A high-salt diet enhances leukocyte adhesion in association with kidney injury in young Dahl salt-sensitive rats

Abstract

Salt-sensitive hypertension is associated with severe organ damage. Generating oxygen radicals is an integral component of salt-induced kidney damage, and activated leukocytes are important in oxygen radical biosynthesis. We hypothesized that a high-salt diet causes the upregulation of immune-related mechanisms, thereby contributing to the susceptibility of Dahl salt-sensitive rats to hypertensive kidney damage. For verifying the hypothesis, we investigated leukocytes adhering to retinal vessels when Dahl salt-sensitive rats were challenged with a high-salt (8% NaCl) diet using acridine orange fluoroscopy and a scanning laser ophthalmoscope. The high-salt diet increased leukocyte adhesion after 3 days and was associated with a significant increase in mRNA biosynthesis of monocyte chemotactic protein-1 and intercellular adhesion molecule-1 (ICAM-1) -related molecules in the kidney. Losartan treatment did not affect increased leukocyte adhesion during the early, pre-hypertensive phase of high salt loading; however, losartan attenuated the adhesion of leukocytes during the hypertensive stage. Moreover, the inhibition of leukocyte adhesion in the pre-hypertensive stage by anti-CD18 antibodies decreased tethering of leukocytes and was associated with the attenuation of functional and morphological kidney damage without affecting blood pressure elevation. In conclusion, a high-salt challenge rapidly increased leukocyte adhesion through the over-expression of ICAM-1. Increased leukocyte adhesion in the pre-hypertensive stage is responsible for subsequent kidney damage in Dahl salt-sensitive rats. Immune system involvement may be a key component that initiates kidney damage in a genetic model of salt-induced hypertension.

Figure 1

(a) Effects of a high salt load on leukocyte adhesion to retinal vessels. The number of leukocytes adhered to retinal vessels was determined using acridine orange fluorescence (see text for details). Open bars represent Dahl S rats fed the low-salt diet, and solid bars represent Dahl S rats fed the high-salt diet. The high-salt diet was given to 4-week-old rats. Rats were tested 3 days after loading and then weekly for 4 weeks. Differences were assessed using the Mann–Whitney U-test with Bonferroni correction. *P<0.01 and ^†P<0.001 vs. the low-salt group at each time point. (b) Characterization of leukocytes adhered to retinal vessels. Cells were stained with fluorescein isothiocyanate conjugated with ConA (top, green), and anti-CD18, CD11a, CD11b and CD45 antibodies (middle, red). Signals of ConA were consistent with those of the antibodies (bottom, Merge), thereby indicating that the ConA-positive cells detected were leukocytes loaded with CD18, CD11a, CD11b and CD45 antigens. Arrowheads indicate leukocytes adhered to the retinal artery. Scale bar, 20 μm. (c) Adhesion molecule mRNA expression in the kidney. Adhesion molecule mRNA expression in the kidney of 7-week-old Dahl S rats was determined 3 weeks after salt loading. Open bars represent Dahl S rats fed the low-salt diet (n=5), and solid bars represent Dahl S rats fed the high-salt diet (n=5). The mRNA content is expressed relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA in the kidney tissue. Differences were assessed using the Kruskal–Wallis test followed by Mann–Whitney U-test with Bonferroni correction. *P<0.02 and ^†P<0.01 vs. Dahl S rats fed the low-salt diet.

Figure 2

(a) Influence of angiotensin receptor antagonism on leukocyte adhesion. Leukocytes adhered to retinal vessels were measured using acridine orange fluorescence (see text for details). Experimental groups: 0.3%DS, Dahl S rats fed a low-salt (0.3%) diet; 8%DS-control, Dahl S rats fed a high-salt (8% NaCl) diet; 8%DS-early, Dahl S rats fed a high-salt diet and treated with losartan for the first 10 days only; 8% DS-late, Dahl S rats fed a high-salt diet and treated with losartan for the last 10 days only; 8%DS-whole, Dahl S rats fed a high-salt diet and treated with losartan throughout the experiment. Differences were assessed using the Kruskal–Wallis test followed by the Mann–Whitney U-test with Bonferroni correction. *P<0.05. (b) Determination of mRNA of adhesion molecules in the kidneys. mRNA levels of adhesion molecules in the kidneys were quantitatively determined by real-time PCR as described in the text. Tissue mRNA contents were standardized relative to the content in Dahl S rats fed a low-salt diet alone. Differences were assessed using the Kruskal–Wallis test followed by the Mann–Whitney U-test with Bonferroni correction. *P<0.05.

Figure 3

Control (LS) represents Dahl S rats fed a low-salt (0.3% NaCl), HS-IgG represents Dahl S rats fed a high-salt (8% NaCl) diet and given a nonspecific IgG fraction, and HS-Anti-CD18 represents Dahl S rats fed a high-salt diet and given an active anti-CD18-specific antibody. (a) Blocking of leukocyte adhesion by anti-CD-specific antibody. We evaluated the effects of anti-CD18 antibody on leukocytes adhered to the arterioles and the smaller veins 1 and 3 weeks after the start of the experiment. Differences were assessed using the Kruskal–Wallis test followed by the Mann–Whitney U-test with Bonferroni correction. *P<0.025, ^†P<0.005. (b) Urinary protein excretion. Differences were assessed using the one-way ANOVA followed by post-hoc analysis using the Scheffe test. *P<0.0001, ^†P<0.01. (c) Creatinine clearance rate. Differences were assessed using the one-way ANOVA followed by post-hoc analysis using the Scheffe test. *P<0.0001, ^†P<0.01, ^‡P<0.05. (d) Glomerular sclerosis pattern 1 week after commencement of the experiment. (e) Glomerular sclerosis pattern 3 weeks after commencement of the experiment. (f) Glomerular sclerosis score. Left and right scales show scores 1 week and 3 weeks after commencement of the experiment, respectively. Differences were assessed using the one-way ANOVA followed by post-hoc analysis using the Scheffe test. *P<0.005, ^†P<0.02, ^‡P<0.025. (g) Correlation between the number of leukocytes adhering to the arterioles and glomerular sclerosis score 3 weeks after commencement of the experiment. Correlation was performed using simple Pearson correlations. ANOVA, analysis of variance.