The Role of Opioid Receptors in Podocyte Injury and Kidney Damage During the Development of Salt‐Induced Hypertension
Authors: Golosova Daria , Levchenko Vladislav , Klemens Christine , El-Meanawy Ashraf , Palygin Oleg , Staruschenko Alexander 
Published: April 2020

The global increase in opioid use requires to better understand the side effects of opioids in order to avoid additional complications. Opioids are common analgesics for patients with hypertension and CKD despite its association with increased albuminuria and reduced glomerular filtration rate. 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 Fluo4/FuraRed and Fluo8 fluorescent dyes. For chronic studies Dahl SS rats were on a 0.4% (LS) or 8% (HS) NaCl diets 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 0 mM calcium 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 Albumin/Creatinine at day 7 of HS), higher mean arterial pressures (MAP, 179 ± 15 mmHg vs. 151 ± 11 mmHg), and nephrinuria (50,232 ± 22,254 vs. 15,230 ± 4,647 a.u./Creatinine) compared to control rats. Podocytes isolated from the glomeruli from the hypertensive opioid treated animals had a significantly higher level of basal calcium (245 ± 23 vs. 139 ± 9 nM) 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 established that stimulation of kappa‐ORs were specifically involved in increasing of intracellular calcium in podocytes through the TRPC6 pathway, which caused calcium overload and cellular damage. The opioid‐induced increase in the calcium flux in podocytes is expected to contribute to podocytopathy, proteinuria, kidney injury and progression of salt‐induced hypertension.