Exposure to High-Fat Style Diet Induced Renal and Liver Structural Changes, Lipid Accumulation and Inflammation in Intact and Ovariectomized Female Rats

Abstract

Purpose: We hypothesized that low estrogen levels aggravate obesity-related complications. Diet-induced obesity can cause distinct pathologies, including impaired glucose tolerance, inflammation, and organ injury that leads to fatty liver and chronic kidney diseases. To test this hypothesis, ovariectomized (OVX) rats were fed a high-fat style diet (HFSD), and we examined structural changes and inflammatory response in the kidney and liver.

Methods: Sprague-Dawley female rats were ovariectomized or sham-operated and divided into four groups: sham-operated rats fed a normal diet (ND); ovariectomized rats fed a normal diet (OVX-ND); sham-operated rats fed a HFSD; ovariectomized rats fed a high-fat style diet (OVX-HFSD). Mean blood pressure and fasting blood glucose were measured on weeks 0 and 10. The rats were sacrificed 10 weeks after initiation of ND or HFSD, the kidney and liver were harvested for histological, immunohistochemical and immunofluorescence studies.

Results: HFSD-fed rats presented a significantly greater adiposity index compared to their ND counterparts. Liver index, fasting blood glucose and mean blood pressure was increased in OVX-HFSD rats compared to HFSD rats at study terminal. Histological and morphometric studies showed focal interstitial mononuclear cell infiltration in the kidney of HFSD rats with mesangial expansion being greater in the OVX-HFSD rats. Both HFSD fed groups showed increased expressions of renal inflammatory markers, namely TNF-alpha, IL-6 and MCP-1, and infiltrating M1 macrophages with some influence of ovarian hormonal status. HFSD-feeding also caused hepatocellular steatosis which was aggravated in ovariectomized rats fed the same diet. Furthermore, hepatocellular ballooning was observed only in the OVX-HFSD rats. Similarly, HFSD-fed rats showed increased expressions of the inflammatory markers and M1 macrophage infiltration in the liver; however, only IL-6 expression was magnified in the OVX-HFSD.

Conclusion: Our data suggest that some of the structural changes and inflammatory response in the kidney and liver of rats fed a HFSD are exacerbated by ovariectomy.

Keywords: hepatic steatosis; macrophage infiltration; mesangial expansion; obesity; ovariectomy.

Figure 1

Adiposity (A) and kidney indices (B) of normal diet (ND)- and high fat style diet (HFSD)-fed rats with and without ovariectomy (OVX). Data are expressed as mean ± SEM (n = 12 per group). ****P < 0.001 vs. ND; ^#P < 0.05 or ^####P < 0.001 vs. OVX-ND.

Figure 2

Fasting blood glucose (A) and mean blood pressure (B) of high-fat style diet (HFSD)-fed intact and ovariectomized rats (OVX). Data are expressed as mean ± SEM (n = 12 per group). ^####P < 0.001 vs. OVX-ND, ^δδP < 0.01 or ^δδδδP < 0.001 vs. HFSD, ^ϕϕϕϕP < 0.001 vs. week 0 of the corresponding group. FBG, fasting blood glucose; MBP, mean blood pressure.

Figure 3

Mesangial expansion (A) and mononuclear cell infiltration (B) of PAS stained kidneys of high fat style diet (HFSD)-fed rats, with and without ovariectomy (OVX) (n = at least 4 per group). Red arrow indicates the mononuclear cell infiltration. Magnification: 400×.

Figure 4

Hepatic steatosis (H&E stained) (A), lipid accumulation (Oil Red O stained) (B), lobular infiltrates (C), hepatocytes ballooning (D) in H&E stained liver of high fat style diet (HFSD)-fed rats, with and without ovariectomy (OVX) (n = at least 4 per group). Magnification: 8× (A), 10× (B). Yellow color closed arrow indicates microvesicular steatosis and blue color closed arrow indicates macrovesicular steatosis.

Figure 5

Quantification of mean pixel intensities of Oil Red O staining in liver (A) and liver index (B) of normal diet (ND)- and high fat style diet (HFSD)-fed rats with and without ovariectomy (OVX) (n = 12 per group). **P < 0.01 or ****P < 0.001 vs. ND; ^####P < 0.001 vs. OVX-ND; ^δδP < 0.01 or ^δδδP < 0.005 vs. HFSD.

Figure 6

Immunohistochemical staining of IL-6 in kidneys in the cortex, outer medulla and inner medulla regions (A) and quantification of mean pixel intensities of kidney cortical IL-6 staining (B) of normal diet (ND)- and high fat style diet (HFSD)-fed rats, with and without ovariectomy (OVX) (n = 12 per group) at 40X magnification. Dark red arrow indicates the positive immunohistochemical stainings. *P < 0.05 or **P < 0.01 vs. ND; ^####P < 0.001 vs. OVX-ND; ^δδδδP < 0.001 vs. HFSD.

Figure 7

Immunohistochemical staining of TNF-α in kidneys in the cortex, outer medulla and inner medulla regions (A) and quantification of mean pixel intensities of kidney cortical TNF- α staining (B) of normal diet (ND)- and high fat style diet (HFSD)-fed rats, with and without ovariectomy (OVX) (n = 12 per group) at 40X magnification. Dark red arrow indicates the positive immunohistochemical stainings. ****P < 0.001 vs. ND; ^####P < 0.001 vs. OVX-ND; ^δδδδP < 0.001 vs. HFSD.

Figure 8

Immunohistochemical staining of MCP-1 in kidneys at the cortex, outer medulla and inner medulla regions (A) and quantification of mean pixel intensities of kidney cortical MCP-1 staining (B) of normal diet (ND)- and high fat style diet (HFSD)-fed rats, with and without ovariectomy (OVX) (n = 12 per group) at 40X magnification. Dark red arrow indicates the positive immunohistochemical stainings. **P < 0.01 vs. ND; ^####P < 0.001 vs. OVX-ND; ^δδδδP < 0.001 vs. HFSD.

Figure 9

Double immunofluorescence staining of p- NF-κB (indicated in green color) and NF-κB (indicated in red color) (A) in kidneys, and quantification of mean pixel intensities of kidney cortical p-NF-κB (B) and NFκB (C) of normal diet (ND)- and high fat style diet (HFSD)-fed rats, with and without ovariectomy (OVX) (n = 12 per group) at 40X magnification. p-NF-κB, phosphorylated nuclear factor kappa-B; NF-κB, total nuclear factor kappa-B. *P < 0.05, **P < 0.01 or ***P < 0.005 vs. ND; ^####P < 0.001 vs. OVX-ND.

Figure 10

Immunohistochemical staining of IL-6 (A), TNF-α (B) and MCP-1 in liver (C) and quantification of mean pixel intensities of hepatic IL-6 (D), TNF-α (E) and MCP-1 (F) staining of normal diet (ND)- and high fat style diet (HFSD)-fed rats, with and without ovariectomy (OVX) (n = 12 per group) at 40X magnification. Dark red arrow indicates the positive immunohistochemical stainings. **P < 0.01 or ****P < 0.001 vs. ND; ^##P < 0.01 or ^####P < 0.001 vs. OVX-ND; ^δP < 0.05 vs. HFSD.

Figure 11

Immunofluorescence staining of M1 (indicated in red color fluorescent) (A), M2 (indicated in green color fluorescent) (B) macrophages in kidneys, and quantification of mean pixel intensities of kidney cortical M1 (C) and M2 markers (D) of normal diet (ND)- and high fat style diet (HFSD)-fed rats, with and without ovariectomy (OVX) (n = 12 per group) at 40X magnification. ****P < 0.001 vs. ND; ^####P < 0.001 vs. OVX-ND; ^δδδδP < 0.001 vs. HFSD.

Figure 12

Immunofluorescence staining of M1 (indicated in red color) (A), M2 (indicated in green color) (B) macrophages in liver, and quantification of mean pixel intensities of hepatic M1 (C) and M2 markers (D) of normal diet (ND)- and high fat style diet (HFSD)-fed rats, with and without ovariectomy (OVX) (n = 12 per group) at 40X magnification. ****P < 0.001 vs. ND; ^####P < 0.001 vs. OVX-ND.