LAUSR

The global increase in opioid use requires practitioners to be aware of the side effects and contraindications of these medications in order to avoid complications, especially in patients with organ dysfunction such as cardiovascular disease and chronic kidney disease. It has been reported that opioid use is strongly associated with predictors of poor cardiovascular outcomes such as increased albuminuria and reduced GFR. Opioids work by binding to kappa-, mu- and delta-opioid receptors (ORs). We hypothesize that stimulation of ORs modulates calcium flux in podocytes ultimately leading to cell apoptosis, development of albuminuria, glomeruli damage and progression of hypertension. To test this hypothesis, we applied specific OR agonists to: 1) in vitro - immortalized cultured human podocytes (hPod), 2) ex vivo - freshly isolated human and rat glomeruli; and 3) in vivo - Dahl salt-sensitive (SS) rats. Calcium response in the podocytes was analyzed via ratiometric confocal fluorescent microscopy using calcium fluorescent dyes. For chronic studies Dahl SS rats were fed a high salt (HS; 8%) NaCl diet for 14 days with or without a daily i.v. bolus infusion of BRL52537, a potent and selective kappa-OR agonist. The hPods displayed a fast increase in fluorescence following an addition of BRL52537 (EC50 = 162 ± 2 µM) in a 2 mM calcium solution. A pre-application of kappa-OR antagonist (NorBNI) disrupted the effect of BRL52537 on calcium flux. The effect of BRL52537 on calcium in podocytes was completely abolished when we used the calcium-free solution or a pre-application of SAR7334 (a TRPC6 channel inhibitor), which revealed that calcium flux is mediated by activation of TRPC6 channels rather than other plasma membrane channels or depletion of intracellular calcium stores. Immunofluorescent analysis of human kidney validated kappa-OR protein expression in podocytes. Podocytes of the freshly isolated human glomeruli also revealed robust increases in fluorescent intensity following a single application of BRL52537, which was abolished by a pre-application of SAR7334. Effect of kappa-OR agonist was specific since application of Damgo (mu-OR agonist) or DPDPE (delta-OR agonist) had either no or minimal effect in normotensive rats. Chronic studies in Dahl SS rats showed that in BRL-treated group animals had elevation of microalbuminuria (13 ± 3 vs. 3 ± 1 Alb/Cre at day 7 of HS), higher mean arterial pressures (179 ± 15 mmHg vs. 151 ± 11 mmHg), and nephrinuria (50,232 ± 22,254 vs. 15,230 ± 4,647 a.u./Cre) compared to control rats. Glomeruli isolated from the hypertensive opioid treated rats had a significantly higher level of basal calcium (245 ± 23 vs. 139 ± 9 nM) in podocytes compared to non-treated group. Additionally, acute application of BRL52537 to podocytes of hypertensive animals resulted in a sustained increase in total [Ca2+]i influx. Our data establish that stimulation of kappa-ORs are specifically involved in increasing of intracellular calcium in podocytes through the TRPC6 pathway, which causes calcium overload and cellular damage. Observed increase in the calcium flux in podocytes most likely further contribute to the kidney injury and progression of salt-induced hypertension.