Higher Concentrations of Folic Acid Cause Oxidative Stress, Acute Cytotoxicity, and Long-Term Fibrogenic Changes in Kidney Epithelial Cells

Abstract

Kidney fibrosis is a common step during chronic kidney disease (CKD), and its incidence has been increasing worldwide. Aberrant recovery after repeated acute kidney injury leads to fibrosis. The mechanism of fibrogenic changes in the kidney is not fully understood. Folic acid-induced kidney fibrosis in mice is an established in vivo model to study kidney fibrosis, but the mechanism is poorly understood. Moreover, the effect of higher concentrations of folic acid on kidney epithelial cells in vitro has not yet been studied. Oxidative stress is a common property of nephrotoxicants. Therefore, this study evaluated the role of folic acid-induced oxidative stress in fibrogenic changes by using the in vitro renal proximal tubular epithelial cell culture model. To obtain comprehensive and robust data, three different cell lines derived from human and mouse kidney epithelium were treated with higher concentrations of folic acid for both acute and long-term durations, and the effects were determined at the cellular and molecular levels. The result of cell viability by the MTT assay and the measurement of reactive oxygen species (ROS) levels by the DCF assay revealed that folic acid caused cytotoxicity and increased levels of ROS in acute exposure. The cotreatment with antioxidant N-acetyl cysteine (NAC) protected the cytotoxic effect, suggesting the role of folic acid-induced oxidative stress in cytotoxicity. In contrast, the long-term exposure to folic acid caused increased growth, DNA damage, and changes in the expression of marker genes for EMT, fibrosis, oxidative stress, and oxidative DNA damage. Some of these changes, particularly the acute effects, were abrogated by cotreatment with antioxidant NAC. In summary, the novel findings of this study suggest that higher concentrations of folic acid-induced oxidative stress act as the driver of cytotoxicity as an acute effect and of fibrotic changes as a long-term effect in kidney epithelial cells.

Figure 1.

Effect of acute exposure to folic acid (FA) on the growth of kidney epithelial cells as measured by the MTT assay (upper panel: A–C) and on the level of intracellular ROS as measured by the DCF assay (lower panel: D–F). Upper panel: Bar graph representing the cell viability (in percentage) in FA-treated Caki-1 cells (A), HK-2 cells (B), and NRK cells (C) as compared to their respective untreated control cells (as 100%). Lower panel: Bar graph representing DCF fluorescence (in percentage) in FA-treated Caki-1 cells (D), HK-2 cells (E), and NRK cells (F) as compared to their respective untreated control cells (as 100%). Different concentrations (as given in the graph) of FA either alone or in combination with 25 μM NAC were used for cotreatment. Error bar showing the standard deviation (±SD) of the mean of triplicate values. Statistically significant (P < 0.05) changes as compared to control are shown by the symbol “*”. A statistically significant change in the FA and NAC cotreated group as compared to FA alone is shown by the symbol “#”.

Figure 2.

Histogram of flow cytometry data showing the effects of FA exposure on the cell population in G0/G1, S, and G2/M phases of the cell cycle from Caki-1 cells. Cells were given acute treatment with different concentrations of FA alone or cotreated with FA and NAC combination, and the population of the cells in various phases of the cell cycle was analyzed by flow cytometry as described in the Materials and Methods.

Figure 3.

Effect of acute and long-term exposure to folic acid (FA) on the expression of marker genes for cell proliferation/survival (cyclin D1, survivin, and Bcl-2), antioxidant (GPx1 and MnSOD), and DNA damage (OGG1 and PARP1) in Caki-1 cells as measured by quantitative real-time PCR. Bar graphs represent the fold changes in the expression of the cell proliferation marker genes (A) and antioxidants as well as DNA repair genes (B) in acute (acute FA) and long-term (LT-FA) FA-treated Caki-1 cells as compared to the untreated control cells (control = 1-fold). Error bar showing the standard deviation (±SD) of the mean of triplicate values. Statistically significant (P < 0.05) changes in treated groups as compared to control are shown by the symbol “*”. The statistical significance of the cotreatment of NAC as compared to folic acid alone acute treatment is shown by the symbol “#”, whereas statistically significant changes in the cotreatment of NAC and FA as compared to FA alone in long-term treated cells are shown by the symbol “%”.

Figure 4.

Effect of long-term exposure to folic acid (FA) on the growth and intracellular ROS production in kidney epithelial cells. Bar graph representing cell viability in percentage (control = 100%) in long-term FA-treated cells as measured by the MTT assay in Caki-1 cells (A), as well as intracellular levels of ROS in Caki-1 cells (B), HK-2 cells (C), and NRK cells (D). Different concentrations of FA as mentioned in the graph were used both alone and in combination with 25 μM of NAC. Error bars represent the standard deviation (±SD) of the mean of triplicate values. Statistically significant (P < 0.05) changes in treated groups as compared to control are shown by the symbol “*”. Statistically significant changes in the NAC and FA cotreatment groups as compared to FA alone are shown by the symbol “#”. Statistically significant changes in the NAC and FA cotreatment groups as compared to the untreated control are shown by the symbol “∞”.

Figure 5.

Effect of acute and long-term exposure to folic acid (FA) on the expression of marker genes transcripts for fibrosis and EMT (in Caki-1 cells as measured by quantitative real-time PCR (qRT-PCR)) (A,B) and on the expression of E-cadherin, β-catenin, and GSTP1 proteins as measured by Western blot analysis (C,D). Bar graphs represent the fold changes in the expression of gene transcripts of fibronectin and alpha-SMA (A), and E-cadherin, N-cadherin, and vimentin (B), in both acute (acute FA) and long-term (LT-FA) FA-treated Caki-1 cells as compared to untreated control cells (control = 1-fold). Images of Western blots of E-cadherin, β-catenin, GSTP1, and GAPDH (internal control) proteins in Caki-1 cells (C), and their band intensities (in arbitrary units) as measured by ImageJ software (D). The band intensity of each protein was normalized by the band intensity of the internal control GAPDH from each sample, and the bar graph was plotted using values in arbitrary units. Error bar showing the standard deviation (±SD) of the mean of triplicate values. Statistically significant (P < 0.05) changes in treated groups as compared to the control are shown by the symbol “*”. A statistical significance of the cotreatment of NAC as compared to folic acid alone acute treatment is shown by the symbol “#”, whereas statistically significant changes in the cotreatment of NAC and FA as compared to FA alone in long-term treated cells are shown by the symbol “%”.

Figure 6.

Microscopic images of the immunofluorescence detection of fibronectin in long-term FA-treated and untreated control Caki-1 and HK-2 cells. Representative immunofluorescence microscopic photographs showing the expression of fibronectin in untreated control and long-term FA-treated cells of Caki-1 (upper two panels) and HK-2 (lower two panels). Fibronectin staining is in red, whereas the nuclear staining by DAPI is in blue. The immunofluorescence staining of fibronectin was performed as described in the Materials and Methods. Scale bar = 100 μm.

Figure 7.

Representative RAPD fingerprints showing genotoxicity at the DNA sequence level in long-term FA-treated Caki-1 cells. RAPD-PCR fingerprints were generated by using primer OPK-17 (lanes 2–9) and OPK-19 (lanes 10–17). Samples in each lane are as follows: lane 1, DNA size marker; lanes 2 and 10, control; lanes 3 and 11, control + NAC; lanes 4 and 12, LT-FA 100 μM; lanes 5 and 13, LT-FA 100 μM + NAC; lanes 6 and 14, LT-FA 200 μM; lanes 7 and 15, LT-FA 200 μM + NAC; lanes 8 and 16, LT-FA 400 μM; and lanes 9 and 17, LT-FA 400 μM + NAC.