Deferasirox and vitamin D3 co-therapy mitigates iron-induced renal injury by enhanced modulation of cellular anti-inflammatory, anti-oxidative stress, and iron regulatory pathways in rat.

BACKGROUND: Chronic iron overload could induce nephropathy via oxidative stress and inflammation, and chelating therapy has limited efficacy in removing excess intracellular iron. Although vitamin D (VD) has shown potent antioxidant and anti-inflammatory effects, as well contribute to iron homeostasis, none of the previous studies measured its potential remedial effects against chronic iron toxicity. AIMS: To measure the alleviating effects of deferasirox (DFX) and/or vitamin D (VD) single and combined therapies against nephrotoxicity induced by chronic iron overload. METHODS: Forty male rats were divided into negative (NC) and positive (PC) controls, DFX, VD, and DFX/VD groups. The designated groups received iron for six weeks followed by DFX and/or VD for another six weeks. Then, the expression pattern of renal genes and proteins including hepcidin, ferroportin (FPN), megalin, transferrin receptor 1 (TfR1), ferritin heavy and light chains, VD receptor (VDR), VD synthesizing (Cyp27b1) and catabolizing (Cyp24a1) enzymes were measured alongside serum markers of renal function and iron biochemical parameters. Additionally, several markers of oxidative stress (MDA/H2O2/GSH/SOD1/CAT/GPx4) and inflammation (IL-1ß/IL-6/TNF-α/IL-10) together with renal cell apoptosis and expression of caspase-3 (Casp-3) were measured. RESULTS: The PC rats showed pathological iron and renal biochemical markers, hypovitaminosis D, increased renal tissue iron contents with increased Cyp24a1/Megalin/ferritin-chains/hepcidin, and decreased Cyp27b1/VDR/TfR1/FPN expression than the NC group. The PC renal tissues also showed abnormal histology, increased inflammatory (IL-1ß/IL-6/TNF-α), oxidative stress (MDA/H2O2), and apoptosis markers with decreased IL-10/GSH/SOD1/CAT/GPx4. Although DFX monotherapy reduced serum iron levels, it was comparable to the PC group in renal iron concentrations, VD and iron-homeostatic molecules, alongside markers of oxidative stress, inflammation, and apoptosis. On the other hand, VD monotherapy markedly modulated renal iron and VD-related molecules, reduced renal tissue iron concentrations, and preserved renal tissue relative to the PC and DFX groups. However, serum iron levels were equal in the VD and PC groups. In contrast, the best significant improvements in serum and renal iron levels, expression of renal iron-homeostatic molecules, oxidative stress, inflammation, and apoptosis were seen in the co-therapy group. CONCLUSIONS: iron-induced nephrotoxicity was associated with dysregulations in renal VD-system together with renal oxidative stress, inflammation, and apoptosis. While DFX reduced systemic iron, VD monotherapy showed better attenuation of renal iron concentrations and tissue damage. Nonetheless, the co-therapy approach exhibited the maximal remedial effects, possibly by enhanced modulation of renal iron-homeostatic molecules alongside reducing systemic iron levels. AVAILABILITY OF DATA AND MATERIALS: All data generated or analysed during this study are included in this published article [and its Supplementary information files].

Enhanced renoprotective actions of Paricalcitol and omega-3 fatty acids co-therapy against diabetic nephropathy in rat.

Introduction: Although the synthetic vitamin D analogue, Paricalcitol, and omega-3 Fatty acids (ω-3) alleviated diabetic nephropathy (DN), their combination was not previously explored. Objectives: This study measured the potential ameliorative effects of single and dual therapies of Paricalcitol and/or ω-3 against DN. Methods: Forty rats were assigned as follow: negative (NC) and positive (PC) controls, Paricalcitol, ω-3 and Paricalcitol + ω-3 groups. Diabetes was generated by high-fat/high-fructose diet and a single streptozotocin injection (40 mg/kg). DN was confirmed by raised fasting blood glucose (FBG), polyuria, proteinuria, and decreased urine creatinine levels. Paricalcitol intraperitoneal injections (0.25 µg/Kg/day; 5 times/week) and oral ω-3 (415 mg/kg/day; 5 times/week) started at week-9 and for eight weeks. Results: The PC group showed hyperglycaemia, dyslipidaemia, abnormal renal biochemical parameters, elevated caspase-3 expression, and increased apoptosis by TUNEL technique. The mRNAs and proteins of the pathogenic molecules (TGF-ß1/iNOS) and markers of tissue damage (NGAL/KIM-1) augmented substantially in the PC renal tissues relative to the NC group. The oxidative stress (MDA/H2O2/protein carbonyl groups) and pro-inflammatory (IL1ß/IL6/TNF-α) markers increased, whereas the anti-inflammatory (IL10) and anti-oxidative (GSH/GPx1/GR/SOD1/CAT) declined, in the PC renal tissues. The monotherapy groups were associated with ameliorated FBG, lipid profile and renal functions, and diminished TGF-ß1/iNOS/NGAL/KIM-1/Caspase-3 alongside the apoptotic index than the PC group. The oxidative stress and pro-inflammatory markers decreased, whilst the anti-oxidative and anti-inflammatory molecules escalated, in the monotherapy groups than the PC group. Although the Paricalcitol renoprotective actions were better than ω-3, all the biomarkers were abnormal than the NC group. Alternatively, the Paricalcitol + ω-3 protocol exhibited the best improvements in metabolic control, renal functions, oxidative stress, inflammation, and apoptosis. However, FBG and tissue damage were persistently higher in the co-therapy group than controls. Conclusions: Both monotherapies showed modest efficacy against DN, whereas their combination displayed boosted renoprotection, possibly by enhancing renal anti-oxidant and anti-inflammatory pathways.

In Vivo and In Vitro Enhanced Tumoricidal Effects of Metformin, Active Vitamin D3, and 5-Fluorouracil Triple Therapy against Colon Cancer by Modulating the PI3K/Akt/PTEN/mTOR Network.

Chemoresistance to 5-fluorouracil (5-FU) is common during colorectal cancer (CRC) treatment. This study measured the chemotherapeutic effects of 5-FU, active vitamin D3 (VD3), and/or metformin single/dual/triple regimens as complementary/alternative therapies. Ninety male mice were divided into: negative and positive (PC) controls, and 5-FU, VD3, Met, 5-FU/VD3, 5-FU/Met, VD3/Met, and 5-FU/VD3/Met groups. Treatments lasted four weeks following CRC induction by azoxymethane. Similar regimens were also applied in the SW480 and SW620 CRC cell lines. The PC mice had abundant tumours, markedly elevated proliferation markers (survivin/CCND1) and PI3K/Akt/mTOR, and reduced p21/PTEN/cytochrome C/caspase-3 and apoptosis. All therapies reduced tumour numbers, with 5-FU/VD3/Met being the most efficacious regimen. All protocols decreased cell proliferation markers, inhibited PI3K/Akt/mTOR molecules, and increased proapoptotic molecules with an apoptosis index, and 5-FU/VD3/Met revealed the strongest effects. In vitro, all therapies equally induced G1 phase arrest in SW480 cells, whereas metformin-alone showed maximal SW620 cell numbers in the G0/G1 phase. 5-FU/Met co-therapy also showed the highest apoptotic SW480 cell numbers (13%), whilst 5-FU/VD3/Met disclosed the lowest viable SW620 cell percentages (81%). Moreover, 5-FU/VD3/Met revealed maximal inhibitions of cell cycle inducers (CCND1/CCND3), cell survival (BCL2), and the PI3K/Akt/mTOR molecules alongside the highest expression of cell cycle inhibitors (p21/p27), proapoptotic markers (BAX/cytochrome C/caspase-3), and PTEN in both cell lines. In conclusion, metformin monotherapy was superior to VD3, whereas the 5-FU/Met protocol showed better anticancer effects relative to the other dual therapies. However, the 5-FU/VD3/Met approach displayed the best in vivo and in vitro tumoricidal effects related to cell cycle arrest and apoptosis, justifiably by enhanced modulations of the PI3K/PTEN/Akt/mTOR pathway.

Vitamin D3 and calcium cosupplementation alleviates cadmium hepatotoxicity in the rat: Enhanced antioxidative and anti-inflammatory actions by remodeling cellular calcium pathways.

Although vitamin D (VD) and calcium (Ca) attenuate cadmium (Cd) metabolism, their combined antioxidant and anti-inflammatory actions against Cd toxicity have not been previously explored. Hence, this study measured the protective effects of VD ± Ca supplements against Cd hepatotoxicity. Forty adult male rats were distributed to: negative controls (NCs), positive controls (PCs), VD, Ca, and VD3 and Ca (VDC) groups. All groups, except NC, received CdCl2 in drinking water (44 mg/L) for 4 weeks individually or concurrently with intramuscular VD3 (600 IU/kg; three times per week) and/or oral Ca (100 mg/kg; five times per week). The PC group showed abnormal hepatic biochemical parameters and increase in cellular cytochrome C, caspase-9, and caspase-3 alongside the apoptotic/necrotic cell numbers by terminal deoxynucleotidyl transferase dUTP nick end labeling technique. The PC hepatic tissue also had substantially elevated pro-oxidants (malondialdehyde [MDA]/H2 O2 /protein carbonyls) and inflammatory cytokines (interleukin 1ß [IL-1ß]/IL-6/IL17A/tumor necrosis factor-α), whereas the anti-inflammatory (IL-10/IL-22) and antioxidants (glutathione [GSH]/GPx/catalase enzyme [CAT]) markers declined. Hypovitaminosis D, low hepatic tissue Ca, aberrant hepatic expression of VD-metabolizing enzymes (Cyp2R1/Cyp27a1/cyp24a1), receptor and binding protein alongside Ca-membrane (CaV 1.1/CaV 3.1), and store-operated (RyR1/ITPR1) channels, and Ca-binding proteins (CAM/CAMKIIA/S100A1/S100B) were observed in the PC group. Both monotherapies decreased serum, but not tissue Cd levels, restored the targeted hepatic VD/Ca molecules' expression. However, these effects were more prominent in the VD group than the Ca group. The VDC group, contrariwise, disclosed the greatest alleviations on serum and tissue Cd, inflammatory and oxidative markers, the VD/Ca molecules and tissue integrity. In conclusion, this report is the first to reveal boosted protection for cosupplementing VD and Ca against Cd hepatotoxicity that could be due to enhanced antioxidative, anti-inflammatory, and modulation of the Ca pathways.

Protective effect of Vitamin D3 against lead induced hepatotoxicity, oxidative stress, immunosuppressive and calcium homeostasis disorders in rat.

Lead (Pb) is an extremely poisonous, non-essential trace element and toxicity develops in humans following frequent exposure to the heavy metal in polluted environmental and occupational settings. Pb induces hepatic damage through the depletion of the antioxidant system, enhancing cellular oxidative stress and stimulation of proinflammatory cytokines. Although the antioxidant and anti-inflammatory actions of vitamin D3 (VD3) are well-established, a minority of studies measured the protective actions of VD3 against Pb toxicity. Therefore, this work studied the effects of vitamin VD3 therapy on the fundamental molecular basis underlying hepatic injury induced by chronic Pb toxicity. Twenty-four adult male rats were distributed equally into the negative controls (NC), positive controls (PC) and VD3 groups. While both the PC and VD3 groups received Pb-acetate in drinking water (1000 mg/L) for four weeks, the latter group also received intramuscular VD3 injections (1000 IU/kg; 3 days/week) simultaneously with Pb. The liver enzymes together with the serum and hepatic tissue Pb concentrations increased markedly in the PC group compared with the NC group. Pb toxicity also drastically induced hepatocyte apoptosis/necrosis, increased the hepatic tissue concentrations of malondialdehyde and the pro-inflammatory cytokines (TGF-ß, IL-4 & TNF-α) as well as reduced the anti-oxidative enzymes (GSH, GPx & CAT) and the anti-inflammatory cytokine, IL-10, compared with the NC group. Pb also significantly decreased the serum concentrations of VD3 and Ca2+. Additionally, the hepatic expressions of VD receptor, Cyp24a1 enzyme, L-type Ca2+-channel, calbindin-D28k & -D29k, calmodulin and calmodulin-dependent protein kinase II were significantly upregulated, whereas the VD binding protein, CYP2R1 enzyme and T-type Ca2+-channel were markedly inhibited at the gene and protein levels following Pb intoxication. VD3 alleviated the hepatic damage, inhibited the oxidative stress and pro-inflammatory molecules as well as upregulated the anti-oxidant and anti-inflammatory markers and restored the expression of the VD/Ca2+ regulatory molecules compared with the PC group. VD3 supplementation discloses promising protective effects against Pb-induced hepatic damage, through its anti-inflammatory and antioxidant actions as well as by modulating the hepatocyte calcium homeostatic molecules.

Enhanced remedial effects for vitamin D3 and calcium co-supplementation against pre-existing lead nephrotoxicity in mice: The roles of renal calcium homeostatic molecules.

BACKGROUND: Lead (Pb) is a toxic heavy metal and nephropathy is common with chronic exposure. Although vitamin D (VD) and calcium (Ca) showed promising protections, their co-administration was not previously investigated in Pb nephrotoxicity. This study measured the potential interactions and remedial effects of VD and/or Ca on established Pb nephropathy. METHODS: Fifty adult male mice were equally distributed into: negative controls (NC), positive controls (PC), Ca, VD and VDC groups. The study duration was seven weeks and all groups, except the NC, received Pb acetate in drinking water (500 mg/L) throughout the study. The Ca, VD and VDC groups also received oral Ca (50 mg/kg; five times/week) and/or intramuscular VD (1000 IU/kg; three times/week) from week four till the end of the study. RESULTS: The PC group showed substantial reduction in serum VD, hypocalcaemia, hypercalciuria and proteinuria alongside marked tissue inflammation, oxidative stress and apoptosis/necrosis. Pathological alterations were also detected in the mRNAs and proteins of the VD-metabolising enzymes, receptor and binding protein alongside several Ca-membrane channels, membrane transporters, intracellular binding proteins and mediators. While both monotherapies equally demonstrated moderate improvements, the VDC showed the utmost corrective actions on serum and tissue Pb concentrations, the inflammatory and antioxidative markers, the expressions of renal VD/Ca-molecules and tissue integrity. Moreover, the results were comparable between the VDC and NC groups. CONCLUSIONS: This report is the first to reveal potential enhanced remedial outcomes for combining VD and Ca against pre-existing Pb nephrotoxicity and the enhancements could be dependent on Ca-regulatory pathways.