Reorganization and Suppression of Store-Operated Calcium Entry in Podocytes of Type 2 Diabetic Rats

Abstract

Type 2 diabetes mellitus (DM2) is a widespread metabolic disorder that results in podocyte damage and diabetic nephropathy. Previous studies demonstrated that TRPC6 channels play a pivotal role in podocyte function and their dysregulation is associated with development of different kidney diseases including nephropathy. Here, using single channel patch clamp technique, we demonstrated that non-selective cationic TRPC6 channels are sensitive to the Ca²⁺ store depletion in human podocyte cell line Ab8/13 and in freshly isolated rat glomerular podocytes. Ca²⁺ imaging indicated the involvement of ORAI and sodium-calcium exchanger in Ca²⁺ entry induced upon store depletion. In male rats fed a high-fat diet combined with a low-dose streptozotocin injection, which leads to DM2 development, we observed the reduction of a store-operated Ca²⁺ entry (SOCE) in rat glomerular podocytes. This was accompanied by a reorganization of store-operated Ca²⁺ influx such that TRPC6 channels lost their sensitivity to Ca²⁺ store depletion and ORAI-mediated Ca²⁺ entry was suppressed in TRPC6-independent manner. Altogether our data provide new insights into the mechanism of SOCE organization in podocytes in the norm and in pathology, which should be taken into account when developing pharmacological treatment of the early stages of diabetic nephropathy.

Keywords: ORAI channels; TRPC6 channels; diabetic nephropathy; sodium–calcium exchanger (NCX); store-operated calcium entry (SOCE); type 2 diabetes mellitus.

Figure 1

Ca²⁺ store depletion activates TRPC6 channels in human culture podocytes Ab8/13. (A) Representative traces of currents recorded in cell-attached configuration in culture podocytes Ab8/13 at holding potential of −40 mV. Shown are basal currents and currents after bath application of 1 µM thapsigargin (Tg). Fragments of the recordings and corresponding amplitude histogram after Tg application are shown at the bottom on an expanded time scale. (B) Representative traces of currents recorded in inside-out configurations in Ab8/13 podocytes at holding potential of −40 mV. Shown are basal currents and currents after bath application of 10 µM hyperforin 9 (Hyp9). (C) The current–voltage relationship of observed TRPC6 channels in response to Tg and Hyp9. (D) The open channel probability collected during 30 s of maximal activity (NPomax30) before and after bath application of 1 µM Tg. Data presented as mean bar plots and paired data points were statistically analyzed using paired sample Wilcoxon signed-rank test. (E) The frequency of TRPC6 channel observation as portion of experiments. In bar plot are shown the number of experiments with TRPC6 activity over total number of performed experiments. Data were statistically analyzed by Barnard’s exact test. * and ** indicate p values of <0.05 and <0.01, respectively.

Figure 2

TRPC6 channels modulate store-operated Ca²⁺ entry mediated by ORAI and NCX in human culture podocytes Ab8/13. (A,C) An averaged traces demonstrating ratio of fura-2 fluorescence at 340 and 380 nm, which reflect changes in intracellular Ca²⁺ concentration in human podocytes Ab8/13. Cells were incubated with 2 µM Tg in 0 mM Ca²⁺ solution for 10 min and afterwards 2 mM Ca²⁺ solution was added to register store-operated Ca²⁺ entry. (B,D) Bar plots showing the normalized fura-2 fluorescence ratio integrated over 100 s right after addition of 2 mM Ca²⁺. (A,B) Shown are control Ca²⁺ response (black), Ca²⁺ response in the presence of 1 µM CM4620 (green), Ca²⁺ response in the presence of 1 µM SAR7334 (blue) and Ca²⁺ response in the presence of 5 µM SEA0400 (light blue). (C,D) Ca²⁺ entry recorded in the presence of 5.4 mM KCl used as control (black), 0.5 mM KCl (gold), 50 mM KCl (dark red). Data were statistically analyzed by Kruskal–Wallis test followed by Dunnett’s test. *, and *** indicate p values of <0.05, and <0.001, respectively.

Figure 3

High-fat diet and STZ injection results in the glomerular hypertrophy and mesangial expansion. (A) Masson Trichrome stained slices from control (Ctr) and diabetic (DM2) right kidney. Scale bar equal 50 µm. (B) The averaged data obtained from analysis of right kidney slices (N = 9 rats per group; n= 450 glomeruli per group). Data were statistically analyzed by Student t-test. n.s., not significant, ** and *** indicate p values of <0.01, and <0.001, respectively.

Figure 4

Diabetes development cancels the regulation of TRPC6 channels by Ca²⁺ store depletion in rat podocytes. (A,B) Representative traces of TRPC6 currents recorded in cell-attached configuration in freshly isolated rat glomerular podocytes from control (A) and diabetic (B) groups. The membrane potential was held at −40 mV. Shown are basal currents and currents after bath application of 1 µM thapsigargin (Tg). Fragments of the recordings are shown at the bottom on an expanded time scale. (C) The current–voltage relationship and reversal potential of observed TRPC6 channels. Data were analyzed by two-tailed Student t-test. (D) Left—the open channel probability collected during 30 s of maximal activity (NPomax30) before and after bath application of 1 µM Tg. Data are presented as mean bar plots and paired data points. Line indicates the median value. Data were statistically analyzed by Kruskal–Wallis test followed by Dunnett’s test. (D) Right—the frequency of TRPC6 channel activity observation as percentage of all patches with TRPC6 activity in the group. In the bars are shown the number of active recordings at these conditions over total number of recordings with activity. Data were statistically analyzed by Barnard’s exact test. * indicate p values of <0.05.

Figure 5

Diabetes leads to the increase in TRPC6 mRNA expression level. Shown are averaged data and individual data points for expression of Nephrin, Orai1, TRPC6, STIM1 and STIM2 mRNA. Data were analyzed by two-tail Mann–Whitney test. * indicate p values of <0.05.

Figure 6

Store-operated Ca²⁺ entry is decreased and receptor-activated Ca²⁺ entry is increased in glomerular podocytes from diabetic rats. (A,B) An averaged ratio of fura-2 fluorescence at 340 and 380 nm, which reflect changes in intracellular Ca²⁺ concentration in control (black) and diabetic (red) podocytes. Podocytes were stimulated with 2 µM Tg or 10 µM Ang II for 4 min in 0 mM Ca²⁺ solution prior to 2 mM Ca²⁺ addition. The bars above the graph indicate the time when 0 or 2 mM Ca²⁺ was used, when 2 µM Tg, 10 µM Ang II or 50 µM 2-APB were added. (C) Bar plots summarizing the analyzed parameters from control (Ctr, black) and diabetic (DM2, red) groups measured from the data shown in panel A and B. Indicated are resting fluorescence ratio (Frest, which reflects the basal level of [Ca²⁺]_i), an averaged amplitude of Tg-induced Ca²⁺ entry (dF-Tg, measured from experiments shown in panel A), an averaged amplitude of Ang II-induced Ca²⁺ entry (dF-Ang, measured from experiments shown in panel B), and averaged fluorescence obtained after application of 50 µM 2-APB (F-APB, measured from experiments shown in panel A). Data were analyzed by Mann–Whitney test. * indicate p values of <0.05.

Figure 7

An inhibitory analysis of Ca²⁺ influx into the rat glomerular podocytes. (A–C) Shown are averaged fura-2 fluorescence ratio in response to Ca²⁺ store depletion by application of 2 µM Tg for 4 min in glomerular podocytes from control (Ctr, black) and diabetic (DM2, red) rats. Left—original traces aligned to the fluorescence ratio level before Ca²⁺ addition; shown are traces recorded in the presence of DMSO or indicated inhibitor. Right—difference between Ca²⁺ response in the presence of DMSO minus Ca²⁺ response in the presence of indicated inhibitor, the ordinate axis reflects the positive and negative contribution of the indicated pathway, estimated by the effect of corresponding inhibitor. Inhibitors were applied in 0 mM Ca²⁺ for 4 min together with 2 µM Tg and kept till the end of experiment. 1 µM SAR7334 (panel A), 5 µM SEA0400 (panel B) and 1 µM CM4620 (panel C) were used. The scale bars shown on the left are the same for all panels. Traces shown as Mean ± S.E.M. values.