Nitric oxide (NO) is a potent signaling molecule involved in many physiological and pathophysiological processes in the kidney. NO plays a complex role in glomerular ultrafiltration, vasodilation, and inflammation. Changes in NO bioavailability in pathophysiological conditions such as hypertension or diabetes may lead to podocyte damage, proteinuria, and rapid development of chronic kidney disease (CKD). Despite the extensive data highlighting essential functions of NO in health and pathology, related signaling in glomerular cells, particularly podocytes, is understudied. Several reports indicate that NO bioavailability in glomerular cells is decreased during the development of renal pathology, while restoring NO level can be beneficial for glomerular function. At the same time, the compromised activity of nitric oxide synthase (NOS) may provoke the formation of peroxynitrite and has been linked to autoimmune diseases such as systemic lupus erythematosus. It is known that the changes in the distribution of NO sources due to shifts in NOS subunits expression or modifications of NADPH oxidases activity may be linked to or promote the development of pathology. However, there is a lack of information about the detailed mechanisms describing the production and release of NO in the glomerular cells. The interaction of NO and other reactive oxygen species in podocytes and how NO-calcium crosstalk regulates glomerular cells’ function is still largely unknown. Here, we discuss recent reports describing signaling, synthesis, and known pathophysiological mechanisms mediated by the changes in NO homeostasis in the podocyte. The understanding and further investigation of these essential mechanisms in glomerular cells will facilitate the design of novel strategies to prevent or manage health conditions that cause glomerular and kidney damage.

Background: Angiotensin Receptor blockers (ARBs) are the first-line treatment for hypertension and other chronic kidney diseases and act by inhibiting signaling through the angiotensin 1 receptor (AT1R). AT1R activation initiates signaling through G protein-coupled receptor (GPCR) effector signaling and β-arrestins. Recently, a novel AT1R agonist TRV120027 (TRV) that selectively activates the β-arrestin cascade without impacting the GCPR pathway has become available. Therefore, we hypothesized that β-arrestin and G-protein signaling pathways via AT1R have distinct downstream effects in podocytes. Furthermore, we propose that β-arrestin signaling initiates TRPC6-mediated Ca2+ entry. Methods: We used a conditionally immortalized human podocyte cell line to determine β-arrestin’s involvement in podocyte calcium signaling and cytoskeletal reorganization. Intracellular Ca2+ influx (Fluo-4 AM) and NO response (DAF-FM) to Ang II or TRV applications were analyzed with confocal microscopy. The role of TRPC channels was tested by single-channel electrophysiology and confocal imaging using a variety of commercially available inhibitors (AC1903 for TRPC5 and GSK283350A, SAR7334 for TRPC6). Western blotting for apoptosis-associated proteins, TUNEL staining to detect DNA cleavage, and actin cytoskeleton rearrangements were performed in podocytes exposed to TRV or Ang II treatment. Results: Our experiments determined that TRV-mediated β-arrestin pathway activation in podocytes promotes rapid elevation of intracellular Ca2+ in a dose-dependent manner (IC50=15μM, ligand-receptor binding fit modeled with Levenberg–Marquardt algorithm, adj. R2=0.93). Interestingly, the amplitude of β-arrestin-mediated Ca2+ influx was four times higher than the response to similar Ang II concentrations (t-test, n≥30, p<0.05). Moreover, the TRV response was significantly increased under hyperglycemic stress conditions, and promoted rapid apoptosis in podocytes (one-way ANOVA, n≥21, p=0.003). The pharmacological blockade of TRPC6, but not TRPC5, significantly attenuated Ca2+ influx in response to β-arrestin. In contrast to Ang II, TRV does not induce AT1R-mediated NO production, suggesting that β-arrestin activation only targets AT1R, and not AT2R. Single-channel analyses show rapid activation of TRPC activity in response to acute TRV application in podocytes (one-way ANOVA, n=8, p=0.004). Overall prolonged activation of the β-arrestin pathway in podocytes results in enhanced actin bundle thickness, abnormal actin cytoskeleton distribution, and increased apoptotic cell markers. Conclusions: TRV-mediated β-arrestin signaling in podocytes promotes high ionotropic TRPC6 channel-mediated Ca2+ influx, cytoskeleton rearrangement, and apoptosis, possibly leading to severe defects in glomerular filtration barrier integrity and kidney health. Under these circumstances, the potential therapeutic application of TRV for hypertension treatment is controversial and requires further investigation. DK126720 (to OP), DK129227 (to AS and OP), and NIH/NCATS/SCTR UL1TR001450/SCTR 2214 (to OP), endowed funds from the SC SmartState Centers of Excellence (to OP), Veterans Affairs Support Veterans Affairs (Merit Award I01 BX000820 to JL), research grant from Dialysis Clinic, Inc (to JL). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Introduction: Although nicotine’s harmful effects on renal function are established, the precise cellular mechanisms of smoking-related damage are understudied. Smoking-Related Glomerulopathy (SRG) is a renal disease phenotype associated with smoking. This condition histologically mimics diabetic nephropathy, however, SRG patients present with proteinuria and renal insufficiency without diabetes. Here we investigated the acute and chronic nicotine-related oxidative and nitrosative stress in glomerular podocytes. We hypothesized that nicotine may promote nitrosative stress in the kidney cells through the rapid production of peroxynitrite (ONOO-) and a decline in nitric oxide (NO) bioavailability. Methods: To test the hypothesis, we used a conditionally immortalized human podocyte line and confocal imaging to detect the production of ONOO- (Hydroxyl Radical and Peroxynitrite Sensor; HPF), intracellular Ca2+ (Fluo-8), and NO (DAF-FM). The presence of nicotinic acetylcholine receptors (nAChR) receptor subunits in podocytes was confirmed with immunocytochemistry and live imaging using commercially available pharmacology. NOS activity was analyzed in response to Ang II with the specific blockers for NOS1 (NΩ-Propyl-L-arginine hydrochloride) and NOS2 (L-NIL) subunits. One-way ANOVA (OriginPro) was used for statistical analysis. Results: Immunostaining indicated that human and rat kidneys express nicotinic acetylcholine receptors (nAChR). Notably, we detected specific expression of α7 nAChR in glomerular podocytes. In podocytes, acute nAChR activation promoted the mobilization of intracellular Ca2+ controlled by intracellular store activation, and fast ONOO- transients. Multiple consecutive applications of nicotine resulted in repeated intracellular Ca2+ and ONOO- transients. Nicotine-mediated ONOO- response was efficiently blocked in the presence of superoxide dismutase (SOD). The application of specific α7 or α4β2, α2β4, α4β4 and α3β4 nAChR agonists elicited Ca2+ transients but did not reproduce the ONOO- response to nicotine, suggesting that nitrosative processes may occur independently from Ca2+ influx or nAChR function. Chronic exposure to nicotine (12-hrs) resulted in a significant decline in podocytes’ NO bioavailability (p<0.05). While under normal conditions, NO is produced primarily by NOS1 (70%), and the rest is attributed to NOS2, chronic nicotine exposure led to an elevation of NOS2 (75%) and a decline in NOS1 activity (p<0.001). We observed similar NOS remodeling under hyperglycemic conditions, suggesting similar nitrosative processes in response to nicotine and high glucose. Conclusions: Nicotine promotes superoxide-stimulated nitrosative stress and peroxynitrite formation in podocytes leading to decline in NO bioavailability and pathological activation of NOS2. R01 NIDDK DK126720 (OP), DK129227 (OP), HL148114 (DVI), NIH/NCATS/SCTR UL1TR001450/SCTR 2214 (OP). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

INTRODUCTION. Mesangial cells provide structural support to the glomerular tuft and modulate the glomerular capillary flow via their contractile properties. Mesangial cells undergo phenotypic changes into myofibroblast-like cells, such as proliferation, mesangial expansion, abnormal glomerular tuft formation, and reduced numbers of capillary loops, are present in several glomerular diseases, including diabetic nephropathy and glomerulonephritis. In addition, thrombin-induced mesangial remodeling was found in diabetic patients, and expression of the corresponding protease-activated receptors (PARs) in the renal mesangium was reported. However, the functional PAR-mediated signaling and mechanisms in mesangial cells were not examined. This study aims to investigate protease-activated mechanisms regulating mesangial cell contraction and glomerular capillary flow. METHODS. We used primary human renal mesangial cells (HRMC) to determine the signaling mechanisms mediated by PAR1 thrombin-activated receptors. Confocal fluorescent microscopy was utilized to detect changes in intracellular Ca2+ response to specific PAR1 modulators. Pharmacology and patch clamp electrophysiology was further applied to reveal downstream signaling mechanisms responsible for intracellular Ca2+ oscillations. RESULTS. PAR1-mediated Ca2+ response displayed high sensitivity to specific agonist (TFLLR-NH2) with EC50 values of 3 and 6.3 nM for male and female-derived cultured cells, respectively (the competition of a ligand for receptor binding fit converged; adj. R2=0.98). The response to PAR1 activation promoted initial cytosolic Ca2+ increase followed by synchronized, damped Ca2+ oscillations with a lag of 6.74±0.84 min between peaks. The pre-application of a specific inhibitor (RWJ56110) eliminated PAR1-mediated response, and oscillations were blocked by the changes of an extracellular solution to zero Ca2+. The specific inhibitors for store-operated calcium (SOCs) (Pyr6) and TRPC3 (GSK 2833503A) channels strongly attenuated oscillation behavior (up to 40% when added separately and up to 65% when both were applied; two-way ANOVA, * p<0.0001). In addition, the effect of a specific inhibitor of TRPC6 channels (BI-749327) had a minimal impact on Ca2+ flux. Further single-channel electrophysiology experiments in HRMC cells confirmed the involvement of SOC and TRPC3 channels in PAR1-mediated GPCR activation. CONCLUSION. Our results indicate that coagulation proteases like thrombin may strongly regulate mesangial cell contraction and corresponding glomerular capillary flow by PAR1 GPCRs-related activation. The contraction mechanism is mediated presumably through SOCs entry and TRPC3 channels. Since high thrombin levels are linked to poor diabetic control, the described signaling may play a crucial role in the development of diabetic glomerular disease. DK126720 (to OP), DK129227 (to AS and OP), Veterans Affairs Support Veterans Affairs (Merit Award I01 BX000820 to JL), research grant from Dialysis Clinic, Inc (to JL). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.