Dysregulation of Podocyte BK Channels and Nephrosis: Effects of Circulating Factors and Auxiliary β4 Subunits

Abstract

Podocytes express large-conductance Ca²⁺-activated K⁺ channels (BK channels) and at least two different pore-forming KCa1.1 subunit C-terminal splice variants, known as VEDEC and EMVYR, along with auxiliary β and γ subunits. Podocyte KCa1.1 subunits interact directly with TRPC6 channels and BK channels become active in response to Ca²⁺ influx through TRPC6. Here, we confirmed that Ca²⁺ influx through TRPC channels is reduced following the blockade of BK channels by paxilline. The overall abundance of KCa1.1 subunits, as well as that of β4 and γ3 subunits, were increased in glomeruli isolated from Sprague Dawley rats during chronic puromycin aminonucleoside (PAN) nephrosis. Exposing cultured mouse podocytes for 24 h to recombinant TNFα, a circulating factor implicated in pediatric nephrotic syndromes, did not affect the total abundance of KCa1.1, but did evoke significant increases in both β4 and γ3. However, TNFα evoked a marked increase in the surface abundance of KCa1.1 subunits, similar to that of its previously reported effects on TRPC6 channels. The effect of TNFα on the surface expression of KCa1.1 was eliminated following siRNA knockdown of the β4 subunits, suggesting a role for this subunit in KCa1.1 trafficking to the cell surface. By contrast, treating podocytes with suPAR did not affect the total or surface expression of KCa1.1. The coordinated activation of KCa1.1 channels may promote Ca²⁺ influx through TRPC channels during normal and abnormal podocyte function by maintaining a membrane potential that allows for the efficient permeation of divalent cations through TRPC pores.

Keywords: BK channel; TNFα; TRPC6; calcium; circulating factor; nephrosis; podocyte.

Figure 1

Quantitative fluorescence measurement of cytosolic free Ca²⁺ in immortalized podocytes in response to a hypoosmotic stretch stimulus known to cause robust and sustained activation of TRPC6. The Ca²⁺ signal was measured using the indicator dye Calybryte-520™: (a) a hypoosmotic stretch stimulus caused a marked increase in cytosolic free Ca²⁺ that was maintained for as long as the stretch stimulus was maintained, as we have reported previously for TRPC6 channels [13]; (b) pre-exposure for 10 min to the TRPC6 blocker SAR-7334 (100 nM) caused a marked reduction in the amplitude of this signal; and (c) a similar reduction was observed in podocytes pre-treated for 10 min with the BK channel inhibitor paxilline (20 μM). Points represent means amplitudes relative to baseline fluorescence, error bars represent SEM, and the number of cells examined is indicated. The inhibition by paxilline suggests that BK channels allow for enhanced Ca²⁺ influx through TRPC6.

Figure 2

Evidence of kidney disease in Sprague Dawley rats 30-days after treatment with PAN or saline, as indicated. This experimental design is the chronic PAN nephrosis model: (a) increase in spot urine albumin:creatinine ratios (ACR) following PAN treatment; (b) increase in serum creatinine in PAN-treated rats compared to the control, indicating a decrease in the glomerular filtration rate; (c) glomerulosclerosis in PAN-treated rats compared to the saline-treated controls shown in representative PAS-stained section; and (d) mean glomerular score in saline- and PAN-treated rats based on the analyses of PAS-stained sections by an observer blind to the treatment group being scored. Points are results from individual animals. Error bars represent SD. Data were analyzed by Student’s unpaired t-test. In this experiment, there were N = 4 animals in each group.

Figure 3

Increase in the expression of pore-forming and auxiliary subunits of BK channels in whole glomeruli isolated from saline- and PAN-treated rats during chronic PAN nephrosis. Representative immunoblots measuring pore-forming KCa1.1 subunits and auxiliary β4 and γ3 subunits are shown on the left (all from the same animal); quantitative densitometric analyses from the two groups of animals are shown on the right. Graphs show individual values and SD for N = 4 animals in each group.

Figure 4

Effects of TNFα on BK channel subunits. Exposing cultured podocytes to 10 ng/mL TNFα for 24 h does not increase overall abundance of KCa1.1 (a) but increases overall abundance of auxiliary β4 and γ3 subunits (b). Representative blots are shown to the left. Points in graphs on the right are the results from each repetition and error bars represent SD. Data were analyzed by Student’s unpaired t-test.

Figure 5

Exposing cultured podocytes to 20 ng/mL suPAR for 24 h had no effect on the overall abundance of pore-forming KCa1.1 subunits and caused a decrease in auxiliary β4 and γ3. Data were analyzed by Student’s unpaired t-test. n.s. denotes statistically non-significant difference.

Figure 6

Exposing cultured podocytes to TNFα for 24 h increased cell surface expression of KCa1.1 (a); whereas suPAR had no effect (b). Graphs show individual values of the relative expression of surface and total KCa1.1 based on cell-surface biotinylation assays. Error bars represent SD. Data were analyzed by Student’s unpaired t-test. n.s. denotes statistically non-significant difference.

Figure 7

The stimulatory effect of TNFα on the surface expression of KCa1.1 subunits in podocytes was inhibited by siRNA knockdown of β4 subunits: (a) immunoblot analysis showing reduced expression of the β4 subunit by the siRNA targeting that subunit compared to the control siRNA; and (b) representative cell-surface biotinylation assays are shown above the quantitative analyses of these types of experiments. There was a significant difference in the groups by one-way ANOVA (F = 11.05, p < 0.01); the siRNA targeting the β4 subunit significantly reduced the effects of TNFα (p < 0.01, Tukey’s honestly significant difference post hoc test).