Metformin prevents renal stone formation through an antioxidant

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Metformin prevents renal stone formation through an antioxidant mechanism in vitro and in vivo

Metformin prevents renal stone formation through an antioxidant mechanism in vitro and in vivo Presenter: Ms. Sajida Abdul Kadar Reg no. : 226 / July 2016 3/10/2021 1

3/10/2021 2

3/10/2021 2

Backgound • Calcium oxalate (Ca. Ox) – most prevalent stones • Oxalate excretion -

Backgound • Calcium oxalate (Ca. Ox) – most prevalent stones • Oxalate excretion - lower in healthy subjects compared with idiopathic Ca. Ox renal stone patients • Oxalate - associated with renal tubular cell injury • Stone formation depends - response of renal tubular epithelium to oxalate 3/10/2021 3

Cont. • ROS induces renal tubular cell injury • Antioxidants protect renal epithelial cells

Cont. • ROS induces renal tubular cell injury • Antioxidants protect renal epithelial cells from oxidative injury induced by oxalate • Metformin is a potent antioxidant; suppresses ROS production, scavenges free radicals and changes antioxidant defense pathways. • Inhibits injuries caused by oxidative stress 3/10/2021 4

Aim To investigate the preventive effects of metformin on renal tubular cell injury induced

Aim To investigate the preventive effects of metformin on renal tubular cell injury induced by oxalate and stone formation in a hyperoxaluric rat model 3/10/2021 5

Methodology • Cell Cultured: MDCK and HK-2 cells • MTT Assay: Viability of MDCK

Methodology • Cell Cultured: MDCK and HK-2 cells • MTT Assay: Viability of MDCK cells and HK-2 cells • Assessment of superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels in vitro • Animal Model and Experimental Design: Male Sprague-Dawley rats divided into three groups; normal controls, EG treated (0. 75% in drinking water) and EG + metformin treated (200 mg/kg/day) for 8 weeks 3/10/2021 6

Cont. • Measurement of SOD and MDA levels in vivo (Kidney tissues) • Serum

Cont. • Measurement of SOD and MDA levels in vivo (Kidney tissues) • Serum and urinary biochemistry • Detection of kidney crystal formation • Statistical Analysis: IBM SPSS, version 20. Twotailed unpaired �� -test or one-way ANOVA was performed for statistical analysis 3/10/2021 7

Results 3/10/2021 8

Results 3/10/2021 8

Metformin Protected Renal Tubular Cells from Oxalate - Induced Cytotoxicity Fig 1. Effect of

Metformin Protected Renal Tubular Cells from Oxalate - Induced Cytotoxicity Fig 1. Effect of various concentrations of oxalate and metformin on cytotoxicity in cultured MDCK cells. (a) MDCK cells were treated with the indicated concentrations (0. 05– 6. 4 m. M) of sodium oxalate for 2 days. (b) MDCK cells were treated with the indicated concentrations (0. 5– 64 m. M) of metformin for 2 days. Values represent mean ± SD of 3 independent experiments. ∗�� < 0. 05; ∗∗�� < 0. 001, versus control. 3/10/2021 9

Metformin Protected Renal Tubular Cells from Oxalate - Induced Cytotoxicity Fig 2. Cell viability

Metformin Protected Renal Tubular Cells from Oxalate - Induced Cytotoxicity Fig 2. Cell viability by MTT assay and representative dose response viability data. a) MDCK cells were pretreated with different concentrations of metformin (0, 0. 5, 1, 2, 4, and 8 m. M) for 2 h and then incubated with or without 1. 6 m. M sodium oxalate for an additional 24 h. Viability of cells was assessed by MTT assay. Percentage of cell viability was relative to the untreated control cells. b) HK-2 cells were pretreated with different concentrations of metformin (0, 0. 5, and 1 m. M) for 2 h and then incubated with or without 1. 6 m. M sodium oxalate for an additional 24 h. 3/10/2021 10

Metformin Protected Renal Tubular Cells from Oxalate- Induced Oxidative Stress Fig 3. Effect of

Metformin Protected Renal Tubular Cells from Oxalate- Induced Oxidative Stress Fig 3. Effect of metformin on SOD and MDA levels in cultured MDCK cells treated with oxalate. (a) MDA levels of MDCK cells were treated with 1. 6 m. M sodium oxalate with or without 8 m. M metformin for the indicated time points. (b) SOD activities of MDCK cells were treated with 1. 6 m. M sodium oxalate with or without 8 m. M metformin for the indicated time points. Values represent mean ± SD of 3 independent experiments. ∗�� < 0. 05; ∗∗�� < 0. 001, versus control. 3/10/2021 11

Metformin Protected Renal Tubular Cells from Oxalate- Induced Oxidative Stress Fig 4. Effect of

Metformin Protected Renal Tubular Cells from Oxalate- Induced Oxidative Stress Fig 4. Effect of metformin on SOD and MDA levels in cultured HK-2 cells treated with oxalate. (a) MDA levels of HK-2 cells were treated with 1. 6 m. M sodium oxalate with or without 1 m. M metformin for the indicated time points. (b) SOD activities of HK-2 cells were treated with 1. 6 m. M sodium oxalate with or without 1 m. M metformin for the indicated time points. Values represent mean ± SD of 3 independent experiments. ∗�� < 0. 05, versus control. 3/10/2021 12

Metformin Relieved EG-Induced Oxidative Stress in Animal Model Fig 5. Quantitative assessment shows that

Metformin Relieved EG-Induced Oxidative Stress in Animal Model Fig 5. Quantitative assessment shows that MDA level and SOD activity in renal tissue. (a) MDA level and (b) SOD activity. ∗�� < 0. 05, versus the control group; #�� < 0. 05 versus the EG treated group. 3/10/2021 13

Serum and Urinary Biochemistry Results Table 1: Serum and urinary biochemistry 3/10/2021 14

Serum and Urinary Biochemistry Results Table 1: Serum and urinary biochemistry 3/10/2021 14

Metformin Ameliorated EG-Induced Renal Crystal Formation in Rat Model Fig 6. Morphologic distribution of

Metformin Ameliorated EG-Induced Renal Crystal Formation in Rat Model Fig 6. Morphologic distribution of renal Ca. Ox crystals: (a–c) Representative micrographs of renal sections and crystal deposits were presented in the control group, the EG treated group, and the EG + metformin treated group, respectively, using HE staining and polarized light optical microphotography. (d–f) Representative micrographs of the control group, the EG treated group, and the EG + metformin treated group, respectively. 3/10/2021 15

Metformin Ameliorated EG-Induced Renal Crystal Formation in Rat Model Fig 7. Quantitative estimation of

Metformin Ameliorated EG-Induced Renal Crystal Formation in Rat Model Fig 7. Quantitative estimation of renal Ca. Ox crystals: (g)The ratios of areas with renal crystal deposition per low-powered field were estimated. (h) Grade of calcium oxalate deposits per high-powered field was assessed. ∗�� < 0. 05, versus the EG treated group. 3/10/2021 16

Discussion • Previous experiments in vivo and in vitro reported that high concentrations of

Discussion • Previous experiments in vivo and in vitro reported that high concentrations of oxalate result in increased intracellular oxidative stress • Previous prevented investigations oxidative showed stress-induced - metformin injuries in several cell types and tissues 3/10/2021 17

Cont. • This study demonstrated the effect of metformin on renal injury induced by

Cont. • This study demonstrated the effect of metformin on renal injury induced by oxalate and on kidney crystal sedimentation • This study showed - crystal formation was considerably prevented in the metformin group compared to the stone forming group 3/10/2021 18

Conclusions • Metformin has potent antioxidant effects and could effectively reduce renal tubular injury

Conclusions • Metformin has potent antioxidant effects and could effectively reduce renal tubular injury resulting from lipid peroxidation production induced by oxalate • Metformin inhibits renal crystal deposition in rats • These findings establish metformin as a new prospective therapeutic agent for treatment of calcium oxalate stone formation and might benefit individuals with primary hyperoxaluria or recurrent calcium oxalate stone formation 3/10/2021 19

Thank You 3/10/2021 20

Thank You 3/10/2021 20