, in the present study Ang II

ameliorated the massive infiltration typically seen on chinery counteracts the attack by offenders at multiplelevels. Several “self-cooling” devices are present in resi-day 1 of the ATS nephritis (from 2.7 to 1.2 monocytes/

glomerular cross section). Antiproliferative effects have dent cells [22]. These may include cyclin kinase inhibitorsthat inhibit cyclin-dependent kinases (CDK). The CDKbeen described for the AT2 receptor in endothelial cells

Wenzel et al: Ang II and glomerulonephritis1028

inhibitor p27KIP1 has been shown to play an important present study by less inflow of disease activators or lessrole in cell cycle regulation. Expression of the CDK immunological inflow. First, we have carefully controlledinhibitor p27KIP1 has been shown by us and others to be in the present study induction of the nephritis by control-up-regulated by Ang II in vitro [23, 24]. The role of ling glomerular binding of IgG and activation of comple-cell cycle inhibitor in the glomerulus has been recently ment. Second, we recently were able to show completeinvestigated using knockout mice. When anti-GBM ne- induction and development of the ATS nephritis in thephritis was induced the p27KIP1 knockout mice developed stenosed kidney of renovascular hypertensive rats, indi-more severe disease compared with wild-type mice [25]. cating that a reduced blood flow does not necessarilyThese results suggested that p27KIP1 functions as a “safe- ameliorate the acute phase of the ATS nephritis [9].guard molecule” of the glomerulus against inflammation. However, since starting the administration of Ang IITherefore, it is appealing that glomerular induction of after disease induction as well as starting 12 hours beforep27KIP1 by Ang II found in the present study might influ- injection of the antiserum were less effective times thanence glomerular proliferation as well as matrix expansion administration for several days, we cannot completelyin the nephritic animals. rule out that Ang II somehow reduced the immunologi-

In the dynamic process of fibrogenesis, growth pro- cal inflow to the glomerulus.cesses such as proliferation of intrinsic glomerular cells Our data on the beneficial effects of Ang II at firstmay precede the development of matrix expansion and sight are at odds with the data in the literature [7, 8, 28].sclerosis. Previous studies have established a relationship However, it is unlikely that interference with a localbetween mesangial proliferation and matrix production endogenous renin-angiotensin system using an ACE-[26]. Ang II stimulates collagen synthesis in renal cells inhibitor or AT1-receptor blocker can be compared within vitro. The reason for the decreased matrix expansion the infusion of exogenous Ang II. In addition, activationin Ang II infused nephritic animals remains unclear. of the renin-angiotensin system might have beneficialHowever, maneuvers that reduce mesangial prolifera- renal effects in certain pathophysiological situations.tion, such as low protein diet or blocking platelet-derived One such situation is renal artery stenosis, where block-growth factor, also have been associated with the reduc- ade of the renin-angiotensin system might have detri-tion in matrix production. Therefore, the decreased ma- mental effects for the stenosed kidney [29]. A salutarytrix production might be the result of reduced prolifera- effect of Ang II also was shown recently in partial urinarytion. tract obstruction [30]. The remnant kidney is another

Studies were performed to determine if another form model of renal injury with an activated renin-angiotensinof pharmacologically induced hypertension, induced by system, and it has been shown recently that the remnantinfusion of norepinephrine, exhibited similar antiprolif- kidney is surprisingly protected against ischemia/reper-erative effects. Norepinephrine did not change glomeru- fusion injury [31]. In addition, tissue adaptation to re-lar proliferation. In addition, no difference in glomerular peated stress has long been known [32]. Endotoxin-proliferation was found after induction of the ATS ne- induced cross-tolerance to subsequent renal ischemia/phritis in rats with renovascular hypertension [9]. These reperfusion injury was recently described [33]. We pro-findings are compatible with the concept that hyperten- pose from our data that Ang II infusion induces a cross-sion per se does not ameliorate the nephritis, but that tolerance to a subsequent inflammatory injury. Althoughthe found effects are specific for Ang II. Removal of the it seems less likely from our data, we cannot completelyosmotic minipumps 12 hours before induction of the rule out that reduction of renal blood flow by Ang IInephritis lowered blood pressure to normotensive levels contributed to the protection of the kidney due to lesswithin hours. However, there was still an antiprolifera- immunological inflow.tive effect five days after induction of the nephritis. Clearly, the surprising effect of Ang II on the ATSShort-term delivery of Ang II starting 12 hours before nephritis has begotten a series of questions that will beinduction of the nephritis or even starting with Ang II instrumental in our understanding of the pathogenesisinfusion after injection of the antiserum showed a weaker of inflammatory glomerular disease and the therapeuticantiproliferative effect than the administration of Ang role of converting enzyme inhibitors or Ang II antago-II for five days. This indicates that the presence of Ang II nists in acute glomerulonephritis. However, our data dois not necessary for inhibition of glomerular proliferation not question the well-known and established beneficialand that Ang II needs some time to develop its maximal effect of ACE inhibitors in chronic renal disease [34].effect. These observations suggest that Ang II inducessome factors in the glomerulus that, once induced, exert ACKNOWLEDGMENTSthe positive effects. Ang II reduces renal blood flow.

This study was supported by the Deutsche ForschungsgemeinschaftThere are reports about unilateral renal disease with an(We 1688/4-3, Wo 460/2-6, 8-2, 12-1). Parts of this study were presented

undiseased kidney behind a renal artery stenosis [27]. at the Annual Meeting of the American Society of Nephrology, MiamiBeach, 1999 and published in abstract form (J Am Soc Nephrol 10:564,One could argue that Ang II protects the kidney in the

Wenzel et al: Ang II and glomerulonephritis 1029

1999). We acknowledge the excellent technical assistance of Ms. Ivonne lial cells. Role of the angiotensin type 2 receptor. J Clin Invest100:1047–1058, 1997Jacob.

17. Stoll M, Steckelings UM, Paul M, et al: The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endo-Reprint requests to Ulrich O. Wenzel, M.D., Division of Nephrologythelial cells. J Clin Invest 95:651–657, 1995(Pav. 61), Department of Medicine, University of Hamburg, Marti-

18. Chevalier RL, Thornhill BA, Wolstenholme JT: Renal cellularnistrasse 52, 20246 Hamburg, Germany.response to ureteral obstruction: Role of maturation and angioten-E-mail: wenzel@uke.uni-hamburg.desin II. Am J Physiol 277:F41–F47, 1999

19. Song XY, Gu ML, Jin WW, et al: Plasmid DNA encoding trans-forming growth factor beta 1 suppresses chronic disease in a strep-REFERENCEStococcal cell wall-induced arthritis model. J Clin Invest 101:2615–

1. Anderson S, Rennke HG, Brenner B: Therapeutic advantage of 2621, 1998converting enzyme inhibitors in arresting progressive renal disease 20. Kitamura M: Identification of an inhibitor targeting macrophageassociated with systemic hypertension in the rat. J Clin Invest production of monocyte chemoattractant protein-1 as TGF-beta1.77:1993–2000, 1986 J Immunol 159:1404–1411, 1997

2. The GISEN Group: Randomised placebo-controlled trial of effect 21. Wolf G, Mueller E, Stahl RAK, Ziyadeh FN: Angiotensin II-of ramipril on decline in glomerular filtration rate and risk of induced hypertrophy of cultured murine proximal tubular cells is

mediated by endogenous transforming growth factor-beta. J Clinterminal renal failure in proteinuric, nondiabetic nephropathy.Invest 92:1366–1372, 1993Lancet 349:1857–1863, 1997

22. Kitamura M, Fine LG: The concept of glomerular self-defense.3. Wolf G: Angiotensin II: A pivotal factor in the progression ofKidney Int 55:1639–1671, 1999renal diseases. Nephrol Dial Transplant 14(Suppl 1):42–44, 1999

23. Braun Dullaeus RC, Mann MJ, Ziegler A, et al: A novel role4. Border WA, Okuda S, Languino LR, et al: Suppression of experi-for the cyclin-dependent kinase inhibitor p27Kip1 in angiotensin II-mental glomerulonephritis by antiserum against transforming growthstimulated vascular smooth muscle cell hypertrophy. J Clin Investfactor beta 1. Nature 346:371–374, 1990104:815–823, 19995. Floege J, Johnson RJ, Gordon K, et al: Increased synthesis of

24. Wolf G, Stahl RAK: Angiotensin II-stimulated hypertrophy ofextracellular matrix in mesangial proliferative nephritis. KidneyLLC-PK1 cells depends on the induction of the cyclin-dependentInt 40:477–488, 1991kinase inhibitor p27Kip1. Kidney Int 50:2112–2119, 19966. Wenzel U, Schneider A, Valente AJ, et al: Monocyte chemoat- 25. Ophascharoensuk V, Fero ML, Hughes J, et al: The cyclin-kinasetractant protein 1 mediates monocyte/macrophage influx in anti- inhibitor p27 Kip1 safeguards against inflammatory injury. Nat

thymocyte antibody-induced glomerulonephritis. Kidney Int 51: Med 4:575–580, 1998770–776, 1997 26. Johnson RJ, Raines EW, Floegge J, et al: Inhibition of mesangial

7. Peters H, Border WA, Noble NA: Angiotensin II blockade and cell proliferation and matrix expansion in glomerulonephritis inlow-protein diet produce additive therapeutic effects in experimen- the rat by antibody to platelet-derived growth factor. J Exp Medtal glomerulonephritis. Kidney Int 57:1493–1501, 2000 175:1413–1416, 1992

8. Kiyma S, Nanishi F, Tomooka S, et al: Inhibitory effects of antihy- 27. Palmer JM, Eversole SL, Stamey TA: Unilaterale glomerulone-pertensive drugs on mesangial cell proliferation after anti-thymo- phritis. Virtual absence of nephritis in a kidney with partial occlu-cyte serum-induced mesangiolysis in spontaneously hypertensive sion of the main renal artery. Am J Med 40:816–822, 1966rats. Life Sci 54:1891–1990, 1994 28. Zoja C, Abbate M, Corna D, et al: Pharmacologic control of

9. Wenzel UO, Thaiss F, Panzer U, et al: Effect of renovascular angiotensin II ameliorates renal disease while reducing renal TGF-beta in experimental mesangioproliferative glomerulonephritis.hypertension on experimental glomerulonephritis in rats. J LabAm J Kidney Dis 31:453–463, 1998Clin Med 134:192–203, 1999

29. Jackson B, Franze C, Sumithran E, Johnston I: Pharmacologic10. Schneider A, Panzer U, Zahner G, et al: Monocyte chemoattrac-nephrectomy with chronic angiotensin converting enzyme inhibitortant protein 1 mediates collagen deposition in experimental glo-treatment in renovascular hypertension in the rat. J Lab Clin Medmerulonephritis by transforming growth factor beta. Kidney Int115:21–27, 199056:135–144, 1999

30. Fujinaka H, Miyazaki Y, Matsusaka T, et al: Salutary role for11. Wolf G, Wenzel U, Ziyadeh FN, Stahl RAK: Angiotensin con-angiotensin in partial urinary tract obstruction. Kidney Int 58:2018–verting-enzyme inhibitor treatment reduces glomerular p16INK4 and2027, 2000p27Kip1 expression in diabetic BBdp rats. Diabetologia 42:1425–

31. Vercauteren SR, Ysebaert DK, De Greef KE, et al: Chronic1432, 1999reduction in renal mass in the rat attenuates ischemia/reperfusion12. Wenzel UO, Wolf G, Thaiss, F, et al: Renovascular hypertension injury and does not impair tubular regeneration. J Am Soc Nephroldoes not influence repair of the glomerular lesions induced by anti- 10:2551–2561, 1999

thymocyte glomerulonephritis. Kidney Int 58:1135–1147, 2000 32. Gerner EW, Schneider MJ: Induced thermal resistance in HeLa13. Cattel V: Macrophages in acute glomerular inflammation. Kidney cells. Nature 256:500–502, 1975

Int 45:945–952, 1994 33. Heemann U, Szabo M, Hamar P, et al: Lipopolysaccharide pre-14. Johnson RJ, Alpers CE, Yoshimura A, et al: Renal injury from treatment protects from renal ischemia/reperfusion injury. Possible

angiotensin II-mediated hypertension. Hypertens 19:464–474, 1992 connection to an interleukin-6-dependent pathway. Am J Pathol15. Westerhuis R, Van Straaten SC, Van Dixhoorn MGA, et al: 156:287–293, 2000

Distinctive roles of neutrophils and monocytes in anti-thy-1 nephri- 34. Wenzel UO: ACE-inhibitor and progression of renal disease:tis. Am J Pathol 156:303–310, 2000 evidence from clinical studies, in Contributions To Nephrology.

16. Wolf G, Thaiss F, Ziyadeh FN, et al: Angiotensin II stimulates The Renin Angiotensin System and Progression of Renal Disease,expression of the chemokine RANTES in rat glomerular endothe- Basel, Karger, 2001