Obesity-Associated NAFLD Coexists with a Chronic Inflammatory Kidney Condition That Is Partially Mitigated by Short-Term Oral Metformin

Abstract

Background/Objectives: Chronic kidney disease (CKD) is twice as prevalent in individuals with obesity-associated non-alcoholic fatty liver disease (Ob-NAFLD), highlighting the need to determine the link and mechanisms of kidney injury as well as explore therapies. Metformin, a first-line treatment for type 2 diabetes, shows promise in managing NAFLD, but its renal benefits in Ob-NAFLD remain unclear. This study investigates the impact of Ob-NAFLD on kidney injury and assesses the potential protective effects of metformin. Methods: Five-week-old female Zucker rats (obese fa/fa and lean Fa/Fa) were fed an AIN-93G diet for 8 weeks to induce Ob-NAFLD, then fed the diet with Metformin for 10 weeks. Kidneys were collected for histopathological and biochemical analyses. Results: Histopathological studies showed increased tubular injury, mesangial matrix expansion, and fibrosis in kidneys with Ob-NAFLD compared to lean control (LC) rats. Immunohistochemistry further revealed an elevated macrophage and neutrophil infiltration and increased levels of nitrotyrosine and p22phox in Ob-NAFLD kidneys. Furthermore, Ob-NAFLD rat kidneys showed upregulation of TNF-α and CCL2 genes and increased levels of caspase-3 (total and cleaved). Interestingly, metformin treatment significantly decreased TNF-α mRNA and blunted nitrotyrosine levels, and modestly reduced immune cell infiltration in Ob-NAFLD. Conclusions: These findings indicate that Ob-NAFLD promotes CKD as evidenced by tubular injury, oxidative stress, inflammation, and fibrosis. While short-term metformin treatment showed anti-oxidative and anti-inflammatory effects in Ob-NAFLD, its impact on structural kidney damage was limited, highlighting the need for longer treatment or alternative therapeutics such as oxidant scavengers and anti-inflammatory drugs to effectively mitigate renal pathologies.

Keywords: inflammation; kidney injury; metformin; obesity-NAFLD; oxidative stress.

Figure 1

Effects of Ob-NAFLD and metformin treatment on rat kidney histopathology. Rat kidneys were isolated and processed for formalin-fixed paraffin-embedded sections. Three groups, namely lean control (LC), obesity associated NAFLD (Ob-NAFLD), and metformin treated Ob-NAFLD (Ob-NAFLD + M) were considered for histological evaluations. (A) Representative images of Periodic Acid–Schiff (PAS) (a–h) and Masson’s Trichrome-stained kidney sections (i–l). (a,e,i) LC rats showing normal renal histology with intact tubular structure (a), no mesangial expansion (e), and absence of fibrosis (i). (b,f,j) Metformin-treated lean rats exhibiting tubular injury (b), no mesangial matrix expansion (f), and absence of interstitial fibrosis (j). (c,g,k) Ob-NAFLD rats exhibiting evident kidney damage, including tubular injury (c), mesangial matrix expansion (g), and interstitial fibrosis (k). (d,h,l) Metformin-treated Ob-NAFLD rat kidneys showing pathological features similar to untreated Ob-NAFLD rats, including moderate tubular injury (d), mesangial expansion (h), and fibrosis (l), indicating a lack of renal protective effect from short-term metformin treatment. Scale bars = 100 μm. (B–D) Graphs showing pathological evaluation performed by a blinded renal pathologist. (B) Tubular injury scores, (C) mesangial matrix expansion score, and (D) interstitial fibrosis score across experimental groups. Data are expressed as mean ± SEM (n = 5 per group). Statistical significance between the two groups was determined by a non-parametric Mann–Whitney U test (* p < 0.05; ** p <0.01).

Figure 2

Evaluation of oxidative stress in the kidneys of Ob-NAFLD rats with or without metformin treatment. Rat kidney sections from lean control (LC), lean treated with metformin (L + M), obesity associated NAFLD (Ob-NAFLD), and metformin-treated Ob-NAFLD (Ob-NAFLD + M) groups were employed for immunohistochemistry of nitrotyrosine (a marker of reactive oxygen species). (A) Representative immunohistochemical images of nitrotyrosine staining (a–d). Scale bars = 100 μm. (B) Graph showing semi-quantitative analysis of nitrotyrosine levels across experimental groups. Data are expressed as mean ± SEM (n = 5 per group). Statistical significance was determined by two-way ANOVA (* p < 0.05).

Figure 3

Immune cell infiltration in the kidneys of Ob-NAFLD rats with or without metformin treatment. Rat kidney sections from lean control (LC), lean treated with metformin (L + M), obesity associated NAFLD (Ob-NAFLD), and metformin-treated Ob-NAFLD (Ob-NAFLD = M) groups were employed for immunohistochemistry of CD68, a macrophage marker, and neutrophil elastase, a neutrophil marker. (A) Representative immunohistochemistry images showing CD68+ cells in rat kidneys of the following: (a) LC; (b) L + M; (c) Ob-NAFLD; and (d) Ob-NAFLD + M groups. Scale bars = 100 μm. (B) Representative immunohistochemistry images for neutrophil elastase+ cells in rat kidneys of the following: (a) LC; (b) L + M; (c) Ob-NAFLD; and (d) Ob-NAFLD + M groups. Scale bars = 100 μm. (C) Graph showing quantification of CD68+ cells within 10 microscopic fields (40×) across experimental groups. (D) Graph showing quantification of neutrophil elastase+ cells within 10 microscopic fields (40×) across experimental groups. Data are expressed as mean ± SEM (n = 5 per group). Statistical significance between the two groups was determined by a non-parametric Mann–Whitney U test (* p < 0.05; ** p < 0.01).

Figure 4

Expression of p22phox, a subunit of NADPH oxidase, in kidney tissues of Ob-NAFLD rats. Rat kidney sections from lean control (LC), lean treated with metformin (L + M), obesity associated NAFLD (Ob-NAFLD), and metformin-treated Ob-NAFLD (Ob-NAFLD = M) groups were employed for immunohistochemistry of p22phox, a subunit of NAPDH complex and a marker of reactive oxygen species production. (A) Representative immunohistochemistry images showing p22phox+ cells in rat kidneys of the following: (a) LC; (b) L + M; (c) Ob-NAFLD; and (d) Ob-NAFLD + M groups. Scale bars = 100 μm. (B) Graph showing quantification of p22phox+ cells within 10 microscopic fields (40×) across experimental groups. Data are expressed as mean ± SEM (n = 5 per group). Statistical significance between the two groups was determined by a non-parametric Mann–Whitney U test (** p < 0.01).

Figure 5

Antioxidant enzyme levels in the kidneys of Ob-NAFLD rats and the impact of metformin treatment. RIPA lysates were prepared from rat kidney homogenates followed by SDS-PAGE Western blotting. (A) Representative Western blot images of glutathione-S-transferase-P (GST-P), Cu/Zn-superoxide dismutase (Cu/Zn-SOD), and Mn-superoxide dismutase (Mn-SOD) in renal lysates from lean control (LC) and obesity associated NAFLD (Ob-NAFLD) groups. (B-D) Graphs showing densitometry analysis of protein bands of GST-P (B), CuZnSOD (C), and MnSOD (D) normalized to β-actin. (E) Representative Western blot images of glutathione-S-transferase-P (GST-P), Cu/Zn-superoxide dismutase (Cu/Zn-SOD), and Mn-superoxide dismutase (Mn-SOD) in renal lysates from lean treated with metformin (L + M), Ob-NAFLD, and metformin-treated Ob-NAFLD (Ob-NAFLD + M) groups. (F-H) Graphs showing densitometry analysis of protein bands of GST-P (F), CuZnSOD (G), and MnSOD (H) normalized to β-actin. Data are presented as mean ± SEM (n = 5 per group). Statistical significance between the two groups was determined by a non-parametric Mann–Whitney U test (* p < 0.05, ** p < 0.01).

Figure 6

Expression of inflammatory cytokine and chemokine genes in the kidneys of Ob-NAFLD rats with or without metformin treatment. Kidney tissues from lean control (LC), lean treated with metformin (L + M), Ob-NAFLD, and Ob-NAFLD treated with metformin (Ob-NAFLD + M) groups were used for mRNA isolation followed by complementary DNA (cDNA) synthesis. (A–C) SYBR green PCR was used to quantify gene expression of tumor necrosis factor (TNF-α), a pro-inflammatory cytokine, chemokine ligand 2 (Ccl2), a chemoattractant for immune cell infiltration, and the CXC chemokine receptor 2 (CXCR2). Gene expression levels were normalized to β-actin and expressed as fold change relative to the LC group. Graphs showing quantitative real-time PCR analysis of TNF-α (A), Ccl2 (B), and CXCR2 (C) mRNA expression. Data are presented as mean ± SEM (n = 5 per group). Statistical analysis was performed using two-way ANOVA (* p < 0.05; **p < 0.01).

Figure 7

Effects of Ob-NAFLD on renal apoptosis and the impact of metformin treatment. RIPA lysates were prepared from rat kidney homogenates, followed by SDS-PAGE Western blotting. (A) Representative Western blot images of total caspase-3 and cleaved caspase-3 proteins in renal lysates from lean control (LC) and obesity associated NAFLD (Ob-NAFLD) groups. (B,C) Graphs showing densitometry analysis of protein bands of total caspase-3 (B) and cleaved caspase-3 proteins (C) normalized to β-actin. (D) Representative Western blot images of total caspase-3 and cleaved caspase-3 proteins in renal lysates from lean treated with metformin (L + M), Ob-NAFLD, and metformin-treated Ob-NAFLD (Ob-NAFLD + M) groups. (E,F) Graphs showing densitometry analysis of protein bands of total caspase-3 (E) and cleaved caspase-3 proteins (F) normalized to β-actin. Data are presented as mean ± SEM (n = 5 per group). Statistical significance between the two groups was determined by a non-parametric Mann–Whitney U test (* p < 0.05; **p < 0.01).