Functional distinctions in cytosolic calcium regulation between cells of the glomerular filtration barrier

Abstract

The importance of intracellular calcium ([Ca(2+)]i) regulation in the glomerular filtration barrier (GFB) has recently been highlighted by mutations in the cation channel TRPC6, resulting in a renal-specific phenotype. We examined the effects of FFA, a tool that can activate TRPC6, on [Ca(2+)]i in human conditionally immortalised glomerular endothelial cells (ciGEnC) and human podocytes (ciPod) that form the GFB. Changes in [Ca(2+)]i stimulated by FFA were measured in Fura 2-AM loaded cells. In GEnC, cell activation by FFA was dependent on external Ca(2+), yet in ciPod it was not. Depletion of internal Ca(2+) stores with thapsigargin did not affect cell activation by FFA in ciGEnC, but inhibited it in ciPod in a nephrin-dependent manner, demonstrated using nephrin deficient (ND) ciPod in conjunction with nephrin rescue experiments. FFA induced [Ca(2+)]i store release in ciPod, but not in ciGEnC or ND ciPod. In parallel, there were differences in the localisation of overexpressed TRPC6 between ciGEnC and ciPod. Furthermore, co-transfection of nephrin with TRPC6 in HEK293 cells reduced the FFA-induced increase in [Ca(2+)]i and nephrin clustering altered TRPC6 distribution. In conclusion, cell activation by FFA in podocytes stimulates the opening of a Ca(2+) channel, probably TRPC6, in a nephrin-dependent manner with a different activation profile to GEnC.

Fig. 1

The FFA induced Ca²⁺ signalling is dependent on external Ca²⁺ in ciGEnC, but not in ciPod. ciGEnC and ciPod were loaded with Fura-2AM and stimulated with 200 μM FFA in either Krebs-Ringer phosphate buffer containing normal or minimal. (A) Representative traces of ciGEnC stimulated with FFA in normal and minimal extracellular Ca²⁺. (B) Summary of area under the curve for experiments in (A) (paired t-test, n = 9 and 6 respectively). (C) Representative trace of ciPod stimulated with FFA in normal and minimal extracellular Ca²⁺. (D) Summary of area under the curve for experiments in (C) (unpaired t-test, n = 7 and 6 respectively). *p < 0.05, **p < 0.01.

Fig. 2

The FFA induced Ca²⁺ activation is store-independent in GEnC but is store-dependent in ciPod. GEnC and ciPods loaded with Fura-2AM were pre-incubated with TG to deplete stores, then stimulated with FFA. (A) An example of an entire experiment in ciGEnC showing response to vehicle, TG then FFA, ionomycin (IM) then quench (MnCl). (B) Representative trace of the response to FFA in GEnC after store depletion, taken from (A). (C) Representative trace of the response to FFA in ciPod after store depletion. (D) Summary of data represented in (B) and (C) presented as area under the curve above 1, unpaired t-test, n = 6 and 5 respectively *p < 0.05.

Fig. 3

The FFA induced Ca²⁺ activation is store-independent in the absence of nephrin in ciPods. ND ciPods were loaded in Fura-2AM, then stimulated with FFA in normal or minimal extracellular Ca²⁺ with or with out pre-incubation with thapsigargin (TG). (A) Representative trace of ND ciPod stimulated with FFA in normal Ca²⁺ compared to previous results in WT ciPod and ciGEnC (dashed lines). (B) Representative trace of ND ciPod stimulated with FFA in minimal Ca²⁺ compared to previous results in WT ciPod and ciGEnC (dashed lines). (C) Summary of mean area under the curve for ND ciPod experiments in (A) and (B), n = 6 and 6. Unpaired t-test. (D) Representative trace demonstrating the effect of FFA after depletion of stores in ND ciPod compared to previous results in WT ciPod and ciGEnC (dashed lines). (E) Summary of area under the curve above 1 for experiments shown in (D), including previous results for WT ciPod and ciGEnC, One way ANOVA n = 5, p < 0.05. ***p < 0.001.

Fig. 4

Nephrin rescue in ND ciPod modifies FFA signalling to that of WT ciPod in normal and minimal Ca²⁺ conditions. Proliferating ND ciPods are readily transfectable compared to differentiated ND ciPods and were therefore used to rescue nephrin expression in these cells. (A) Proliferating ND ciPods were transfected with plasmid alone or with nephrin. Cell and glomerular (glom) lysates were western blotted for nephrin. Proliferating ND ciPods, transfected with nephrin or empty plasmid were loaded with Fura-2AM. (B) Representative traces of the effect of FFA in ND ciPods with and without nephrin rescue. (C) Representative traces of the effect of FFA in minimal Ca²⁺ on ND ciPod with and without nephrin rescue Experiments from (B) and (C) are summarised in (D), n = 5, 4, 5 and 5 respectively. One way ANOVA p < 0.0001, Bonferroni post hoc tests indicated. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5

Nephrin modifies the Ca²⁺ signal induced by FFA in ciGEnC. ciGEnC were successfully transfected with wild type nephrin (n = 3). (A) Glomeruli were used to indicate the expected MW of nephrin (180 kDa) and actin was used to demonstrate equal loading. GEnC, transfected with nephrin or pcDNA3 were loaded with Fura-2AM. (B) Representative traces of the effect of FFA in GEnC with and without transfected nephrin in normal Ca²⁺. (C) Representative traces of the effect of FFA in minimal Ca²⁺ on GEnC with and without transfected nephrin. Experiments from (B) and (C) are summarised in (D), n = 6, 4, 6 and 4 respectively. One way ANOVA p < 0.01, Bonferroni post hoc tests indicated. *p < 0.05, **p < 0.01, n.s. = non significant.

Fig. 6

Cell activation by FFA does not affect release from Ca²⁺ stores in GEnC, but induces release from Ca²⁺ stores in ciPod in a nephrin dependent manner. GEnC, WT ciPod and ND ciPod, loaded in Fura-2AM were stimulated first with FFA, then TG. (A) A representative trace of ciGEnC stimulated with FFA followed by TG. (B) Representative traces of TG in ciGEnC, WT ciPod and ND ciPod that were first stimulated with FFA (store release after FFA) as in A. (C) All cell types were stimulated with TG in minimal Ca²⁺ without FFA to assess total store capacity. The amount of store release by TG after FFA treatment was then expressed as a percentage of total store capacity for each cell type, n = 5, n = 7 and 5 respectively. One way ANOVA p < 0.001. Bonferroni post hoc test significances indicated ***p < 0.001, **p < 0.01 n.s. = not significant.

Fig. 7

Expression and distribution of TRPC6 in ciGEnC and ciPod compared to a platelet positive control. Lysates from ciPod and cGEnC were Western blotted and probed using anti-TRPC6 and anti-actin antibodies. (A) Representative images of ciGEnC (Bi) and ciPod (Bii) were microinjected with pcDNA3TRPC6 and immunofluorescence was carried out using an anti-TRPC6 antibody. Cells were imaged using confocal microscopy. X–Y stacks of ciGEnC and ciPod in (B) are shown in (Ci) and (Cii) respectively.

Fig. 8

Nephrin clustering affects the distribution of TRPC6 in HEK293 cells and potentially FFA induced changes in [Ca²⁺]_i. HEK293 were transiently transfected with TRPC6 and CD16-nephrin for 24 h. Cells were incubated with either secondary antibody alone (Ai–iii) or 1 mg/ml anti-CD16, then secondary antibody (Bi–iii) before fixing and immunostaining for TRPC6. Dapi nuclear staining is also shown in the merged images. Transfected HEKs, loaded with Fura-2AM were incubated in 1 mg/ml anti-CD16 for 10 min or vehicle, then incubated with 1 mg/ml mouse IgG for 10 min and finally stimulated with 200 μM FFA. Representative traces are shown for cells stimulated with FFA that were transfected with TRPC6 and pcDNA3 or TRPC6 and CD16-nephrin under control and experimental conditions (C). Data from C are summarised in (D) as area under the curve (p < 0.01 One way ANOVA, n = 3, 6 and 5 respectively, Bonferroni post hoc tests indicated *p < 0.05, **p < 0.01).