Effects of diabetes and hypertension on macrophage infiltration and matrix expansion in the rat kidney

Abstract

Background: In experimental models of diabetes mellitus, aggravation of renal injury by concomitant hypertension has been described. Inflammatory mechanisms contribute to renal damage in both diseases. We investigated whether hypertension and diabetes mellitus act synergistically to induce macrophage infiltration and matrix expansion in the kidney.

Methods: Insulin-dependent diabetes mellitus was induced by streptozotocin injections to hypertensive mRen2-transgenic rats (TGR) and normotensive Sprague-Dawley control rats. Quantitative immunohistochemical examination of kidney tissue sections was used to measure macrophage infiltration and matrix expansion. The expression of MCP-1, Osteopontin, RANTES, ICAM-1 and VCAM-1 was evaluated by real-time RT-PCR. The localization of MCP-1 was studied by immunohistochemistry.

Results: Macrophage infiltration was present in the kidney of normotensive diabetic rats. Hypertensive rats exhibited a more marked infiltration of macrophages, regardless of whether diabetes was present or not. Gene expression of ICAM-1, VCAM-1 and RANTES was unaltered whereas Osteopontin and MCP-1 were induced by hypertension. Immunoreactive MCP-1 was slightly increased in diabetic rat kidney podocytes, and more markedly increased in hypertensive animals. Glomerular matrix accumulation was induced by diabetes and hypertension to a similar degree, and was highest in hypertensive, diabetic animals.

Conclusion: Diabetes mellitus caused a mild, and angiotensin-dependent hypertension a more marked infiltration of macrophages in the kidney. Combination of both diseases led to additive effects on matrix expansion but not on inflammation. Hypertension appears to be a much stronger stimulus for inflammation of the kidney than STZ diabetes, at least in mRen2-transgenic rats.

Figure 1

Blood glucose (panel A), arterial blood pressure (panel B) and albuminuria (panel C) of diabetic and hypertensive rats. Blood glucose was measured 24 hours before termination of the experiment. Mean arterial blood pressure was determined in awake rats via indwelling catheters inserted into the femoral artery. For determination of albuminuria, urine was collected 24 hours before sacrifice. SD, normotensive normoglycemic control rats; TGR, transgenic hypertensive rats; STZ, streptozotocin treatment. * indicates p < 0.05 versus normotensive normoglycemic SD control rats.

Figure 2

Macrophage infiltration of glomeruli (panel A) and interstitial space (panel B) of the kidney from rats with diabetes mellitus and/or hypertension. Glomerular macrophages are expressed as ED-1-positive cells per glomerular cross-section, interstitial macrophages as ED-1-positive cells per square mm. SD, normotensive normoglycemic control rats; TGR, transgenic hypertensive rats; STZ, streptozotocin treatment. Data are mean ± SEM of n = 5 rats. * indicates p < 0.05 versus normotensive normoglycemic SD control rats.

Figure 3

Real-time RT-PCR analysis of MCP-1 (A) and osteopontin (B) mRNA expression in the renal cortex. Data are expressed as fold control compared to SD control rats. Data are mean ± SEM of n = 5 rats. * indicates p < 0.05 versus normotensive normoglycemic SD control rats. SD, normotensive normoglycemic control rats; TGR, transgenic hypertensive rats; STZ, streptozotocin treatment.

Figure 4

Western blot analysis for MCP-1 protein in cortical protein preparations of two rats of each experimental group, yielding the characteristic MCP-1 double band (14 and 16 kDa). SD, normotensive normoglycemic control rats; TGR, transgenic hypertensive rats; STZ, streptozotocin treatment.

Figure 5

Immunohistochemistry for MCP-1 and the macrophage marker ED-1. Panel A-D: examples of photomicrographs of MCP-1 staining (brown) in glomeruli, hematoxylin counterstain (blue nuclei) Panel A (glomerulus of a normotensive normoglycemic rat) represents score 0, panel B (from a normotensive diabetic rat) score 1, panel C (from a hypertensive normoglycemic rat kidney) score 2, and panel D (from a hypertensive diabetic rat) score 3. Panel E, High power magnification of MCP-1 immunohistochemistry (brown) in a hypertensive diabetic rat. MCP-1 staining localized to spindle-shaped cells, probably fibroblasts, surrounding tubules in a double immunohistochemistry with the macrophage marker ED-1 (blue cytoplasm, arrows), methyl-green counterstain. Macrophages were often localized in close proximity to the MCP-1 positive cells surrounding tubules (asterisk). Scale bars (50 μm) are indicated. Note the identical scale for panels A-D but different scale for panel E.

Figure 6

Semiquantitative evaluation of MCP-1 immunohistochemistry. For glomerular staining (A) more than 100 glomeruli per kidney section were classified 0 to 3 (see methods and figure 5 for details). The percentage of glomeruli assigned to a given score value is shown. For interstitial MCP-1 scores (B), 20 low-power cortical fields were classified 0 to 4 (see methods for details). The percentage of interstitial fields assigned to a given score value is shown. SD, normotensive normoglycemic control rats; TGR, transgenic hypertensive rats; STZ, streptozotocin treatment. Data are mean ± SEM of n = 5 rats. * indicates p < 0.05 versus normotensive normoglycemic Sprague-Dawley (SD) control rats.

Figure 7

Matrix expansion in the renal cortex and the glomerulus. A: measurement of cortical collagen I staining and B: measurement of glomerular collagen IV staining. Data are mean ± SEM, * indicates p < 0.05 versus normotensive normoglycemic SD controls. # indicates p < 0.05 versus SD-STZ. § indicates p < 0.05 versus normoglycemic TGR.