In Vitro Effects of Natrum muriaticum in Kidney Cell Lines MDCK and LLC-PK1

Authors

  • Rafael Harduim
  • Venício Veiga
  • Vanessa Baldez
  • Marcelo Einicker-Lamas
  • Carla Holandino

Keywords:

Homeopathy, Natrum muriaticum, hypertension, MDCK, LLC-PK1.

Abstract

Previous papers have indicated that homeopathic solutions modify the cellular and biochemical aspects of cells maintained in culture. In this study, the effects of Natrum muriaticum, a medicine used in the homeopathic clinic for the treatment of hypertension, were evaluated in kidney MDCK and LLC-PK1 cell lines. The following cellular parameters were analyzed: viability, morphology and expression of the (Na+-K+)-ATPase and the angiotensin II receptors AT1 and AT2. The cell lines were plated (5.0 x 104 cells/mL) in DMEM supplemented with 10% fetal calf serum (FCS). After 24 hours at 37°C, DMEM was re-fed with the addition of 10% (V/V) and 1% (V/V) of the following samples: Natrum muriaticum 30CH, water 30CH and non-dynamized sterile distilled water to do the MTT assay. The results obtained from these groups were compared to those obtained by incubation of the cells in culture medium free of these solutions (Control). Cell viability was assessed by a colorimetric MTT ELISA assay (490nm). The values from four independent experiments performed in quintuplicate were plotted and statistically analyzed by Sigma Plot v.11 (Jandel Scientific). The morphology of MDCK cells was evaluated by optical microscope after Giemsa’s staining. The expression of the (Na++K+)-ATPase and AT1/AT2 of LLC-PK1 cells was evaluated by Western Blot (WB) analysis. For this experimental set, 5.0x104 cells/mL were incubated in DMEM supplemented with 10% FCS and daily culture medium was replaced by a new one, containing: Natrum muriaticum 30CH and water 30CH. Additionally, cells were treated for 5, 10 and 15 days with 1% of specific solutions and the total protein was measured by the Lowry method, after cell lysis. The samples were analyzed by electrophoresis in SDS-PAGE (12% gel) and transferred to nitrocellulose membrane. This membrane was incubated with specific primary antibodies (anti-(Na++K+)-ATPase, anti-AT1 and AT2 or anti-anti-beta-actin). The detection was performed using ECL system and Hyperfilm. MTT assays showed a statistically significant reduction in cellular mitochondrial activity (p<0.001) probably attributed to an osmotic effect due to the use of 10% (V/V) concentration rather than 1% (V/V). The optical microscopy analysis revealed no significant morphological changes in MDCK and LLC-PK1 cells submitted to the different treatments when compared to controls groups. The WB analysis indicated a proportional increase in (Na++K+)-ATPase content according to an increase in the homeopathic stimuli. Although preliminary, these results show for the first time, that the homeopathic medicine is able to modify the expression of important physiological markers for LLC-PK1 cells, which are directly involved in the genesis of hypertension.

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Published

2011-09-13

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