Vitamin B12 modulates D-galactose-induced renal dysfunction

Abstract

Background & objectives Age-related renal impairment presents a significant challenge in contemporary clinical practice. Cellular senescence and oxidative stress are the key contributors to chronic kidney disease (CKD) during aging. Senescence is triggered by advanced glycation end products (AGEs), hyperphosphatemia, and higher glucose levels, which lead to renal dysfunction by inducing inflammation, endoplasmic reticulum (ER) stress, fibrosis, and apoptosis. Further, vitamin B12 is known to influence biological ageing and has been suggested to improve kidney function in the elderly; however, the underlying mechanisms require further investigation. In this study, we investigated the potential of vitamin B12 in mitigating renal dysfunction using a D-galactose-induced aging rat model. Methods Twelve-month-old male Wistar rats were grouped into Control, D-galactose (300 mg/kg/day), and D-galactose + vitamin B12 supplementation groups (n=6). Renal dysfunction was evaluated by kidney function markers (creatinine, albumin, urea, and BUN), renal damage markers (kidney injury molecule-1 [KIM-1], lipocalin-2 [LCN-2], fatty-acid binding protein-1 [FABP-1], and tissue inhibitor of metalloproteinase-1 [TIMP-1]), and histopathology (glomerular changes). Signalling mechanisms of cellular senescence, phosphate metabolism, inflammation, fibrosis, and renal apoptosis were analysed by qRT-PCR and immunoblotting. Results Vitamin B12 supplementation attenuated renal dysfunction by alleviating the senescence-induced accumulation of AGEs and hyperphosphatemia. Furthermore, vitamin B12 administration conferred renal protection by subsiding inflammation, fibrosis, and apoptosis through modulation of the RAGE-NFkB, pPERK-GSK3β, and JNK signalling pathways. Vitamin B12 supplementation mitigated hyperphosphatemia by mediating the Klotho-FGF23 axis. Interpretation & conclusions The findings provide evidence for vitamin B12 supplementation in managing renal aging.

Keywords: AGEs; chronic kidney disease; fibrosis; inflammation; phosphate; renal aging.

Fig. 1.

Impact of vitamin B12 supplementation on (A) plasma total cobalamin and transcobalamin, (B) urinary urea, albumin creatinine ratio, and estimated glomerular filtration rate (eGFR). (C) Transcript levels of KIM-1, LCN-2, Fabp-1, and Timp-1, respectively. C, Control; G, D-Gal; G+B12, Galactose with B12 (50 µg/Kg) supplementation. Data are mean±SEM (n=4 to 8). P^*<0.05.

Fig. 2.

Vitamin B12 supplementation inhibits the accumulation of advanced glycation end products markers. (A) Representative immunoblots of CML, RAGE, and their quantification. (B) Plasma CML levels. (C) Representative microscopic images of H&E stained rat kidney sections and histology scoring of endothelial, glomerular, tubular, and interstitial cell damage. Black arrow- normal corpuscular structure (normal glomerular capillary tuft, mesangium and Bowman’s capsule) and normal proximal convoluted tubule (PT). Green arrow-thickened basement membrane. Blue arrow- atrophy of the glomerular tuft. Scale bar = 100 µm. Data are presented as the mean±SEM (n=4). P^*<0.05. C, Control; G, D-Gal; G+B12, Gal with B12 supplementation.

Fig. 3.

Vitamin B12 supplementation alleviates phosphate toxicity by regulating the Klotho-FGF23 axis. (A) Plasma phosphate levels and vitamin D3 levels. (B) Densitometry quantification of Klotho, FGF23 immunoblots after normalisation with GAPDH. (C) Transcript levels of vitamin D receptor, CYP27b and PTH. Data are mean±SEM (n=4). C, Control; G, D-Gal; G+B12, Galactose with B12 supplementation. P^*<0.05.

Fig. 4.

Vitamin B12 attenuates renal inflammation. (A) Gene expression of TLR-4, TGFB-1, MCP-1, ICAM-1, and VCAM. (B) Densitometry quantification of pNFkB, NFkB, TNF-α, and IL-6 immunoblots after normalisation with GAPDH. Data are presented as the mean±SEM (n=4). C, Control; G, D-Galactose (300 mg/Kg body wt/day); G+B12, Galactose with B12 (50 µg/Kg) supplementation. P^*<0.05.

Fig. 5.

Vitamin B12 supplementation attenuates renal fibrosis. (A) Representative microscopic images of MT-stained rat kidney sections. Green arrow- positive staining, Scale bar = 20 µm. (B) Transcript levels of TGF-β and, (C) Transcript levels of TERT, TERC, and TERF normalised with GAPDH. Data are mean±SEM (n=4). P^*<0.05. C, Control group; G, D-Galactose group; G+B12, Galactose with B12 supplementation.

Fig. 6.

Vitamin B12 supplementation prevented renal apoptosis. (A) Quantification of expression of podocin and nephrin. GAPDH was used as a loading control. (B) Quantification of Bax, Bcl2, and cleaved Caspase-3 immunoblots. Data are mean±SEM (n=4). P^*<0.05. C, Control group; G, D-Galactose group; G+B12, Galactose with B12 supplementation.