Intercellular Adhesion Molecule-1–Deficient Mice Are Resistant Against Renal Injury After Induction of Diabetes

ICAM-1^−/− mice (C57BL/6J background) (17) were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6J mice (ICAM-1^+/+ mice) were used as controls.

Male ICAM-1^+/+ and ICAM-1^−/− mice aged 8 weeks were divided into four groups: 1) nondiabetic ICAM-1^+/+ mice (n = 10), 2) nondiabetic ICAM-1^−/− mice (n = 10), 3) STZ-induced diabetic ICAM-1^+/+ mice (n = 10), and 4) diabetic ICAM-1^−/− mice (n = 10). Mice in the diabetic groups received an intraperitoneal injection of STZ (Sigma Chemical, St. Louis, MO) at 200 mg/kg in citrate buffer (pH 4.5). Nondiabetic ICAM-1^+/+ and ICAM-1^−/− mice received citrate buffer injections only. All mice had free access to standard diet and tap water. All procedures were performed according to the Guidelines for Animal Experiments at Okayama University Medical School, Japanese Government Animal Protection and Management Law (no. 105), and Japanese Government Notification on Feeding and Safekeeping of Animals (no. 6). Six months after the induction of diabetes, all mice were killed, and the kidneys were harvested. Kidneys were weighed and fixed in 10% formalin for periodic acid–methenamine silver (PAM) and azan staining, and the remaining tissues were embedded in OCT compound (Sakura Finetechnical, Tokyo, Japan) and immediately frozen in acetone cooled on dry ice.

Blood glucose, urinary creatinine, urinary albumin excretion (24 h), systolic blood pressure, and body weight were measured at 0, 1, 3, and 6 months. Urine collection was performed for 24 h with each mouse individually housed in a metabolic cage with free access to food and water. Serum creatinine was measured at 6 months after the onset of diabetes. Blood glucose was measured by the glucose oxidase method. Creatinine was measured by the enzymatic method. Urinary albumin concentration was measured by nephelometry (Organon Teknika-Cappel, Durham, NC). Blood pressure was measured by the tail-cuff method (Softron). HbA_1c was measured at 6 months after induction of diabetes by latex-agglutination assay. Creatinine clearance was calculated in individual mice.

Renal tissues were fixed in 10% formalin and embedded in paraffin in routine fashion. Tissue sections were cut at 4-μm thickness, dewaxed, and stained with PAM and azan. To evaluate glomerular size, 10 randomly selected glomeruli in the cortex per animal (totaling 100 glomeruli for each group) were examined under high magnification (×400). Glomerular tuft area was measured by manually tracing the glomerular tuft using PhotoShop version 6 (Adobe Systems, San Jose, CA) and analyzed by National Institutes of Health (NIH) Image version 1.6. Mesangial matrix area was defined as the PAM-positive area within the tuft area. The mesangial matrix index represented the ratio of mesangial matrix area divided by the tuft area (Fig. 1). The results are expressed as means ± SE (in micrometers squared).

Tissue preparation was performed as previously described (18). Briefly, renal cortical specimens were immersed in 2.5% glutaraldehyde in 0.1 mol/l cacodylate buffer (pH 7.2, 2 h, 4°C), postfixed in 0.1 mol/l osmium tetroxide (2 h, 4°C), and then embedded in Epon following dehydration in a graded series of ethanol preparations. Thin sections were stained with uranyl acetate and lead citrate and examined with an electron microscope (Hitachi H7100) at 100 kV. Glomeruli were photographed at a magnification of ×1,000. Mesangial matrix index was defined as the proportion of the glomerular tuft occupied by the mesangial matrix area (excluding nuclei). Mesangial matrix areas of five glomeruli in each kidney were analyzed. Mesangial matrix areas were selected using PhotoShop and measured with NIH Image.

Immunoperoxidase and immunofluorescence staining were performed as previously described (12). Fresh frozen sections were cut at 4-μm thickness using a cryostat. To evaluate infiltration of macrophages, a specific rat monoclonal antibody (mAb) against mouse monocyte/macrophage (F4/80) was applied to the fresh frozen sections as the primary reaction, followed by a second reaction with biotin-labeled goat anti-rat IgG antibody. Finally, the avidin-biotin coupling reaction was performed on the sections using a Vectastain Elite kit (Vector Laboratories, Burlingame, CA). Intraglomerular F4/80-positive cells were counted in 20 glomeruli per animal (totaling 200 glomeruli for each group). The average number per glomerulus was used for the estimation. To evaluate the expression of TGF-β, a rabbit antibody to TGF-β was used as a primary antibody, followed by a second reaction with biotin-labeled donkey anti-rabbit IgG antibody. Ten glomeruli were examined per animal (totaling 100 glomeruli for each group) under high magnification (×400), and the TGF-β–positive area in a glomerulus was measured using NIH Image. The TGF-β–positive area of each glomerulus was expressed relative to the mean value of TGF-β–positive areas in nondiabetic ICAM-1^+/+ mice. To visualize the expression of ICAM-1 and vascular cell adhesion molecule (VCAM)-1, goat anti-mouse ICAM-1 antibody and rat anti-mouse VCAM-1 mAb were used for the primary reactions, and fluorescein isothiocyanate (FITC)-labeled anti-goat IgG antibody and anti-rat IgG antibody were applied to the sections, respectively. To characterize the change in mesangial matrix proteins, rabbit anti–type IV collagen antibody was used for the primary reactions followed by a second reaction with FITC-labeled anti-rabbit IgG. Micrographic fluorescence photos were obtained using a confocal laser fluorescence microscope (LSM-510; Carl Zeiss, Jena, Germany). Quantification of type IV collagen immunofluorescence intensity was performed as follows. Color images were obtained as PICT files from the LSM-510. Image files in PICT format were inverted and opened in gray-scale mode using NIH Image. The type IV collagen expression was calculated using the formula, [x(density) × positive area (in micrometers squared)], where the staining density is indicated by a number from 0 to 255 in gray scale. The type IV collagen expression of each glomerulus was estimated as the ratio to the mean expression in nondiabetic ICAM-1^+/+ mice. Ten glomeruli were examined per animal for type IV collagen expression (totaling 100 glomeruli for each group).

Renal cortex was minced by leather blades and put into 1.5-mm³ Eppendorf tubes, and then Laemmli’s sample buffer (containing 30% glycerol, 0.9% SDS, 1 mol/l Tris-HCl, and 15% 2-mercaptoethanol) was added. Tissues in each tube were homogenized by passing through needles from 18 to 26 gauges, 20 times for each needle, in numerical order. The homogenates were then cleared by centrifugation, and the protein concentration of each resulting supernatant was determined using an RC DC protein assay kit (Bio-Rad, Hercules, CA).

Immunoblotting was fundamentally performed as previously described (19). Briefly, samples containing 15 μg protein were boiled for 3 min and then subjected to 12.5% SDS-PAGE. The separated proteins were transferred to polyvinylidene difluoride membranes by electrotransfer. The blots were subsequently blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween-20 at room temperature for 1 h and then incubated at 4°C overnight with 1:100 rabbit anti-human type IV collagen antibody. After washing three times for 10 min with Tris-buffered saline containing 0.1% Tween-20, the membrane was incubated with a 1:1,000 dilution of horseradish peroxidase–linked anti-rabbit IgG secondary antibody at room temperature for 1 h. The blots were then visualized with the enhanced chemiluminescence Western blot detection system (Amersham Biosciences, Amersham Place, U.K.). The primary antibody recognizes the α1-subunit of human type IV collagen and shows a distinct cross-reactivity with mouse type IV collagen. The amount of type IV collagen was analyzed using Image QuaNT analysis software version 4.2-J (Molecular Dynamics, Sunnyvale, CA).

Rat anti-mouse monocyte/macrophage mAb (F4/80) was purchased from Jackson Immunoresearch Laboratories (West Grove, PA). Goat anti-mouse ICAM-1 polyclonal antibody (M-19); rabbit anti-human TGF-β1, -β2, and -β3 rabbit polyclonal antibody (H112); and rabbit anti-human type IV collagen antibody (H234) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rat anti-mouse VCAM-1 mAb was purchased from Endogen (Woburn, MA). Rabbit anti-mouse type IV collagen antibody was purchased from Chemicon International (Temecula, CA). Biotin-labeled goat anti-rat IgG antibody, FITC-labeled donkey anti-goat IgG antibody, biotin-labeled donkey anti-rabbit IgG antibody, and FITC-labeled goat anti-rat IgG antibody were purchased from Jackson Immunoresearch Laboratories.

All data are expressed as means ± SE. One-way ANOVA followed by the Scheffe method was used to compare the means of group data. A P value <0.05 denoted the presence of a statistically significant difference.

At 6 months after induction of diabetes, HbA_1c and creatinine clearance in both diabetic ICAM-1^+/+ and ICAM-1^−/− mice were significantly higher than in nondiabetic ICAM-1^+/+ and ICAM-1^−/− mice. There was no significant difference in HbA_1c and creatinine clearance between diabetic ICAM-1^+/+ and ICAM-1^−/− mice (Table 1). Body weight was lower in both diabetic ICAM-1^+/+ and ICAM-1^−/− mice compared with nondiabetic mice. Kidney weights were higher in diabetic ICAM-1^+/+ mice compared with nondiabetic mice. On the other hand, there was no difference in kidney weight between nondiabetic and diabetic ICAM-1^−/− mice. In addition, kidney weight per body weight was significantly increased in both diabetic groups; however, the ratio was significantly lower in diabetic ICAM-1^−/− mice compared with diabetic ICAM-1^+/+ mice (Table 1).

ICAM-1 protein expression was prominent in the glomeruli of diabetic ICAM-1^+/+ mice but was undetectable in nondiabetic and diabetic ICAM-1^−/− mice (Fig. 2A–D). The staining with VCAM-1 in the glomeruli was weak in all groups. The differences in glomerular VCAM-1 expression between nondiabetic and diabetic mice and between diabetic ICAM-1^+/+ and ICAM-1^−/− mice were minimal (data not shown). The number of macrophages in the glomeruli was higher in both diabetic groups at the end of the 6-month follow-up period compared with the respective nondiabetic groups (3.19 ± 0.12 vs. 0.56 ± 0.18 per glomerulus for diabetic vs. nondiabetic ICAM-1^+/+ mice, respectively, P < 0.0001; 1.35 ± 0.09 vs. 0.79 ± 0.06 per glomerulus for diabetic vs. nondiabetic ICAM-1^−/− mice, respectively, P < 0.0005) (Fig. 2E–I). Infiltration of macrophages into glomeruli was significantly suppressed in diabetic ICAM-1^−/− mice compared with diabetic ICAM-1^+/+ mice (P < 0.0001).

As shown in Fig. 3, urinary albumin excretion increased progressively during the study in diabetic mice. However, urinary albumin concentrations in diabetic ICAM-1^−/− mice were significantly lower than in diabetic ICAM-1^+/+ mice at 3 months (47.3 ± 5.1 vs. 84.6 ± 5.1 μg/day, P < 0.0001) and at 6 months (31.4 ± 6.7 vs. 83.6 ± 10.9 μg/day, P < 0.05) after the induction of diabetes (Fig. 3).

Representative glomeruli in PAM-stained sections are shown in Fig. 4A–D. Glomerular hypertrophy and mesangial matrix expansion were observed in diabetic ICAM-1^+/+ mice at the end of the 6-month observation period. These changes, however, were not prominent in diabetic ICAM-1^−/− mice as compared with diabetic ICAM-1^+/+ mice. We also investigated changes in the interstitium in azan-stained sections. Interstitial fibrosis was prominent in diabetic ICAM-1^+/+ mice, whereas it was mild in diabetic ICAM-1^−/− mice as compared with diabetic ICAM-1^+/+ mice (Fig. 4E–H). There was no histological change between nondiabetic ICAM-1^−/− and ICAM-1^+/+ mice. Both glomerular area and mesangial matrix index were increased in diabetic ICAM-1^+/+ mice. These values were significantly lower in diabetic ICAM-1^−/− mice (tuft area, 3,829 ± 83 vs. 4,370 ± 137 μm², P < 0.005; mesangial matrix index, 5.6 ± 0.4 vs. 7.6 ± 0.6%, P < 0.01) (Fig. 4I and J).

Representative electron microscopic photos are shown in Fig. 5. Mesangial matrix index was significantly increased in diabetic groups (8.18 ± 0.63 vs. 18.1 ± 1.34% for nondiabetic vs. diabetic ICAM-1^+/+ mice, respectively, P < 0.0001; 6.64 ± 0.60 vs. 10.5 ± 0.82% for nondiabetic vs. diabetic ICAM-1^−/− mice, respectively, P = 0.022). However, the index was significantly lower in diabetic ICAM-1^−/− mice compared with diabetic ICAM-1^+/+ mice (P < 0.0001).

TGF-β–positive areas in glomeruli were larger in diabetic mice compared with nondiabetic mice. Furthermore, the TGF-β–positive area in diabetic ICAM-1^−/− mice was lower than in diabetic ICAM-1^+/+ mice (212 ± 20 vs. 316 ± 21% of nondiabetic ICAM-1^+/+ mice, P < 0.0005) (Fig. 6A–D and I). The intensity of type IV collagen in glomeruli was higher in diabetic mice compared with nondiabetic mice, and the intensity in diabetic ICAM-1^−/− mice was lower than in diabetic ICAM-1^+/+ mice (111 ± 2 vs. 147 ± 3% of nondiabetic ICAM-1^+/+ mice, P < 0.0001) (Fig. 6E–H, and J).

As shown in Fig. 7, Western blot analysis revealed increased type IV collagen protein expression in the kidneys of diabetic ICAM-1^+/+ mice, whereas the type IV collagen expression was significantly suppressed in diabetic ICAM-1^−/− mice compared with diabetic ICAM-1^+/+ mice (73 ± 17 vs. 236 ± 62% for diabetic ICAM-1^−/− and diabetic ICAM-1^+/+ mice, respectively, P = 0.048).

We induced diabetes in ICAM-1^−/− and ICAM-1^+/+ mice and compared the progression of renal injury associated with hyperglycemia in the present study. Diabetic ICAM-1^+/+ mice (C57BL/6 mice) developed mild diabetic nephropathy as previously described (20). Our results showed that glomerular infiltration of macrophages, renal and glomerular hypertrophy, overproduction of TGF-β, accumulation of type IV collagen in glomeruli, and albuminuria were significantly suppressed in ICAM-1^−/− mice when compared with ICAM-1^+/+ mice at 6 months after induction of diabetes.

The phenotypes of ICAM-1^−/− mice used in this study were originally reported by Sligh et al. (17). ICAM-1^−/− mice are fertile and show no apparent developmental defects. Their immune responses are impaired, i.e., their neutrophil emigration in response to chemical peritonitis and decreased contact hypersensitivity is impaired, and with the exception of an increased level of circulating neutrophils and a slight increase in body weight, no obvious abnormal phenotype is seen in normal conditions (21,22). Under normal conditions, no obvious abnormalities in histology and function of the kidneys are observed in these mice. However, ICAM-1 is a critical molecule for leukocyte infiltration in inflammatory processes, and amelioration of experimentally induced autoimmune glomerulonephritis was noted in ICAM-1^−/− mice, which was associated with reduced glomerular leukocyte infiltration (23,24).

ICAM-1 was upregulated in glomeruli of diabetic ICAM-1^+/+ mice. Although it is still controversial whether hyperglycemia per se can induce the expression of ICAM-1 on vascular endothelial cells, recent studies have suggested several possible mechanisms of ICAM-1 induction in diabetic renal tissues. 1) ICAM-1 is induced by inflammatory cytokines such as tumor necrosis factor-α, interleukin-1, and interferon-γ (25). 2) Activation of protein kinase C results in upregulation of ICAM-1 on endothelial cells (26). 3) AGEs enhance the expression of cell adhesion molecules, including ICAM-1 on vascular endothelial cells (27,28). 4) Shear stress could also stimulate the induction of ICAM-1 (29). We previously reported (9,12) that shear stress, which is increased by hyperfiltration of glomerular endothelial cells, is one of the mediators of ICAM-1 induction in glomeruli. 5) Oxidative stress has been documented to increase ICAM-1 expression (30). 6) Osmotic agents upregulate ICAM-1 expression in rat mesangial cells (31) and in human umbilical vein endothelial cells (32).

Our results showed that the infiltration of macrophages into the glomeruli was lower in diabetic ICAM-1^−/− mice compared with diabetic ICAM-1^+/+ mice (reduction rate 78.5%), suggesting that ICAM-1 is a critical molecule for the infiltration of macrophages into glomeruli in diabetic nephropathy. This finding is consistent with our previous study (12), using anti–ICAM-1 antibody in STZ-induced diabetic rats.

Several adhesion molecules are involved in leukocyte infiltration in inflammatory lesions. VCAM-1, in addition to ICAM-1, is a major adhesion molecule. We examined the expression of VCAM-1 in this study, but VCAM-1 expression in glomeruli was low, and little difference was seen between diabetic ICAM-1^−/− and ICAM-1^+/+ mice. Although we could not detect a compensatory upregulation of VCAM-1, it may be possible that VCAM-1 is involved, at least in part, in macrophage infiltration in diabetic ICAM-1^−/− mice.

Glomerular hypertrophy associated with increased mesangial matrix proteins mainly composed of type IV collagens is a characteristic feature of diabetic nephropathy (33). In this regard, TGF-β is thought to play a central role in the enhancement of glomerular extracellular matrix production in diabetic nephropathy (34,35). Albuminuria is the most established marker of glomerular injury in the early stages of diabetic nephropathy. Increased mesangial matrix, glomerular hypertrophy, and albuminuria were significantly suppressed in diabetic ICAM-1^−/− mice compared with diabetic ICAM-1^+/+ mice. Expression levels of TGF-β and type IV collagen were also lower in diabetic ICAM-1^−/− mice compared with diabetic ICAM-1^+/+ mice. These results suggest that ICAM-1–dependent infiltration of macrophages plays a critical role in the development of diabetic glomerulosclerosis. There was no significant difference in urinary albumin excretion between diabetic ICAM-1^+/+ mice and diabetic ICAM-1^−/− mice at 1 month after induction of diabetes, suggesting that ICAM-1 is not involved in increased albuminuria in the very early stages of diabetic nephropathy. A possible explanation is that albuminuria is related to a rapid increase in urine volume and changes in glomerular hemodynamics over the month after induction of diabetes in this study.

Macrophages produce various types of cytokines (which stimulate cell proliferation) and ECM, and they induce tissue injury. Recent studies have reported that the depletion of leukocytes by irradiation decreased the gene expression of TGF-β and type IV collagen in the glomeruli of diabetic rats at 4 weeks after induction of diabetes, suggesting the pathological role of macrophages in the increased expression of ECM proteins (36). ECMs that include type IV collagen are produced by mesangial cells. With respect to the interaction between macrophages and mesangial cells, Pawluczyk and Harris (37) reported that the culture supernatant of macrophages stimulates mesangial cells to produce fibronectin in vitro. When considering the above findings with the present results, we can speculate that infiltrated glomerular macrophages stimulate mesangial cells to secrete TGF-β through certain cytokines. TGF-β can in turn induce ECM overproduction from mesangial cells in autocrine and paracrine fashions. On the other hand, previous studies (38) have indicated that macrophages themselves can induce TGF-β. Thus, macrophages could secrete TGF-β, which stimulates mesangial cells to produce ECM proteins in diabetic glomeruli.

Interstitial fibrosis is another important histopathological change that correlates with renal function in diabetic nephropathy (39). ICAM-1 is expressed in peritubular capillaries and tubular epithelial cells in ICAM-1^+/+ mice, whereas the density of macrophages in the interstitium and interstitial fibrosis was minimal in diabetic ICAM-1^−/− mice in the present study, suggesting that ICAM-1 plays a major role in interstitial infiltration of macrophages and subsequent interstitial fibrosis.

In conclusion, we have demonstrated that renal and glomerular hypertrophy, mesangial matrix expansion, and albuminuria were ameliorated in diabetic ICAM-1^−/− mice compared with ICAM-1^+/+ mice. These results suggest that ICAM-1 plays a critical role in the progression of diabetic nephropathy.

This study was supported in part by a Grant-in-Aid for Scientific Research (C11671036 and C13671116 to K.S. and 12770582 to M.M.) from the Ministry of Education, Science, Culture, Sports, and Technology of Japan.

We thank Atsuko Yuasa (Okayama Central Hospital, Okayama, Japan) and Toshiyo Hashimoto (Okayama University Graduate School) for excellent technical assistance.