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.

Ageing is associated with a higher risk of chronic diseases such as cancer, cardiovascular disease, diabetes, sarcopenia, dementia, and chronic kidney disease (CKD). Ageing leads to functional and structural changes in the kidneys. The kidney is vulnerable to altered homeostasis and senescence; thus, its condition is a major predictor of ageing. Cellular senescence occurs due to changes in environmental and systemic factors like hyperglycaemia, oxidative stress, accumulation of advanced glycation end products (AGEs), and hyperphosphatemia^1-3. High glucose levels and AGEs trigger a hyperosmolar environment that causes premature senescence in glomerular mesangial cells³. Deposition of AGEs in the renal compartments contributes to glomerulosclerosis, progressive nephropathies, interstitial fibrosis, tubular atrophy, podocyte damage, and inflammation. Hyperphosphatemia is a risk factor for CKD progression and accelerates ageing⁴. A compromised vitamin D-parathyroid-fibroblast growth factor 23 (FGF23)-Klotho axis enhances serum phosphate levels. The excess phosphate alters glucose homeostasis, insulin sensitivity, and oxidative stress, which are closely linked with ageing⁵.

The D-galactose-induced ageing model is widely used to study age-related complications and anti-ageing research^6-10. D-galactose triggers renal ageing by enhancing the accumulation of AGEs and free radicals, reducing antioxidant enzymes, and attenuating immune responses, similar to natural kidney ageing. Accumulated AGEs interact with RAGE and alter intracellular signalling pathways, like inflammation (NF-kB and JNK), pro-fibrotic pathways (TGF-β), and apoptosis (JNK)^6,7. Additionally, glycogen synthase kinase 3β (GSK3β) is implicated in glomerular ageing, podocyte senescence, and renal fibrosis. Given the complexity of renal ageing, these signalling pathways offer potential targets for therapeutic interventions in age-related renal dysfunction.

Deficiency of vitamins B12, B6, and folate could lead to hyperhomocysteinemia, hyperphosphatemia, and accumulation of AGEs. These B-vitamins regulate energy metabolism/homeostasis, methylation, DNA repair, oxidative stress-antioxidant balance, and immunity; their malnutrition in the ageing population may lead to neurodegenerative, renal, and cardiovascular disorders. Vitamin B12 deficiency is commonly observed in the elderly. Subclinical vitamin B12 deficiency induces oxidative stress through the accumulation of homocysteine (Hcy), reduced reactive oxygen species (ROS) scavenging, and glutathione (GSH) levels. This leads to low-grade inflammation, creating a positive feedback cycle of AGEs and B12 deficiency¹¹. Nevertheless, the molecular mechanism by which B12 modulates renal senescence, particularly in relation to hyperphosphatemia and AGEs accumulation, remains unclear. Hence, the present study was planned to investigate the role of vitamin B12 in mitigating renal dysfunction in a D-galactose-induced ageing rat model.

Materials & Methods

The study was conducted at the department of Biochemistry, ICMR-National Institute of Nutrition, Hyderabad, India. The study protocols were approved by the Institute’s Animal Ethics Committee.

Animals and treatment

Twelve-month-old male Wistar rats with an average body weight of 350 g were procured from the Animal Facility of the Institute (December 2022). The animals had unrestricted access to standard food and water in an environment with controlled humidity (50-60%), temperature (22-24°C), and a 12-h light and dark cycle. They were randomly divided into three groups (n=6): the Control (C) group, the D-galactose (G) group, and the D-galactose + B12 intervention (G+B12) group. Rats in both G and G+B12 groups were intraperitoneally injected with D-galactose at 300 mg/kg body weight/day (HiMedia, PA, USA) for 120 days. The dose of D-galactose injection was based on previously reported studies^7,12-14. Rats in G+B12 group also received supplementation of B12 at 50 µg/kg diet from the first day of D-galactose injection. Control group (C) rats were intraperitoneally injected with normal saline for 120 days. The body weight of rats was measured weekly, and after 120 days of the experimental period, the rats were sacrificed following an overnight fast, and the kidneys were collected for analysis.

Experimental diets

During the experimental period, rats were given a control or a B12-supplemented diet ad libitum. While the B12-supplemented diet contained 50 µg/kg, the control diet contained 25 µg/kg of vitamin B12 (Supplementary Table I).

Kidney function assessment

Blood was collected at the end of the experimentation period, and plasma was separated for further assays. Rats were housed in metabolic cages for 24-h urine collection. The creatinine, albumin, urea, BUN, cholesterol, and triglyceride levels were estimated using Biosystems kits (Barcelona, Spain) per the manufacturer’s instructions.

Enzyme-linked immunosorbent assay (ELISA)

The plasma was used to estimate total cobalamin (GE Bioscience), transcobalamin, carboxymethyl lysine (CML), lactate dehydrogenase (LDH), and vitamin D (Krishgen Biosystems, CA, USA) by ELISA, according to manufacturer instructions.

Histological analysis

The right kidney (near the right hindlimb) from each animal was preserved in formaldehyde, encased in paraffin, sliced into approximately 5 μm-thick sections, and stained with haematoxylin and eosin (H&E) to visualise kidney morphology and Masson’s trichrome (MT) to assess kidney fibrosis and collagen deposition. The sections were then examined under a microscope. The renal damage was assessed by three blind reviewers. The histopathological changes were calculated by the EGTI scoring system, considering the endothelial, glomerular, tubular, and interstitial cell damage¹⁵.

qRT-PCR expression analysis

Total RNA was extracted from the cortex of the left kidney using the Trizol method. Two µg of total RNA was used to synthesise cDNA employing the SuperScript III First-Strand System (Invitrogen, Waltham, MA, USA). Expression analysis of various markers was performed on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) using SYBR Green Master mix (Takara) as per the manufacturer’s instructions. GAPDH transcript levels were used as an internal control for normalising the expression of various genes listed in the supplementary table II.

Immunoblotting

The left kidney tissue homogenate was prepared using Tris lysis buffer (20 mM and pH 7.5) containing protease inhibitors for immunoblotting analysis. The homogenates were resolved on SDS-PAGE and transferred onto a nitrocellulose membrane. Blocking was done with 5% skimmed milk powder in PBS (20 mM; 0.9% NaCl). The membrane was left overnight at 4°C with the respective primary antibodies [pNF-kB, NF-kB, Bax, caspase 3, p53, pGSK3β, pβ-Catenin, N-Cadherin and GAPDH obtained from Cell Signaling Technology, Inc (Massachusetts, USA)., CML from Abcam (Cambridge, UK), pPERK, TNFα, Bcl2, nephrin, RAGE, IL-6, β-galactosidase, and FGF23 from ThermoFisher Scientific (Massachusetts, USA), Klotho and podocin from Sigma Chemicals (St. Louis, MO, USA)]. The membrane was washed with PBST and incubated with secondary antibodies at specific concentrations. Immunoblots were developed using an ECL reagent (Bio-Rad, CA, USA). GAPDH was used as the internal control for normalising protein expression.

Statistical analysis

The statistical analysis was conducted using GraphPad Prism 10.3. Results were shown as mean±standard error of the mean (SEM) (n=4 rats/group). Significance of differences between groups was evaluated using a one-way analysis of variance (ANOVA) followed by a post hoc Tukey test for pairwise comparisons. Values of P<0.05 were considered statistically significant.

Results

Vitamin B12 supplementation ameliorated D-galactose-induced renal injury

No significant differences in food intake and body weight were observed between the experimental groups. Compared to group C, lower plasma cobalamin and transcobalamin-2 levels were observed in group G. In comparison, group G+B12 showed significantly higher levels of both these vitamers compared to the other two groups (P<0.05) (Fig. 1A). We next evaluated the renal functional markers to evaluate B12 role in renal protection. The renal functional markers: 24-h urinary creatine, urea, albumin levels and albumin creatinine ratios were abnormal in group G compared to group C, indicating poor renal function. The estimated glomerular filtration rate (eGFR) was higher in group G than in group C, which may represent an initial, temporary compensatory mechanism against galactose-induced renal injury. However, in the G+B12 group, these abnormalities were significantly prevented (Fig. 1B), suggesting improved renal function. Further, we investigated renal damage by assessing mRNA expression levels of 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) by qRT-PCR. Transcript levels of KIM-1, LCN-2, Fabp-1, and Timp-1 were significantly upregulated in group G compared with group C, indicating renal dysfunction in the D-galactose-induced aging rat model (P<0.05) (Fig. 1C). Vitamin B12 protected the kidneys by significantly preventing the upregulation of KIM-1, LCN-2, Fabp-1, and Timp-1 compared to group G.

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.

Vitamin B12 supplementation prevented D-galactose-induced protein glycation and cellular senescence

D-Galactose treatment resulted in significant accumulation of carboxymethyl-lysine (CML) and increased expression of RAGE compared to group-C. At the same time, B12 supplementation decreased CML and RAGE levels significantly compared to group-G (Fig. 2A). Similarly, higher plasma levels of CML were detected in group-G compared to controls, which were partially lowered by B12 supplementation (Fig. 2B). Further, we explored the AGEs-related renal injury by investigating changes in the signalling molecules associated with cellular senescence. The expression of senescence markers, β-galactosidase and p53, was increased in the group-G rats compared to group-C (P<0.05). Vitamin B12 intervention significantly prevented these changes (Supplementary Fig. 1A). The histological examination and scores of the H&E-stained kidney sections of the galactose-treated group revealed glomerular atrophy, basement membrane thickening, mesangial expansion, loss of brush border, and glomerular fibrosis (Fig. 2C, Supplementary Fig. 1B). Vitamin B12 supplementation protected against these abnormal histopathological changes and these observations indicated a protective role for vitamin B12 against the formation of AGEs and renal senescence in the D-galactose-induced ageing rats.

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.

Vitamin B12 supplementation ameliorates D-galactose-induced hyperphosphatemia

The serum phosphate levels were influenced by dietary intake, intestinal absorption, and kidney excretion, which are mainly regulated by vitamin D, parathyroid hormone (PTH), FGF23, and αKlotho. Hyperphosphatemia accelerates cellular senescence and causes CKD (tubular damage). In the present study, high phosphate levels were detected in the plasma of group G compared to the control group. Vitamin B12 supplementation significantly restricted phosphate levels compared to Group G (Fig. 3A). While D-Gal treatment lowered the plasma vitamin D levels compared to controls, B12 supplementation significantly prevented the reduction in vitamin D levels (Fig. 3A). Klotho and FGF23 act as a bone-kidney endocrine axis regulating phosphate metabolism. Thus, we also evaluated the impact of vitamin B12 supplementation on the Klotho and FGF23 axis by analysing their protein expression levels using immunoblotting. Elevated FGF23 and decreased Klotho were detected in Group G compared to the control group. Vitamin B12 supplementation decreased FGF23 and upregulated Klotho levels (Fig. 3B, Supplementary Fig. 2A), indicating the B12 potential role against hyperphosphatemia in ageing. In addition, vitamin D receptor (VDR) and CYP27b1 gene expression were downregulated in group G and considerably upregulated by B12 supplementation. In contrast, PTH gene expression levels were upregulated in group G and significantly downregulated in B12 supplementation (Fig. 3C).

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.

Vitamin B12 exerts anti-inflammatory potential

Inflammation plays a key role in kidney dysfunction, particularly fibrotic changes. D-galactose induces inflammation by triggering ROS, which stimulates TNF-α. Hence, in the present study, the pro-inflammatory gene response was determined. Group G showed significantly higher expression of pro-inflammatory cytokines, TLR4, MCP-1, ICAM, and VCAM, which were downregulated in the B12 supplementation group (Fig. 4A). Further, we analysed the protein expression levels of NFkB, TNF-α, and IL-6 by immunoblotting. The group G showed higher NFkB, TNF-α, and IL6 levels in the kidney. Vitamin B12 supplementation significantly prevented the rise (Fig. 4B, Supplementary Fig. 2B).

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.

Vitamin B12 supplementation prevents kidney fibrosis

MT staining indicated a moderate fibrosis in Group G, whereas B12 supplementation reduced the fibrotic area (Fig. 5A), which was further confirmed by TGF-β gene expression. Group_G showed significantly upregulated TGF-β mRNA levels compared to Group C, whereas B12 supplementation downregulated TGF-β expression (Fig. 5B). We also evaluated the B12 effect on the telomerase enzyme, which is strongly associated with renal fibrosis. The mRNA expression of telomere enzyme components- TERT, TERC, and TERF was downregulated in Group G compared to Group C. Treatment with B12 upregulates the expression of telomerase components (Fig. 5C). We next investigated the changes in signalling mechanisms leading to fibrosis. The protein expression of pPERK, pGSK3β, and N-cadherin was significantly upregulated in Group G compared to Group C. Whereas, pβ-catenin showed decreased protein expression levels in Group G compared to Group C. Vitamin B12 supplementation enhanced the pβ-catenin and reduced the fibrosis by decreasing the protein expression of pPERK, pGSK3β, and N-cadherin (Supplementary Fig. 3A). This indicates that treatment with vitamin B12 prevents fibrosis and replicative senescence induced by telomere shortening.

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.

Vitamin B12 supplementation prevents renal apoptosis

There was no significant change in nephrin, but the podocin expression was decreased in Group G compared to Group C. Vitamin B12 supplementation prevented podocin loss and increased the expression of nephrin and podocin compared to Group G (Fig. 6A, Supplementary Fig. 3B).Group G also exhibited elevated Bax, cleaved caspase 3, and decreased Bcl2 protein. Vitamin B12 supplementation significantly decreased the pro-apoptotic markers Bax and cleaved caspase-3, while markedly upregulating the anti-apoptotic marker Bcl-2. These findings suggest that B12 mitigates renal apoptosis by shifting the balance towards cell survival, thereby reducing tissue damage (Fig. 6B, Supplementary Fig. 3C).

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.

Discussion

The present study provides convincing evidence that vitamin B12 supplementation exerts renoprotection against D-galactose-induced renal injury in aged rats through multiple interconnected mechanisms, including inhibition of protein glycation and cellular senescence, correction of phosphate metabolism, anti-inflammatory and anti-fibrotic actions, and thereby prevention of renal apoptosis. In the model of D-galactose-induced ageing, renal dysfunction was evident through increased urinary creatinine, albuminuria, and altered albumin-creatinine ratios. These abnormalities were significantly attenuated with B12 supplementation. Ohnishi et al⁴ reported elevated serum phosphate levels in ageing. A decline in renal function further contributes to the accumulation of phosphate. Defects in the Klotho-FGF23 axis can alter serum phosphate, calcium, and vitamin D levels, triggering ageing. Hyperphosphatemia, vascular calcification, and elevated FGF23 exacerbate cardiovascular disease, contributing to 60 per cent of deaths among patients with CKD on dialysis. The FGF23 and PTH regulate the sodium-dependent phosphate transporters (NaPi2) and inhibit the calcitriol to maintain the phosphate homeostasis¹⁶. In the present investigation, B12 ameliorated hyperphosphatemia by targeting FGF23, PTH, and upregulating the cytochrome P27B. Deficiency of Klotho, the receptor for FGF-23, has been linked to elevated oxidative stress and kidney fibrosis. Klotho plays a significant role in negatively regulating downstream Akt signalling and enhancing antioxidant capacity, which are vital in counteracting ageing-induced abnormalities¹⁷. The observations of protection against renal aging by B12 supplementation through increased Klotho levels and decreased FGF23 are consistent with previously published reports on Polygonatum sibiricum effects on D-galactose-induced ageing rats¹⁸. These findings suggest that B12 supplementation maintains phosphate homeostasis via regulation of the Klotho-FGF23 axis.

AGEs mediate renal ageing through cellular senescence, inflammation, oxidative stress, apoptosis, and fibrosis. In the D-galactose-induced renal ageing model^8-10, AGEs promote glomerulosclerosis by cross-linking with matrix proteins, leading to structural changes in the glomerulus and thickening of the basement membrane. The AGE–RAGE axis activates MAPK, NF-κB, and TLR4 pathways, promoting inflammation, oxidative stress, and renal fibrosis. In this study, vitamin B12 reduced IL-6, TNF-α, MCP-1, and TGF-β levels by inhibiting RAGE–NF–κB, JNK, and GSK3β signalling, thereby decreasing inflammation, apoptosis, and fibrosis. B12 also modulated TGF-β–ERK–GSK3β–β-catenin axis to suppress fibrotic responses. These results are consistent with a study that reported that resveratrol protected against AGEs-induced renal dysfunction by reducing cellular senescence, apoptosis, and fibrosis-related EMT in D-galactose-induced renal aging¹⁹. B12 supplementation also preserved podocyte integrity by maintaining nephrin and podocin, and reduced apoptosis by downregulating Bax and caspase-3 while upregulating Bcl-2.

Telomere attrition mediates senescence via the p53/p21^CIP1/WAF1 pathway in renal ageing. Treatment with resveratrol or aminoguanidine, rheinlysinate, and maltol ameliorates the AGEs-triggered cellular senescence by downregulating these pathways^19-21. Similarly, in the present study, B12 supplementation reduced the AGEs-triggered senescence and upregulated the telomerase enzyme components, thereby mitigating replicative senescence and kidney fibrosis. Telomerase, TERT, TERC, and telomere-associated proteins are emerging therapeutic targets for improving renal function²².

While vitamin B12 exhibited reno-protective effects, concerns regarding its safety, particularly prolonged use, and potential interactions with other medications, warrant careful consideration. Cyanocobalamin, the most widely used form of vitamin B12, poses a specific risk to patients with renal impairment because it contains aluminium, which contributes to neurological and skeletal damage. Hence, active forms such as methylcobalamin or hydroxocobalamin are preferred for this population. Common drugs such as proton pump inhibitors, H2-receptor antagonists, and metformin significantly reduce B12 absorption. Additionally, interactions between B12 and folate are critical in managing cognitive and vascular outcomes, especially in patients with CKD. Moreover, elevated Hcy levels in CKD patients necessitate balanced vitamin B12 and folate supplementation, as both nutrients are key in its metabolism.

Overall, the study provides evidence that vitamin B12 supplementation exerts significant reno-protective effects in a D-galactose rat model. The benefits include amelioration of renal dysfunction and injury markers, attenuation of AGEs accumulation and cellular senescence, normalisation of phosphate homeostasis, suppression of inflammatory pathways, inhibition of key fibrotic mediators, and prevention of renal apoptosis. These pleiotropic actions collectively interrupt critical pathological processes associated with renal aging. These findings highlight the potential importance of maintaining adequate vitamin B12 status for kidney health during the ageing process. Further research, including studies in naturally aged models and clinical investigations, is warranted to confirm these benefits.

Financial support & sponsorship

The study received funding support from the postdoctoral fellowship by Science and Engineering Research Board, Government of India (SERB, PDF/2020/001907) awarded to the first author (MN).

Conflicts of Interest

None.

Use of Artificial Intelligence (AI)-Assisted Technology for manuscript preparation

The authors confirm that there was no use of AI-assisted technology for assisting in the writing of the manuscript and no images were manipulated using AI.