Figure 15: Upregulation of NLRP3 mRNA expression of mDCs after stimulation with NaOx. mDCs prestimulated for 3 hours with LPS (10 ng/ml) (20 µg/ml) and for 6hours with NaOx (0, 6 and 100 µg/ml). Data is representative of one experiment, n=1. Values are ± SEM from 2 replicates/group. *p<0.05; vs cells cultured in LPS only medium without NaOx.
6.9 Physiological role of oxalate transporter SCL26A6
MΦs play an important role in inflammatory kidney diseases. In the setting of crystal nephropathy, MΦs are known to accumulate in the renal tissue where they surround, internalize and degrate the crystals [52]. During these degradation steps, cytokine production of the MΦs can be detected. In order to understand oxalate nephropathy, the transport of oxalate into and out of the cells becomes a target of interest. As mentioned above, several transporters from the Slc26 gene family are able to transport oxalate [43]. Existing data has demonstrated a wide expression of Slc26a6. My group found out that murine MΦs are able to transport oxalate across their cell membrane. Through RNA-messenger analysis of potential oxalate transporters as well as oxalate uptake experiments, Slc26a6, could be isolated as the transporter with the highest specificity for oxalate [85].The activity of Slc26a6 in the kidney and the gut is well investigated and understood to date. Deficiency of the Slc26a6 transporter in mice was found to be a model of enteric hyperoxaluria, as it leads to increased net absorption of ingested oxalate. The elevated plasma oxalate itself leads to increased filtration and ultimately to hyperoxaluria with CaOx nephrolithiasis.
6.9.1 SLC26A6 expression on human DCs
As aforementioned, studies of my group have shown expression of Slc26a6 transporter on the cell membrane of murine MΦs. To extend this finding, SCL26A6 expression on the cell membrane of human immune cells was screened. Cell lysates of hDCs were collected and tested in a Western Blot by use of an antibody directed against SLC26A6 (called R29). Two controls were included, one sample of a murine duodenum lysate that is known to express Slc26a6, as a positive control and one duodenum lysate of a Slc26a6-/- mouse, as a negative control. As shown in Figure 16, we were able to detect SLC26A6 expression in the cell lysates of hDC.
6.9.2 Testing a hypothesis of enhanced inflammasome activation by elevated oxalate in Slc26a6-/- macrophages
Recent findings of uptake experiments with radiolabeled C14 oxalate in my work group demonstrate a significantly higher accumulation of oxalate in MΦs from Slc26a6-/- compared to WT MΦs after 18 hours of oxalate stimulation. This finding leads to the assumption that the Figure 16: Detection of SLC26A6 protein in cell lysates of hDCs. Western analysis of hDCs lysates isolated from two healthy donors (A, B) and two small intestine homogenates isolated from one WT mouse (C) and one Slc26a6-/- mouse (D). The upper portion of the blot was probed with the anti SLC26A6 antibody R29 in a 1:100’000 dilution, the lower portion was probed with a GAPDH antibody in a 1:5000 dilution for use as a protein loading control. Exposure time 1sec.
Ponceau S staining in bottom part.
Slc26a6 transporter mediates oxalate efflux that might prevent the cells from an oxalate overload under long term stimulation conditions. Regarding these results, we hypothesized that stimulation of Slc26a6-/- MΦs with NaOx leads to a comparably higher intracellular oxalate and therewith amplifies the inflammasome activation and with it the concentration of proinflammatory cytokines in the cell supernatant. To test the hypothesis, MΦs of both WT and Slc26a6 -/-background were isolated and seeded in 24 well plates. Referring to previous stimulation experiments, cells were first pre-stimulated for 3 hours with LPS, then for 6 hours with various concentration of sodium oxalate.
Figure 17: Hypothesis of elevated intracellular oxalate in cells of Slc26a6-/- mice leading to an amplified inflammasome activation and increased cytokine secretion. (A) Slc26a6 transporter is responsible for oxalate efflux in exchange with chloride ions. (B) In cells from Slc26a6-/- mice, soluble oxalate accumulates in the cells.
As shown in Figure 18 this experiment was not able to prove the hypothesis as illustrated above.
While IL-1α release of Slc26a6-/- MΦs was significantly higher than in WT MΦs, IL-1β release was reduced.
Figure 18: Comparison of cytokine release of MΦs from WT and Slc26a6-/- mice after stimulation with NaOx. Fold change IL-1α (A.) and IL-1β (B) release of MΦs from WT and Slc26a6-/- mice after pre-stimulation with 10 ng/ml of LPS and stimulation with NaOx in 3 different concentrations (6, 12, 100 µg/ml) for 6 hours. All values were normalized to the LPS only change. Data is representative of three experiments, n=3. Values are ± SEM from 2 replicates/group.
6.9.4 Comparison of macrophage cell viability of cells from WT and Slc26a6-/- mice following incubation with soluble oxalate
In order to further clarify the role of the Slc26a6 transporter in MΦs and to demonstrate a difference in the effect of oxalate on cells of WT and Slc26a6-/- mice, a new experiment was designed. In this experiment cells of WT and Slc26a6-/- mice were seeded in equal concentrations of 1*10^4 cells as well as 2*20^4 per well in a 96-well plate. Cells were stimulated in 3 different conditions. Medium only, NaOx in a concentration of 30 µg/ml and NaOx in a concentration of 100 µg/ml. After 12 hours of stimulation WST-1 reagent was added and absorbance was measured at two different time points, 1 hour and 2 hours after addition of the reagent. As shown in Figure 19 we were able to observe a significantly elevated cell viability of the cells of WT mice as compared to the cells isolated from Slc26a6-/- mice, and a greater loss of viability of the cells from Slc26a6-/- mice.
Figure 19: MΦs isolated from Slc26a6-/- mice show less cell viability after incubation with NaOx compared to MΦs from WT mice. Comparison of cell viability of cells of WT and Slc26a6-/- mice after 12 hours stimulation with NaOx (100 µg/ml). (A) Relative cell viability after 2 hours of incubation with WST-1, cell density: 1*10^4 per well. (B) Loss of viability after 2 hours of incubation with WST-1, cell density: 1*10^4 cells per well. For each condition 5-6 replicates were used. Data is representative of three experiments, n=3. Values are ± SEM from 4 replicates/group_**p<0.01;_***p<0.001.