Oxalate induces mitochondrial dysfunction and disrupts redox homeostasis in a human monocyte derived cell line

Abstract

Monocytes/macrophages are thought to be recruited to the renal interstitium during calcium oxalate (CaOx) kidney stone disease for crystal clearance. Mitochondria play an important role in monocyte function during the immune response. We recently determined that monocytes in patients with CaOx kidney stones have decreased mitochondrial function compared to healthy subjects. The objective of this study was to determine whether oxalate, a major constituent found in CaOx kidney stones, alters cell viability, mitochondrial function, and redox homeostasis in THP-1 cells, a human derived monocyte cell line. THP-1 cells were treated with varying concentrations of CaOx crystals (insoluble form) or sodium oxalate (NaOx; soluble form) for 24h. In addition, the effect of calcium phosphate (CaP) and cystine crystals was tested. CaOx crystals decreased cell viability and induced mitochondrial dysfunction and redox imbalance in THP-1 cells compared to control cells. However, NaOx only caused mitochondrial damage and redox imbalance in THP-1 cells. In contrast, both CaP and cystine crystals did not affect THP-1 cells. Separate experiments showed that elevated oxalate also induced mitochondrial dysfunction in primary monocytes from healthy subjects. These findings suggest that oxalate may play an important role in monocyte mitochondrial dysfunction in CaOx kidney stone disease.

Keywords: Calcium oxalate; Kidney stones; Mitochondria; MnSOD; Monocytes; Sodium oxalate.

Graphical abstract

Fig. 1

The effect of (A) calcium oxalate (CaOx) crystals, (B) calcium phosphate (CaP) crystals, (C) cystine crystals, and (D) sodium oxalate (NaOx) on THP-1 cell viability. Results are means ± SEM; n = 3–5 individual experiments. *p < 0.05 compared to untreated monocytes.

Fig. 2

The effect of calcium oxalate (CaOx) crystals on monocyte mitochondrial function. Cells were pretreated with CaOx crystals (0, 50, 100, 200, 500, 1000 µg/ml) for 24 h prior to being seeded on Seahorse XF96 plates. Mitochondrial function was determined using the “Mito Stress Test” inhibitors: oligomycin (Oligo), FCCP, and antimycin A (AA). The effect of CaOx crystals on (A) mitochondrial OCR and (B) individual parameters in THP-1 cells. Results are means ± SEM; n = 5–6 replicates per group; n = 3–5 individual experiments. *p < 0.05 compared to untreated monocytes.

Fig. 3

The effect of sodium oxalate (NaOx) on monocyte mitochondrial function. Cells were pretreated with NaOx (0, 0.1, 0.5, 1, 1.5, and 2 mM) for 24 h prior to being seeded on Seahorse XF96 plates. Mitochondrial function was determined using the “Mito Stress Test” inhibitors: oligomycin (Oligo), FCCP, and antimycin A (AA). The effect of NaOx on (A) mitochondrial OCR and (B) individual parameters in THP-1 cells. Results are means ± SEM; n = 5–6 replicates per group; n = 3–5 individual experiments. *p < 0.05 compared to untreated monocytes.

Fig. 4

The effect of calcium oxalate (CaOx) crystals and sodium oxalate (NaOx) on MnSOD gene expression and protein levels and glutathione levels in THP-1 cells. (A and B) Real-time PCR was performed to determine MnSOD and GAPDH expression in THP-1 cells treated with CaOx crystals or NaOx. MnSOD was normalized to GAPDH (housekeeping gene). Relative fold change was calculated using the ΔΔCT method relative to control samples. (C and D) Representative western blot of MnSOD (25 kDa) and GAPDH (37 kDa) protein levels in THP-1 cells treated with CaOx crystals or NaOx. (E and F) GSH (reduced glutathione)/GSSG (oxidized glutathione) ratio in THP-1 cells treated with CaOx crystals or NaOx. Results are means ± SEM; n = 3–5 individual experiments. *p < 0.05 compared to untreated monocytes.

Fig. 5

The effect of calcium oxalate (CaOx) crystals on mitochondrial function in primary monocytes. Monocytes were treated with or without CaOx crystals (50 µg/ml) for 40 min prior to assessing mitochondrial function using the “Mito Stress Test” inhibitors: oligomycin (Oligo), FCCP, and antimycin A (AA). The effect of CaOx crystals on (A) mitochondrial OCR and (B) individual parameters in primary monocytes. Results are means ± SEM; n = 5–6 replicates per group; n = 7 healthy subjects.

Fig. 6

The effect of sodium oxalate (NaOx) on mitochondrial function in primary monocytes. Monocytes were treated with or without NaOx (0.1 mM) for 40 min prior to assessing mitochondrial function using the “Mito Stress Test” inhibitors: oligomycin (Oligo), FCCP, and antimycin A (AA). The effect of NaOx on (A) mitochondrial OCR and (B) individual parameters in primary monocytes. Results are means ± SEM; n = 5–6 replicates per group; n = 6 healthy subjects.

Fig. S1

The effect of (A) calcium phosphate (CaP) and (B) cystine crystals on monocyte mitochondrial function. Cells were pretreated with CaP or cystine crystals (0, 50, 100, 200, 500, 1000 µg/ml) for 24 h prior to being seeded on Seahorse XF96 plates. Mitochondrial function was determined using the “Mito Stress Test” inhibitors: oligomycin (Oligo), FCCP, and antimycin A (AA). Results are means ± SEM; n = 5–6 replicates per group; n = 3–5 individual experiments.

Fig. S2

The effect of calcium phosphate (CaP) and cystine crystals on MnSOD gene expression and protein levels in THP-1 cells. (A and B) Real-time PCR was performed to determine MnSOD and GAPDH expression in THP-1 cells treated with CaP or cystine crystals. MnSOD was normalized to GAPDH (housekeeping gene). Relative fold change was calculated using the DDCT method relative to control samples. (C and D) Representative western blot of MnSOD (25 kDa) and GAPDH (37 kDa) protein levels in THP-1 cells treated with CaP or cystine crystals. Results are means ± SEM; n = 3–5 individual experiments. *p < 0.05 compared to untreated monocytes.