Dietary addition of magnesium hydride nanoparticles: a breakthrough in combating high-fat diet-induced chronic kidney disease

Abstract

A substantial body of evidence indicates a positive correlation between dyslipidemia and an elevated risk of chronic kidney disease, with renal interstitial fibrosis frequently serving as a common pathway in the advanced stages of chronic kidney disease progression. Hydrogen has anti-inflammatory and antioxidant properties, and magnesium hydride nanoparticle is a material with high hydrogen storage capacity. Magnesium hydride -fortified feed is capable of releasing hydrogen gas steadily and continuously within the digestive tract. A 12-week high-fat diet significantly elevated the serum urea and creatinine levels in mice. In contrast, dietary addition of magnesium hydride demonstrated a notable protective effect against pathological conditions. Additionally, magnesium hydride -fortified feed was found to reduce renal fibrosis and thereby improve renal function. In support of these findings, an in vitro study utilizing human kidney cortical proximal tubule epithelial cells (HK-2 cells) exposed to palmitic acid under conditions mimicking a high-fat diet confirmed the renoprotective effects of magnesium hydride. Furthermore, the primary target phosphatase and tensin homologue deleted on chromosome 10 and the molecular mechanisms underlying the effects of magnesium hydride, specifically its ability to inhibit the transforming growth factor-beta -Smad family member 2 and 3 (Smad2/3) axis through downregulating the expression of phosphatase and tensin homologue deleted on chromosome 10, were elucidated. Additionally, overexpression of Hes family BHLH transcription factor 1 can negate the beneficial effects of magnesium hydride, suggesting that Hes family BHLH transcription factor 1 may serve as an upstream regulatory target in the context of the effects of magnesium hydride. In conclusion, this study demonstrated that magnesium hydride functions as a safe and effective hydrogen source capable of inhibiting the activation of the transforming growth factor-beta/Smad2/3 and protein kinase B/mechanistic target of rapamycin pathways by increasing the expression of phosphatase and tensin homologue deleted on chromosome 10. This mechanism counteracts the progression of high-fat diet-induced chronic renal damage.

Keywords: chronic kidney disease; high-fat diet; magnesium hydride; phosphatase and tensin homolog deleted on chromosome ten; renal fibrosis.

Figure 1

MgH₂ improves HFD/PA-induced renal injury. (A) Animal experiment chart. (B, C) Variations in CREA and BUN levels in the mice. These indicators increased under HFD conditions and decreased after MgH₂ treatment. The data are expressed as the mean ± SD (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). (D, E) Western blotting analysis of the expression of KIM-1 and NGAL in mouse kidney (D) and HK-2 cells (E). The raw data for the quantitative results are shown in Additional Table 1. (F) HE staining and PAS staining of mouse kidney tissue (400× original magnification). A HFD induces changes in renal pathological structure (arrows), and MgH₂ can improve this effect. BUN: Blood urea nitrogen; Con: control group; CREA: creatinine; H&E: hematoxylin and eosin; HFD: high-fat diet; HM: MgH₂ group; KIM-1: kidney injury molecule-1; MgH₂: magnesium hydride; ND: normal control; NGAL: neutrophil gelatinase-associated lipocalin; PA: palmitic acid; PAS: periodic acid-Schiff; PM: PA + MgH₂ group.

Figure 2

MgH₂ ameliorates lipid accumulation and fibrosis in kidneys induced by HFD/PA (A) Western blot analysis of fibrotic protein expression following MgH₂ intervention in mouse kidneys. The raw data for the quantitative results are shown in Additional Table 1. (B) Variation in Vimentin mRNA expression in the mouse kidney. The data were normalized to those of the ND group. (C) FN immunohistochemistry in mouse kidney tissue (100× original magnification (upper), 400× original magnification (lower)), which increased in the HFD group and decreased after MgH₂ treatment. (D, E) Effects of PA and MgH₂ on HK-2 cell cytotoxicity. The data were normalized to those of the 0 μM group. (F) Fluorescence-based assessment of lipid droplets in HK-2 cells (400× original magnification). PA increased the lipid droplet content in HK-2 cells, and MgH₂ treatment resulted in a reduction in lipid droplets. (G) Differential expression of fibrosis markers in HK2 cells as determined by Western blot analysis. The raw data for the quantitative results are shown in Additional Table 1. (H) Changes in Vimentin mRNA expression in HK-2 cells following MgH₂ intervention. The data were normalized to those of the Con group. The data are expressed as the mean ± SD (n = 6). *P < 0.05, **P < 0.01, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). (I, J) Immunofluorescence analysis of Vimentin and E-cadherin in response to MgH₂ stimulation (400× original magnification). Vimentin was elevated in the PA group, whereas E-cadherin was decreased in the PA group. Both of these parameters partially returned to normal levels after MgH₂ treatment. Con: Control group; DAPI: 4′,6-diamidino-2-phenylindole; FN: fibronectin; HFD: high-fat diet; HM: MgH₂ group; MgH₂: magnesium hydride; ND: normal control; PA: palmitic acid; PM: PA + MgH₂ group.

Figure 3

MgH₂ inhibits the activation of the TGF-β pathway. (A) KEGG enrichment of the top 15 signaling pathways. The raw data are shown in Additional Table 2. (B, C) Molecular immunoblotting results of the TGFβ-Smad2/3 pathway after MgH₂ intervention in in vitro (B) and in vivo (C) experiments. The raw data for the quantitative results are shown in Additional Table 1. (D) Molecular immunofluorescence results of TGF-β in HK-2 cells after MgH₂ intervention (400× original magnification). TGF-β increased after PA treatment but decreased after MgH₂ treatment. (E) Molecular immunofluorescence results of Smad2 in HK-2 cells after MgH₂ intervention (400× original magnification). Smad2 levels increased after PA treatment but decreased after MgH₂ treatment. (F) TGF-β immunohistochemistry in mouse kidney tissue (100× original magnification (upper), 400× original magnification (lower)). Compared with that in the ND group, TGF-β was elevated in the HFD group and partially returned to normal levels after MgH₂ treatment. Con: Control group; DAPI: 4’,6-diamidino-2-phenylindole; HFD: high-fat diet; HM: MgH₂ group; KEGG: Kyoto Encyclopedia of Genes and Genomes; ND: normal control; PA: palmitic acid; PM: PA + MgH₂ group; p-Smad2: phospho-Smad family member 2; p-Smad3: phospho-Smad family member 3; Smad2: Smad family member 2; Smad3: Smad family member 3; TGF-β: transforming growth factor-beta.

Figure 4

MgH₂ suppresses AKT/mTOR pathway activation via modulation of PTEN in vitro. (A, B) Western blotting results of the PTEN/AKT/mTOR pathway after MgH₂ intervention in in vitro (A) and in vivo (B) experiments. The raw data for the quantitative results are shown in Additional Table 1. (C) Evaluation of HK-2 cell toxicity induced by SF1670 (a PTEN inhibitor). (D) Analysis of the effects of PTEN inhibition on the expression of proteins in the AKT/mTOR pathway. The raw data for the quantitative results are shown in Additional Table 1. Con: Control group; HFD: high-fat diet; HM: MgH₂ group; MgH₂: magnesium hydride; mTOR: mammalian target of rapamycin; ND: normal control; PA: palmitic acid; p-AKT: phospho-protein kinase B; PM: PA + MgH₂ group; p-mTOR: phospho-mammalian target of rapamycin; PTEN: phosphatase and tensin homolog deleted on chromosome ten.

Figure 5

Mitigation of MgH₂-induced inhibition of the AKT/mTOR and TGFβ-Smad2/3 pathways by HES1. (A) In vivo protein expression levels of HES1. (B) In vitro protein expression levels of HES1. (C) HES1 overexpression was detected via western blot analysis. (D) Impact of HES1 overexpression on the protein expression levels of molecules in the TGFβ-Smad2/3 and AKT/mTOR pathways. The raw data for the quantitative results are shown in Additional Table 1. Con: Control group; HES1: Hes family BHLH transcription factor 1; HFD: high-fat diet; HM: MgH₂ group; MgH₂: magnesium hydride; mTOR: mammalian target of rapamycin; ND: normal control; OE: overexpression; PA: palmitic acid; p-AKT: phospho-protein kinase B; PM: PA + MgH₂ group; p-mTOR: phospho-mammalian target of rapamycin; p-Smad2: phospho-Smad family member 2; p-Smad3: phospho-Smad family member 3; TGFβ: transforming growth factor-beta.