A blueberry-enriched diet improves renal function and reduces oxidative stress in metabolic syndrome animals: potential mechanism of TLR4-MAPK signaling pathway

Abstract

Background: Metabolic syndrome (MetS) is characterized by a cluster of health factors that indicate a higher risk for cardio-renal diseases. Recent evidence indicates that antioxidants from berries are alternative to attenuate oxidative stress and inflammation. We tested the hypothesis that inflammation-induced renal damage is triggered by the activation of TLR4, and subsequent modulation of redox-sensitive molecules and mitogen-activated protein kinase (MAPK) pathway.

Methods: Five-week old lean and obese Zucker rats (LZR and OZR) were fed a blueberry-enriched diet or an isocaloric control diet for 15 weeks. A glucose tolerance test and acute renal clearance experiments were performed. Gene and protein expression levels for TLR4, cytokines and phosphorylation of ERK and p38MAPK were measured. Kidney redox status and urinary albumin levels were quantified. Renal pathology was evaluated histologically.

Results: Control OZR exhibited lower glucose tolerance; exacerbated renal function parameters; increased oxidative stress. Gene and protein expression levels of TLR4 were higher and this was accompanied by increased renal pathology with extensive albuminuria and deterioration in antioxidant levels in OZR. In addition, OZR had increased phosphorylation of ERK and p38MAPK. Blueberry-fed OZR exhibited significant improvements in all these parameters compared to OZR.

Conclusion: TLR4-MAPK signaling pathway is a key to the renal structural injury and dysfunction in MetS and blueberry (BB) protect against this damage by inhibiting TLR4.

Significance: This is the first study to put forth a potential mechanism of TLR4-induced kidney damage in a model of MetS and to elucidate a downstream mechanism by which blueberry exert their reno-protective effects.

Figure 1. Effect of BB feeding on glucose sensitivity in MetS animals.

The glucose sensitivity of rats from all experimental groups as assessed by glucose sensitivity assay (n = 7 per group). All values are presented as mean ± SEM (*p<0.05, **p<0.01).

Figure 2. Effect of BB on renal hemodynamic dysfunctions in MetS animals.

The kidney function of rats from all experimental groups as assessed by renal clearance experiments (n = 6–9 per group) (A) BB supplementation significantly decreased the MAP in obese zucker rats (B) MetS animals exhibited a significantly reduced GFR. BB pretreatment improved the GFR in MetS animals. (C) MetS animals had a reduced renal blood flow. BB treatment normalized the renal blood flow in these animals. (D) The RVR was increased in MetS animals and BB prevented this increase in RVR in them. All values are presented as mean ± SEM (*p<0.05, **p<0.01, ***p<0.001).

Figure 3. Effect of BB on renal renin-angiotensin system in MetS animals.

The mRNA expression of (A) ACE, (B) ACE2, (C) AT1 and (D) AT2 in the renal cortical tissue. The gene expression of ACE and AT1 were significantly increased and ACE2 and AT2 were reduced in MetS rats. The BB enriched diet was able to attenuate these changes in renin-angiotensin system gene expression. All values are presented as mean ± SEM (*p<0.05, **p<0.01, ***p<0.001).

Figure 4. Effect of BB on renal pathology in MetS animals.

(A) Representative images of the renal pathology of rats from all experimental groups as assessed by Masson’s trichrome staining of the kidney sections (n = 6). There are no significant renal structural changes visible in LZRCC and LZRBB groups. However, renal changes in the OZRCC image are evident in glomeruli, tubules and interstitium. There is moderate glomerular fibrosis; tubular dilatation and attenuation of lining epithelium with intraluminal hyaline casts; tubular atrophy; multifocal to coalescing moderate to severe interstitial fibrosis; multifocal interstitial, moderate to severe lymphocytic and plasmacytic nephritis. OZRBB animals exhibited less pathological changes with minimal interstitial fibrosis.

Figure 5. Effect of BB on renal pathology in MetS animals (pathological scoring).

(A) Relative pathological scoring of glomerular sclerosis, interstitial nephritis and proteinuria as evaluated by a veterinary pathologist who was blinded to experimental conditions (n = 6). (B) Urinary albumin levels (mg/dl) in rats from all experimental groups as determined by the albumin assay kit.

Figure 6. Effect of BB on renal TLR4 gene and protein expression in MetS animals.

The (A) mRNA (n = 6) and (B) a representative western blot of TLR4 in the renal cortical tissues. (C) Bands were analyzed and quantified by densitometry (n = 6). The gene and protein expression of TLR4 was increased in the MetS rats. The pretreatment with BB was able to inhibit the increased expression of TLR4 in MetS animals and was comparable to the LZR controls. All values are presented as mean ± SEM (*p<0.05, **p<0.01).

Figure 7. Effect of BB on MAPK signaling in MetS animals.

The protein expression of total and phosphorylated MAPK (ERK and p38) was determined by western blotting. (A) A representative western blot showing ERK and p38MAPK protein expression. Western bands for ERK (B) and p38MAPK (C) were analyzed and quantified by densitometry. All values are presented as mean ± SEM (*p<0.05, **p<0.01, ***p<0.001).

Figure 8. Effect of BB on the renal inflammatory profile in MetS animals.

The (A) mRNA expression (n = 6) of IL-1β, IL-18 and TGF-β in the renal cortical tissues. (B) DNA binding activity of NFκB p65 subunit in renal cortical tissues of rats from each experimental group (n = 6), as determined by ELISA. Increased NFκB p65 DNA binding activity in MetS animals was significantly reduced by BB treatment. (C) A representative western blot showing IL-1β and IL-18 protein expression. Western blot bands for IL-1β (D) and IL-18 (E) were analyzed and quantified by densitometry. All values are presented as mean ± SEM (*p<0.05, **p<0.01).

Figure 9. Effects of BB on antioxidant enzymes and Nrf2-Keap1 signaling in MetS animals.

Renal cortical catalase (A) and SOD (B) levels as determined by commercially available kits (n = 6). (C) A representative western blot showing Nrf2 and Keap 1 protein expression. Western bands for Nrf2 (D) and Keap1 (E) were analyzed and quantified by densitometry. All values are presented as mean ± SEM (*p<0.05, **p<0.01, ***p<0.001).