Role of opioid signaling in kidney damage during the development of salt-induced hypertension

(A) Representative example of specific κ-OR agonist (100 μM, BRL 52537) application in the presence or absence of extracellular calcium (Fluo4 AM fluorescence). Scale bar is 50 μm. Images shown in Fig 2A are taken at 1, 1.5, and 6 min from the start of experiments either in 0 or 2 mM [Ca²⁺]_ex (bottom and top images, respectively). The source data videos are available; 1 min of experiments equals to ∼2 s in the videos. (B) Mean [Ca²⁺]_i transient in human podocytes after κ-OR stimulation in 2 or 0 mM calcium-containing bath solution (top and bottom traces, respectively). (C) Dose–response plot for peak [Ca²⁺]_i increase induced by BRL 52537 (2 mM [Ca²⁺ ]_ex) in human podocyte culture (a number of podocytes analyzed per point on the graph are shown). (D) Representative traces show dose-dependent changes in [Ca²⁺]_i after applications of BRL 52537. (B, E) Peak amplitude of the [Ca²⁺]_i after κ-OR agonist stimulation in experiments shown in (B), or during a preincubation of human podocytes with κ-OR antagonist (norBNI) or TRPC6 inhibitor (100 nM, SAR7334) in 2 mM [Ca²⁺]_ex (n ≥ 38 cells; one-way ANOVA, P < 0.001, Tukey’s post hoc P < 0.05, all pairwise k = 6).

Source data are available online for this figure.

(A) Peak amplitude of the [Ca²⁺]_i transient mediated by κ-OR agonist stimulation shown in (B) (n ≥ 33 cells; ANOVA, P < 0.001). (B) [Ca²⁺]_i response in podocytes from freshly isolated human glomeruli exposed to κ-OR agonist (100 μM, BRL 52537) under control or TRPC6 blockade (50 μM, SAR7334 preincubation) conditions.

(A) An application of κ-OR agonist to human-cultured podocytes promotes significant shrinkage in the cell surface area (top panel, scale bar is 50 μm). Changes in the mean cell surface area were obtained by comparisons between BRL-treated and control cells (number of cells is indicated in the brackets, ANOVA, P < 0.001; panel below). (B) Following BRL-induced podocyte shrinkage, the actin cytoskeleton pattern shows reinforcement and formation of the actin stress fibers. The reorganization of the F-actin cytoskeleton is indicated by the changes in the rhodamine phalloidin staining pattern through the blue line. Scale bar is 50 μm. (C) Surface topography 3D images obtained by scanning ion-conductance microscopy shows lamellipodia dynamics in human podocytes treated with κ-ORs agonist (changes in z-axis indicated by pseudocolor). (D) Summary of scanning ion-conductance microscope topography indicates normal lamellipodial protrusion under control conditions followed by a strong retraction pattern mediated by κ-OR stimulation (n ≥ 3 cells; ANOVA, P < 0.002, Tukey’s post hoc P < 0.05).

(A) Schematic representation of the experimental protocol. (B) The development of mean arterial pressure in SS rats after changing the diet from a normal salt (NS, 0.4%; day 0) to a HS (8% NaCl) and chronic treatment with κ-OR agonist or vehicle (n ≥ 5 rat per group; ANOVA, P < 0.05). (C) Urinary albumin (Alb/Cre, 24 h collection) changes for the experiments shown in (B). (D) Basal calcium levels in podocytes of freshly isolated glomeruli from hypertensive SS rats chronically exposed to κ-OR agonist or vehicle (n ≥ 13 cells; ANOVA, P < 0.003). (E) Western blot analysis of nephrin in the urine samples (24-h collection) from hypertensive (2 wk on HS) and BRL-treated rats (n = 5 rats per group; ANOVA, P < 0.005).

(A) Western blot analyses of the TRPC6 expression in the kidney cortex of normotensive (NS), hypertensive (14DHS), and hypertensive treated with BRL (14DHS+BRL) groups. (B) Summary graph of TRPC6 expression as shown in A (n = 4 rats per group, ANOVA, P < 0.03, Tukey’s post hoc P < 0.05). (C) Mean [Ca²⁺]_i transient in podocytes of glomeruli freshly isolated from salt-sensitive rats fed normal salt (NS) or after 2 wk on HS (14DHS). Note the sustained [Ca²⁺]_i response to acute stimulation of κ-ORs in hypertensive animals.