Docosahexaenoic acid-enriched fish oil attenuates kidney disease and prolongs median and maximal life span of autoimmune lupus-prone mice

Abstract

The therapeutic efficacy of individual components of fish oils (FOs) in various human inflammatory diseases still remains unresolved, possibly due to low levels of n-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) or lower ratio of DHA to EPA. Because FO enriched with DHA (FO-DHA) or EPA (FO-EPA) has become available recently, we investigated their efficacy on survival and inflammatory kidney disease in a well-established animal model of human systemic lupus erythematosus. Results show for the first time that FO-DHA dramatically extends both the median (658 d) and maximal (848 d) life span of (NZB x NZW)F1 (B x W) mice. In contrast, FO-EPA fed mice had a median and maximal life span of approximately 384 and 500 d, respectively. Investigations into possible survival mechanisms revealed that FO-DHA (versus FO-EPA) lowers serum anti-dsDNA Abs, IgG deposition in kidneys, and proteinuria. Further, FO-DHA lowered LPS-mediated increases in serum IL-18 levels and caspase-1-dependent cleavage of pro-IL-18 to mature IL-18 in kidneys. Moreover, FO-DHA suppressed LPS-mediated PI3K, Akt, and NF-kappaB activations in kidney. These data indicate that DHA, but not EPA, is the most potent n-3 fatty acid that suppresses glomerulonephritis and extends life span of systemic lupus erythematosus-prone short-lived B x W mice, possibly via inhibition of IL-18 induction and IL-18-dependent signaling.

Figure 1

Effect of CO, FO-18/12, FO-EPA and FO-DHA diets on median and maximal lifespan of short-lived SLE-prone (NZB × NZW) F1 female mice. At 2-months of age, mice were switched to semi-purified diets containing 10% corn oil (control), and fish oils (FO) enriched in eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA): 1) 18/12 fish oil (FO-18/12) 2) 55/5 EPA-enriched FO (FO-EPA) and 3) 5/60 DHA-enriched FO (FO-DHA). Survival rate was monitored from 2 months onwards. Our results show that (A) FO-DHA significantly increases lifespan of (NZB × NZW)F1 mice compared to FO-EPA and CO control. (B) Both maximum and median survival rates were also increased in FO-DHA fed mice compared to FO-EPA fed mice. Values are expressed as percentage of mice in each dietary group alive. Results were analyzed by Logrank followed by Chi square test.

Figure 2. Effect of CO, FO-18/12, FO-EPA and FO-DHA diets on serum anti-dsDNA antibodies and IgG deposition in kidneys of (NZB × NZW)F1 female mice

At 2-months of age, female (NZB × NZW)F1 mice were fed semi-purified diets containing 10% corn oil (control), and fish oils (FO) enriched in eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA): 1) 18/12 fish oil (FO-18/12), 2) 55/5 EPA-enriched FO (FO-EPA) and 3) 5/60 DHA-enriched FO (FO-DHA). At 5-months of age, mice were challenged with LPS intraperitonelly, and serum anti-dsDNA antibodies were quantified by ELISA. Histological evaluation of IgG deposition was scored on a scale of 0–3 (0, none; 1, weak; 2, moderate; 3, strong) based on fluorescence intensity within glomerular capillary walls (10 glomeruli/mouse and 6 mice/group). (A) FO-DHA fed mice exhibit significantly lower anti-dsDNA antibodies in serum (P<0.01, ANOVA). (B) Representative photomicrographs of fluorescent stained kidneys indicate lower IgG deposition in FO-DHA fed mice compared to FO-EPA and CO fed mice. (C) Histological evaluation of kidney demonstrates significantly decreased IgG deposition in FO-DHA fed mice compared to CO, FO-18/12 and FO-EPA fed mice. Different signs (*†, † and *) indicates significant differences (P< 0.05; ANOVA followed by Newman-Keuls test).

Figure 3. Effect of CO, FO-18/12, FO-EPA and FO-DHA diets on LPS-induced serum IL-18 levels, kidney caspase-1 activity, and pro/mature IL-18 expression in (NZB × NZW)F1 female mice

At 2-months of age, mice were switched to semi-purified diets containing 10% corn oil (control), and fish oils (FO) enriched in eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA): 1) 18/12 fish oil (FO-18/12) 2) 55/5 EPA-enriched FO (FO-EPA) and 3) 5/60 DHA-enriched FO (FO-DHA). At 5 months of age, mice (n=5) were challenged with LPS intraperitonelly to evaluate the IL-18 expression profile in serum and kidney. PBS served as a solvent control. (A) FO-DHA fed mice significantly lowered LPS-induced serum IL-18 levels compared to CO, FO-18/12, FO-EPA and FO-DHA fed mice. (B) LPS-induced caspase-1 activity in kidneys was significantly decreased in FO-DHA fed mice compared to CO, FO-18/12 and FO-EPA fed mice. (C) FO-DHA fed mice expressed lower levels of LPS-induced mature IL-18 in kidneys compared to CO, FO-18/12 and FO-EPA fed mice. (D) Densitometric analysis of pro IL-18 and mature IL-18 after LPS-injection in mice. Results are representative of two independent experiments. Data are expressed as mean ± SEM. Results were analyzed by ANOVA followed by Newman-Keuls test. *LPS-treated FO-DHA fed mice showed significantly (P<0.05) decreased pro IL-18 levels, Caspase-1 activity and mature-IL-18 expression compared to CO, FO-18/12 and FO-EPA fed mice. Different signs (*†, † and *) indicate a significant difference (P<0.05, ANOVA followed by Newman-Keuls test) in LPS-injected groups. Similar sign assigned groups indicates no significant difference between the groups.

Figure 4

Effect of CO, FO-18/12, FO-EPA and FO-DHA diets on PI3K/Akt kinase pathway in (NZB × NZW)F1 female mice. LPS challenged kidney PI3K and Akt activations were determined in mice fed with CO, FO-18/12, FO-EPA and FO-DHA for 3 months. PI3K activation was analyzed by PI3K lipid kinase assays using p85 immunoprecipitates. Akt activation was analyzed by immunoblotting using whole cell homogenates and activation-specific antibodies, and by using a commercially available kinase assay that quantifies Akt-induced glycogen synthase kinase-3 (GSK-3) phosphorylation at Ser21/9. (A) Our results indicated that FO-DHA diet significantly suppressed PI3K activation in kidneys of LPS-injected mice. (B) Western Blot showing significant inhibition of phospho-Akt in kidneys of LPS-injected FO-DHA fed mice. (C) Similarly, immune-complex kinase assays demonstrated significant inhibition of Akt kinase activity in kidneys of LPS-injected FO-DHA fed mice. Results are representative of three independent experiments.

Figure 5

Effect of CO, FO-18/12, FO-EPA and FO-DHA diets on NF-κB activation. Female (NZB × NZW)F1 mice were fed with CO, FO-18/12, FO-EPA and FO-DHA for 3 months, and then challenged with LPS (5 mg/kg body weight, IP) for 4 hrs. Kidneys were harvested and analyzed for NF-κB DNA binding activity by EMSA using nuclear protein extracts (A). Activation of NF-κB was confirmed by analyzing nuclear p65 levels immunoblotting. Immunoblotting (B). Actin served as a loading control. The results show significant inhibition of LPS-mediated NF-κB DNA binding activity (A) and nuclear p65 translocation (B) in FO-DHA-fed mice.