Arterial hypertension is one of the leading causes of end-stage renal failure; its incidence continues to rise despite progress in antihypertensive treatment1. In addition, arterial hypertension is an important factor in the progression of other forms of chronic renal disease2. During the last decade, we3,4,5 and others6,7,8,9 have emphasized the role of mononuclear leukocyte infiltration in experimental hypertensive renal injury. Macrophage (M
) infiltration was observed in angiotensin II (Ang II)-dependent models of hypertensive nephrosclerosis3,6,7,9. The identification of molecular mechanisms of monocyte/M infiltration in hypertensive nephrosclerosis might lead to the development of novel therapies. Recently, we4 and others8 reported evidence for a role of the interstitial cell adhesion molecule-1 in mononuclear leukocyte infiltration in experimental renovascular hypertension.
Chemokines that attract Ms10,11 could also play a role in kidney damage associated with high blood pressure9,10. Wolf et al reported that the chemokine RANTES is associated with M infiltration in Ang II-induced hypertension12. In the present studies, we investigated the expression of monocyte chemoattractant protein-1 (MCP-1)13,14 in the kidney in rat models of hypertension. In vascular smooth muscle cells, MCP-1 was shown to be induced by mechanical strain15 and by Ang II16. The chemokine MCP-1 is expressed in experimental and human forms of immune-mediated glomerulonephritis17,18,19,20,21,22. Several authors provided direct evidence that blockade of MCP-1 reduces monocyte/M infiltration in antibody-induced experimental nephritis18,19,20,21. Our data show that MCP-1 expression is increased in some forms of experimental hypertensive nephrosclerosis, mediated via the Ang II type 1 (AT1) receptor, and is associated with monocyte/M infiltration.
METHODS
Rat models of hypertension
Rats were housed in a room maintained at 22 
2°C and were exposed to a 12-hour dark/light cycle. The animals were allowed unlimited access to chow (#1320; Altromin, Lage, Germany) and tap water. All procedures performed on animals were done in accordance with guidelines of the American Physiological Society and were approved by the local government authorities (Regierung von Mittelfranken, AZ #621-2531.3-10/94).
Two-kidney, one-clip renovascular hypertension (2K1C) was induced in male Sprague-Dawley rats (Charles River, Sulzfeld, Germany) weighing 150 to 170 g by placing a silver clip of 0.2 mm internal diameter around the left renal artery through a flank incision under ether anesthesia, as previously described3,23. Control animals underwent the same procedure without placement of the clip. The animals were then followed by weekly measurements of weight and systolic blood pressure by tail-cuff plethysmography under light ether anesthesia24. Animals were only included in the 2K1C group if systolic blood pressure was above 150 mm Hg, which was achieved in 80 to 90% of all operated animals. Animals that failed to thrive or lost weight were excluded25. Rats were sacrificed, and the kidneys were harvested at 4, 14, and 28 days after clipping (5 2K1C and 5 control animals at each time point).
To test for the role of the AT1, some animals were treated with the AT1 blocker valsartan. The animals received osmotic minipumps (Alzet model 2002; Alza Scientific Products, Palo Alto, CA, USA) intraperitoneally, which delivered 0.5 
L/hour for 14 days. Five 2K1C rats received pumps with 55.8 mg/mL valsartan dissolved in 0.5 mol/L KOH, pH 7.2; eight 2K1C rats and seven sham-operated rats received minipumps filled with solvent (0.5 mol/L KOH, pH 7.2). Fourteen days after implantation, all animals were instrumented with femoral artery catheters for intra-arterial blood pressure measurements under ether anesthesia as described previously26. Measurements were performed on the same day four hours after termination of anesthesia via transducers (Grass Instruments, Quincy, MA, USA) connected to a polygraph (Hellige, Freiburg, Germany), and the animals were sacrificed.
In addition, we studied three models of genetic hypertension. Seven 12-week-old male rats heterozygous for the mouse ren-2 transgene (TGR) with Ang II-dependent hypertension27, and six age-matched Sprague-Dawley-Hannover (SDH) controls (Möllegaard, Eijby, Denmark) were sacrificed after measurement of systolic blood pressure. Four spontaneously hypertensive rats (SHRs)28 and four Wistar-Kyoto control animals (WKR; Charles River, Sulzfeld, Germany) were sacrificed at 12 weeks of age after systolic blood pressure measurement. In addition, we also examined four stroke-prone SHR (SHR-SP) and four respective WKY animals from the Heidelberg colony (Max-Delbrück-Center, Berlin-Buch, Germany). These animals were also studied at 12 weeks of age; systolic blood pressure was determined before the animals were sacrificed.
After weighing kidneys, the organs were decapsulated. Part of each kidney was immediately snap frozen on liquid nitrogen for protein and RNA extraction, while a second part was put in methyl-Carnoy solution (60% methanol, 30% chloroform, and 10% glacial acetic acid) for fixation.
Northern blot detection of mRNA
Total RNA was extracted using a modification of the single-step method of Chomczynski with the TRI reagent (Molecular Research Center Inc., Cincinnati, OH, USA)29. Tissue samples were stored at -80°C after snap freezing. Ice-cold (4°C) TRI reagent was added immediately before processing the samples, and the samples were kept at 4°C during the entire process of RNA extraction. For Northern blot analysis, 20 mg of total RNA were electrophoresed in a 1% agarose/1.8% formaldehyde gel and transferred to positively charged Nylon membranes (Hybond N; Amersham, Braunschweig, Germany) with 20 
standard sodium citrate (SSC) overnight. Ethidium bromide staining of the gel and membrane confirmed equal loading and sufficient blotting of the RNA. The RNA was fixed by baking for two hours at 80°C.
The blots were probed with a full-length rat MCP-1 cDNA14 and a 500 bp GAPDH probe30. DNA fragments were radiolabeled with 
[32]P-dCTP by random prime labeling31 with a commercially available kit (Amersham). Prehybridization was performed at 42°C for two hours with a buffer containing 5 SSC, 0.1% sodium dodecyl sulfate (SDS), 5 Denhardt's solution, 50 mmol/L Na2HPO4 (pH 6.5), 50% deionized formamide, and 250 mg/mL denatured salmon sperm DNA. Hybridization was carried out under the same conditions for 24 hours. The membranes were washed twice with 2 SSC/0.1% SDS at 40°C for 15 minutes and twice with 0.2 SSC/0.1% SDS for 30 minutes at the same temperature. Blots were then exposed to Kodak x-ray films with intensifying screens.
Immunohistochemical detection of MCP-1
After overnight fixation in methyl-Carnoy solution, tissues were dehydrated by bathing in increasing concentrations of methanol, followed by 100% iso-propanol. From the paraffin-embedded tissues, 2 m sections were cut with a Leitz SM 2000 R microtome (Leica Instruments, Nussloch, Germany). After deparaffinization, endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 20 minutes at room temperature.
Sections were then layered with the primary MCP-1 antibody and were incubated at 4°C overnight. After addition of the secondary antibody (dilution 1:500; biotin-conjugated, goat anti-rabbit immunoglobulin G; Dianova, Hamburg, Germany), the sections were incubated with avidin horseradish peroxidase complex and exposed to 0.1% diaminobenzidine tetrahydrochloride and 0.02% H2O2 as a source of peroxidase substrate. The Vectastain DAB kit (Vector Laboratory, Burlingame, CA, USA) was used as a chromogen. Each slide was counterstained with hematoxylin. As negative controls, we used preimmune rabbit IgG and staining with the secondary antibody (goat anti-rabbit IgG) only.
Double stainings for MCP-1 and specific cell markers
Macrophages were counted after staining for the marker ED-1 as described previously3 and, in some experiments, after staining for the M activity marker ED-332. For double stainings of MCP-1 and ED-1, sections were stained for ED-1 first, followed by blocking the peroxidase activity and subsequent staining for MCP-1. To detect ED-1 staining, the Vector VIP substrate kit for peroxidase (Vectastain) was used, resulting in a purple staining.
To localize MCP-1 in specific glomerular cells, the same methods were used for double stainings with MCP-1 antiserum and antibodies against markers for endothelial cells (RECA-1)33 and podocyte nuclei (WT-1)34. In addition, double immunofluorescence was performed on cryostat sections of snap-frozen tissue as described previously3. Immunofluorescence for MCP-1 was combined with immunofluorescence for either the mesangial cell marker Thy-1 or synaptopodin, which stains podocyte foot processes and endothelial cells34. Primary antibodies were applied simultaneously over night at 4°C. After washing, sections were incubated with secondary antibodies, CY2-labeled goat anti-mouse IgG, and CY3-labeled goat anti-rabbit IgG (both from Dianova) at the same time for two hours.
Western blot detection of MCP-1 protein
Rat kidney protein was extracted with TRI reagent by the method of Chomczynski29. The protein content was determined with a protein assay kit (Pierce, Rockford, IL, USA), was made to 1 g/L with SDS sample buffer (50 mmol/L Tris-HCl, pH 6.8, 20% SDS, 2% mercaptoethanol, 0.01% Coomassie Brilliant Blue-G, 12% glycerol), and was boiled for five minutes. Equal amounts of protein (35 g/lane) were loaded onto a denaturing 10 to 18% SDS-polyacrylamide gradient gel in the buffer system described by Schagger and von Jagow35. Sigma Color Ultra Low Range molecular weight markers (1.06 to 26.6 kD; Sigma Chemicals, Deisenhofen, Germany) were used as standards. Proteins were electroblotted onto nitrocellulose (Hybond ECL; Amersham), and membranes were stained with Amido Black staining solution (Sigma Chemicals) to test for complete protein transfer. To prevent nonspecific antibody binding, membranes were blocked with 3% horse serum/bovine serum albumin (BSA) overnight at 4°C. Detection of MCP-1 protein was performed by incubating the membrane in a 1:1000 dilution of a MCP-1 antibody and a peroxidase-conjugated secondary antibody. Peroxidase labeling was detected with luminescence immunodetection (ECL; Amersham).
Antibodies
The antibody used to detect rat MCP-1 by immunohistochemistry and immunofluorescence was kindly provided by Dr. T. Yoshimura (National Cancer Institute/Frederick Cancer Research and Development Center, Frederick, MD, USA). The polyclonal rabbit anti-rat MCP-1 antiserum13,14 was used at a dilution of 1:250. For Western blot, a goat polyclonal anti-rat MCP-1 antibody (Santa Cruz Biotechnologies, Heidelberg, Germany) was used. The mouse monoclonal antibodies against cell markers ED-1, ED-3, RECA, Thy-1, and WT-1 were purchased from Serotec (Biozol, Eching, Germany) or Santa Cruz Biotechnologies, respectively. The synaptopodin antibody was obtained from ProGen Biotechnics GmbH (Heidelberg, Germany). These antibodies were used at a dilution of 1:250, except for the WT-1 antibody, which was diluted 1:50.
Analysis of data
Intraglomerular ED-1–positive cells were counted in all glomeruli of a given kidney section (100 to 300 glomeruli, no selection) and were expressed as cells per glomerular section. Interstitial ED-1–positive cells were counted in 30 medium-power (magnification 25) cortical views per section and were expressed as cells per square millimeter. Counting was begun in a random cortical field and in consecutive nonoverlapping cortical fields to the right of the previous view without selection; if necessary, counting was continued at the opposite (left) edge of the section. In some experimental groups, cells positive for ED-3, a marker for activated Ms32, were counted in the same manner. Two-way analysis of variance (ANOVA), followed by post hoc Newman-Keuls test with adjustment for multiple comparisons, was used to test significance of differences between groups. A P value < 0.05 was considered significant. The procedures were carried out using the Statistica software (StatSoft, Tulsa, OK, USA). Values are displayed as means standard error of the mean (SEM).
RESULTS
In 2K1C animals, systolic blood pressure as measured by tail-cuff plethysmography was increased at 4 days (139 8 vs. 115 8 mm Hg in controls, P < 0.05), 14 days (153 7 vs. 112 4 mm Hg in controls, P < 0.05), and 28 days after clipping (190 13 vs. 115 11 mm Hg in controls, P < 0.05). Similar elevations of systolic blood pressure were found in TGR (189 8 vs. 106 4 mm Hg in SDH, P < 0.05) and SHR-SP (190 9 vs. 110 5 mm Hg in WKY). In SHR, systolic blood pressure was increased to a somewhat lower extent (160 9 vs. 112 5 mm Hg in WKY, P < 0.05).
MCP-1 gene expression
Figure 1 shows the expression of MCP-1 mRNA in individual kidneys of 2K1C, TGR, SHR, SHR-SP, and WKY. Twenty-eight days after clipping, MCP-1 mRNA was clearly increased (approximately fivefold) in the nonclipped, right kidneys of 2K1C animals, whereas there was no consistent increase of MCP-1 mRNA in clipped kidneys Figure 1. Furthermore, MCP-1 mRNA was not increased in the kidneys of SHR and SHR-SP, compared with the respective control animals. In contrast, TGR exhibited 2.8-fold increased mRNA levels for the chemokine, compared with SDH control rats Figure 1.
Figure 1.
Autoradiographs of Northern blots of 20 
g total kidney RNA hybridized with a rat monocyte chemoattractant protein-1 (MCP-1) probe. Each lane represents one individual kidney. The RNA gel stained by ethidium bromide is shown below the autoradiographs. (A) Two kidney-one clip (2K1C) hypertension 28 days after clipping: nonclipped, contralateral kidneys, clipped kidneys, and kidneys from sham-operated (sham) rats. (B) RNA from ren-2 transgenic hypertensive rats (TGR) and Hannover Sprague-Dawley controls (SDH). (C) Spontaneous hypertensive rats (SHR), stroke prone SHR (SHR-SP), and Wistar-Kyoto (WKY) rat kidney RNA.
Localization of MCP-1 protein
Monocyte chemoattractant protein-1 staining was detected in glomeruli, vessels, tubular, and interstitial cells of the contralateral kidneys of 2K1C animals Figure 2. Some degree of staining of the smooth muscle cell layer of interlobular arteries and afferent arterioles was present in control animals Figure 2a and clipped kidneys as well. However, no glomerular, tubular or interstitial MCP-1 staining was observed in clipped kidneys (data not shown) or control animals Figure 2a. Within glomeruli, MCP-1 staining was not detected in mesangial cells Figure 3. However, MCP-1 staining was detected occasionally in endothelial cells, more often in cells with WT-1–positive nuclei, and partly colocalized with synaptopodin staining Figure 3. These findings suggest that glomerular MCP-1 staining was localized to endothelial cells and, more markedly, to podocytes.
Figure 2.
Double immunostaining for MCP-1 (orange brown) and the M
/monocyte marker ED-1 (dark purple cytoplasm). Hematoxylin counterstain (blue nuclei). (A) Kidney section of a sham-operated rat (magnification 400), representative for five rats. Staining of a preglomerular vessel for MCP-1 is indicated by an asterisk. (B–E) From contralateral, nonclipped kidneys of 2K1C rats 28 days after clipping (N = 5). Some examples of ED-1–positive Ms are indicated by black arrows. Occasional Ms that also exhibited MCP-1 staining are indicated by white arrowheads. ED-1–positive Ms were spatially associated with MCP-1 staining in glomeruli (B, magnification 400), peritubular interstitium (C, magnification 400), tubules (D, magnification 400), and injured vessels (E, magnification 600).
Figure 3.
Double stainings for MCP-1 and glomerular cell markers in contralateral kidneys of 2K1C rats 28 days after clipping (examples are representative for 5 rats). (A) Double immunofluorescence for MCP-1 (red) and podocyte cytoplasmic marker synaptopodin (green), showing partial colocalization (yellow, indicated by white arrow). Magnification 400. (B) Double immunofluorescence for MCP-1 (red) and mesangial cell marker Thy-1.1 (green), showing no colocalization. Magnification 400. (C) Double immunohistochemistry for MCP-1 (purple) and rat endothelial cell marker RECA (brown). Hematoxylin counterstain (blue nuclei), magnification 800. Colocalization of MCP-1 and RECA is indicated by a white arrow. (D) Double immunohistochemistry for MCP-1 (purple) and podocyte nuclear marker WT-1 (brown nuclei). Hematoxylin counterstain (blue nuclei), magnification 800. Some podocyte nuclei surrounded by MCP-1–positive cytoplasm are indicated by white arrows.
The most conspicuous MCP-1 staining was observed in peritubular interstitial cells Figure 2c. Staining of tubular cells Figure 2d was occasionally encountered but was quite rare (less than one stained tubule per kidney section) compared with peritubular MCP-1–positive areas similar to the one shown in Figure 2c (
10 to 30 areas per kidney section).
In TGR kidneys, the pattern of MCP-1 staining was essentially the same as in the contralateral kidneys of 2K1C (data not shown). In SHR, no MCP-1 staining was found, except the weak staining of small arteries and arterioles, which was also present in all nonhypertensive control animals, including WKY. In SHR-SP, there was no cortical interstitial or glomerular MCP-1 staining. However, local MCP-1 staining was present at the perimeter of juxtamedullary necrotic lesions Figure 4, which were presumably caused by fibrinoid necrosis of juxtamedullary glomeruli or vessels.
Figure 4.
Juxtamedullary necrotic lesion in SHR-SP kidney, representative for four SHR-SP rats. (A) MCP-1 immunostaining (black) at the perimeter of the lesion (magnification 250). (B) M infiltration (ED-1 staining, black) surrounding the lesion (magnification 250).
MCP-1 and monocyte/macrophage infiltration
In the nonclipped, contralateral kidneys of 2K1C, the development of hypertension was associated with monocyte/M infiltration in both glomeruli and the interstitial space Figure 5. In contrast, clipped kidneys exhibited elevated interstitial ED-1–positive cell counts only at the early days after clipping but not during the development of hypertension Figure 5. Double stainings showed that MCP-1 staining was spatially related to ED-1–positive monocytes/Ms Figure 2. Some ED-1–positive cells appeared to stain for MCP-1 as well, but most MCP-1–positive cells did not stain for ED-1 Figure 2. The extent of glomerular and interstitial monocyte/M infiltration in TGR kidneys Figure 6 was similar to the contralateral kidney of 2K1C Figure 5. A smaller extent of glomerular and interstitial monocyte/M infiltration was seen in SHR-SP Figure 6, whereas no significant infiltration was noted in SHR compared with WKY Figure 6.
Figure 5.
Quantitation of macrophage infiltration in 2K1C kidneys. (A) Glomerular ED-1–positive cells, expressed per glomerular cross-section. (B) Tubulointerstitial ED-1–positive cells per square millimeter tubulointerstitial space. Data are mean SEM of N = 5 animals each. Symbols are: (
) sham; (
) clipped; (
) contralateral nonclipped kidneys. *P < 0.05 vs. sham-operated controls.
Figure 6.
Quantitation of macrophage infiltration in genetic models of hypertension. TGR (N = 7) were compared with SD rats (N = 6), SHR (N = 4), and SHR-SP (N = 4) with the respective WKY strains (N = 4 rats for each strain). (A) Glomerular ED-1–positive cells, expressed per glomerular cross-section. (B) Tubulointerstitial ED-1–positive cells per square millimeter tubulointerstitial space. Data are mean SEM. Symbols are: () control (WKY/SD); () hypertensive rats. *P < 0.05 vs. normotensive controls.
Effects of AT1 antagonism
During treatment of established 2K1C hypertension with valsartan for two weeks, two 2K1C animals in the solvent group but none in the valsartan or sham-operated groups died. Intra-arterial blood pressure measurements demonstrated that mean arterial pressure was significantly lowered but not normalized by valsartan Figure 7. The induction of MCP-1 mRNA in 2K1C right kidneys exposed to high blood pressure was blunted by valsartan Figure 8. Western blot analysis of renal cortical protein for MCP-1 detected several positive bands, in accordance with previous results13,14,36. MCP-1 protein was induced in 2K1C, which was completely abolished by valsartan treatment. Immunohistochemistry confirmed that the induction of MCP-1 protein in cortical interstitial and glomerular tissue of 2K1C right kidneys was almost completely suppressed by valsartan Figure 9.
Figure 7.
Effects of valsartan treatment on mean arterial blood pressure (A) and interstitial macrophage infiltration (B). Total M numbers (ED-1–positive cells per mm2; ) and activated Ms (ED-3–positive cells per mm2; ) were counted. Data represent mean SEM from seven sham-operated, six untreated 2K1C, and five valsartan-treated 2K1C rats. *P < 0.05 vs. sham operated control; §P < 0.05 vs. untreated 2K1C.
Figure 8.
(A) Autoradiograph of Northern blot of 20 g total kidney RNA hybridized with a rat MCP-1 probe. Each lane represents one individual right, nonclipped kidney. The RNA gel stained by ethidium bromide is shown below the autoradiographs. (B) Western blot of kidney cortex protein (right, nonclipped kidneys) for MCP-1 protein. In comparison to sham-operated controls, MCP-1 mRNA and protein are clearly induced in 2K1C rat kidneys. Valsartan (AT1RA) treatment normalizes MCP-1 mRNA and protein levels.
Figure 9.
Representative examples of sections stained for MCP-1 (dark cytoplasm, arrowheads) from sham-operated (upper panel, N = 7), untreated 2K1C (middle panel, N = 6), and valsartan-treated 2K1C rats (lower panel, N = 5). Hematoxylin counterstain (dark nuclei), magnification 400. All sections from right (contralateral) kidneys.
Glomerular infiltration of ED-1–positive total Ms and ED-3–positive activated Ms tended to be higher in untreated 2K1C right kidneys (0.41 0.23 ED-1–positive cells and 0.39 0.15 ED-3–positive cells per glomerular cross-section) than in sham-operated rat kidneys (0.26 0.12 ED-1–positive cells and 0.25 0.08 ED-3–positive cells), whereas valsartan-treated 2K1C right kidneys displayed glomerular M numbers similar to sham-operated controls (0.30 0.11 ED-1–positive cells and 0.26 0.17 ED-3–positive cells per glomerular cross-section). However, there was no overall statistically significant difference by ANOVA. In contrast, interstitial M infiltration was significantly enhanced in 2K1C, which was abolished by valsartan Figure 7.
DISCUSSION
Our results show that MCP-1 expression is increased in glomeruli and tubulointerstitial space in hypertensive nephrosclerosis associated with Ang II-dependent hypertension (2K1C and TGR). The localization of MCP-1 protein was closely associated with M infiltration into glomeruli and tubulointerstitial space of rat kidneys with nephrosclerosis. Blockade of the AT1 receptor abolished the induction of MCP-1 and blunted M infiltration in 2K1C rat kidneys. In contrast, little MCP-1 expression or monocyte/M infiltration was observed in SHR and SHR-SP. Our data show that the AT1 receptor induces MCP-1 expression, which in turn may mediate monocyte/M infiltration in Ang II-dependent forms of hypertensive nephrosclerosis.
The chemoattractant protein MCP-1, cloned by Yoshimura et al in 198913, has been identified as an important mediator of M infiltration in several immune-mediated inflammatory diseases of the kidney: Increased expression of MCP-1 was described in experimental and human forms of glomerulonephritis17,22. Moreover, intervention studies with blocking antibodies demonstrated that MCP-1 is an important chemoattractant for monocytes/Ms in experimental anti-basement–membrane crescentic glomerulonephritis18,19,20 and experimental anti–Thy-1.1 mesangioproliferative glomerulonephritis21. Our data demonstrate that MCP-1 is clearly induced in glomeruli of rats with supposedly nonimmunological hypertensive kidney disease. Furthermore, our results suggest that MCP-1 may play a role for M infiltration into the kidney in these experimental models. Thus, our data further support the hypothesis that inflammatory mechanisms contribute to renal injury in Ang II-dependent forms of hypertension3,4,6,12.
Several lines of evidence suggest that Ang II may directly contribute to the induction of the chemokine MCP-1 in hypertensive nephrosclerosis: Ang II has been shown to increase MCP-1 synthesis in vascular smooth muscle cells in vitro16 and in vivo15. Blockade of Ang II in experimental models of immune-mediated glomerulonephritis reduced the induction of MCP-137,38. Activation of mitogen-activated kinases16 and the transcription factor nuclear factor-
B37 may be involved in the signal transduction. The available evidence suggests that the induction of MCP-1 by Ang II is mediated via the AT1 receptor15,16,38. Interestingly, the AT2 receptor has been implicated in the induction of the chemokine RANTES by Ang II12. Finally, mechanical forces such as glomerular hypertension could directly contribute to MCP-1 expression, which is increased by shear stress in cultured endothelial cells in vitro39. Capers et al investigated MCP-1 expression in aortic tissue of rats, using different models of hypertension, Ang II infusion, and norepinephrine infusion, respectively, as well as different forms of antihypertensive treatment15. These authors elegantly demonstrated that both hypertension and Ang II induce MCP-1 synthesis15; that is, MCP-1 expression was highest when both stimuli were present. Our data do not allow us to decide whether the reduced MCP-1 expression after valsartan treatment was due to a direct effect of AT1 antagonism or to the lowering of blood pressure. However, a direct AT1 effect seems likely in view of the evidence discussed previously in this article. In addition, the absence of MCP-1 expression in SHR animals argues against a major role of hypertension per se for the induction of the chemokine.
We investigated which renal cells express MCP-1 in hypertensive nephrosclerosis. MCP-1 staining was present in interstitial cells and vascular smooth muscle cells. Occasionally, ED-1–positive monocytes/Ms stained for MCP-1, but by far most MCP-1–positive cells were negative for the monocyte/M marker ED-1. In the glomerulus, double stainings did not show MCP-1 in mesangial cells, whereas there was a partial colocalization in podocyte cytoplasm, and occasional staining of endothelial cells. Others have described MCP-1 staining in mesangial cells and/or Bowman's capsule cells in experimental forms of nephritis10,17,22, but most authors did not perform double stainings with specific cell markers. Part of the MCP-1 staining we observed might be localized to extracellular subepithelial or subendothelial deposits, but immunogold electron microscopy will be required to resolve this issue.
We observed a correlation between MCP-1 expression and M infiltration, both with respect to total (ED-1 positive) and activated (ED-3 positive) Ms. Because MCP-1 localization was not restricted to Ms in the kidney, these data suggest but do not prove that MCP-1 does indeed contribute to M infiltration into the kidney tissue. Ang II could play a role in the activation of these Ms40,41. To determine the importance of MCP-1 for M influx and activation in hypertensive nephrosclerosis relative to other factors, blockade of the chemokine would be required. In contrast to the immune-mediated models of glomerulonephritis, which were successfully treated with MCP-1 antibodies18,19,20,21, the initial insult leading to kidney injury is present for weeks or months in hypertensive nephrosclerosis, rather than being restricted to a single temporarily defined injection of an antiserum. Therefore, the use of blocking antibodies is probably not feasible in hypertension. Very recently, mice with targeted deletions of the gene for MCP-142 or its receptor on Ms43 were used to demonstrate the importance of MCP-1 for the development of arteriosclerotic lesions of the wall of large vessels. Such an approach might be helpful in future studies to test the functional relevance of MCP-1 for renal M influx in hypertension.
In summary, our data on MCP-1 add to the evidence that inflammatory mechanisms play a role in hypertensive nephrosclerosis associated with Ang II-dependent models of hypertension. One could speculate that blockade of the chemokine might hold promise as a therapeutic strategy in hypertensive nephrosclerosis, even beyond Ang II blockade, or when the hemodynamic effects of Ang II inhibition are to be avoided. Future experiments in mice with targeted disruption of the gene for MCP-1 or its receptor are necessary to address this issue.
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Acknowledgments
This study was supported by a grant from Novartis, Basel, Switzerland; by the Bundesministerium für Bildung und Forschung (Interdisziplinäres Zentrum für Klinische Forschung Teilprojekt B12), Bonn, Germany; by a grant from the Deutsche Forschungsgemeinschaft (Ge 568/2–3), Bonn-Bad Godesberg, Germany; and by the "Verein zur Förderung der Nieren-und Hochdruckforschung an der Universität Erlangen-Nürnberg" e.V., Nürnberg, Germany. Part of these data were presented at the 1998 Experimental Biology meeting (San Francisco, CA, USA) and the 1998 European Kidney Research Forum (Manchester, UK). We gratefully acknowledge the expert technical assistance of Elisabeth Buder, Miroslava Kupraszewicz, Rainer Wachtveitl, and Rita Zitzmann. We thank Dr. T. Yoshimura for supplying MCP-1 cDNA and antiserum.
