In patients with rhabdomyolysis, the overwhelming release of myoglobin into circulation is the primary cause of kidney injury. Myoglobin causes direct kidney injury as well as severe renal vasoconstriction. An increase in renal vascular resistance (RVR) results in renal blood flow (RBF) and glomerular filtration rate (GFR) reduction, tubular injury, and acute kidney injury (AKI). The mechanisms that underlie rhabdomyolysis-induced AKI are not fully understood but may involve the local production of vasoactive mediators in the kidney. Studies have shown that myoglobin stimulates endothelin-1 (ET-1) production in glomerular mesangial cells. Circulating ET-1 is also increased in rats subjected to glycerol-induced rhabdomyolysis. However, the upstream mechanisms of ET-1 production and downstream effectors of ET-1 actions in rhabdomyolysis-induced AKI remain unclear. Vasoactive ET-1 is generated by ET converting enzyme 1 (ECE-1)-induced proteolytic processing of inactive big ET to biologically active peptides. The downstream ion channel effectors of ET-1-induced vasoregulation include the transient receptor potential cation channel, subfamily C member 3 (TRPC3). This study demonstrates that glycerol-induced rhabdomyolysis in Wistar rats promotes ECE-1-dependent ET-1 production, RVR increase, GFR decrease, and AKI. Rhabdomyolysis-induced increases in RVR and AKI in the rats were attenuated by post-injury pharmacological inhibition of ECE-1, ET receptors, and TRPC3 channels. CRISPR/Cas9-mediated knockout of TRPC3 channels attenuated ET-1-induced renal vascular reactivity and rhabdomyolysis-induced AKI. These findings suggest that ECE-1-driven ET-1 production and downstream activation of TRPC3-dependent renal vasoconstriction contribute to rhabdomyolysis-induced AKI. Hence, postinjury inhibition of ET-1-mediated renal vasoregulation may provide therapeutic targets for rhabdomyolysis-induced AKI.

Rhabdomyolysis is a life‐threatening condition resulting from the breakdown of skeletal muscle fibers leading to the release of myoglobin into the blood. Increased circulating myoglobin can cause kidney damage, and acute kidney injury (AKI) occurs in 33‐50% of patients with myoglobinuria. Persistent constriction of the renal microvessels, which reduces renal blood flow (RBF) and glomerular filtration rate (GFR), contributes to rhabdomyolysis‐induced AKI. Since activation of G‐protein‐coupled receptors (GPCR) and ion channels regulate renal vascular resistance (RVR), rhabdomyolysis may likely modulate RVR via GPCR and vascular ion channel mechanisms. Pretreatment of rats with an endothelin receptor antagonist has been shown to alleviate rhabdomyolysis‐induced AKI. However, the downstream and upstream links between rhabdomyolysis and increased ET‐1 synthesis are unknown. The downstream effectors of ET‐1‐induced vasoconstriction are smooth muscle cell (SMC) Ca2+‐permeable ion channels. Yet, the ion channel mechanisms that trigger renal vasoconstriction in rhabdomyolysis‐induced AKI remain unknown. Also, most cases of rhabdomyolysis‐induced AKI cannot be predicted. Whether post‐injury inhibition of the vascular mechanisms of ET‐1 ameliorates, rhabdomyolysis‐induced AKI is unknown. This study investigates post‐injury renal production and vascular regulation of the ET system in rhabdomyolysis‐induced AKI. Six h of rhabdomyolysis increased peptidase endothelin converting enzyme (ECE‐1)‐dependent biosynthesis of ET‐1 in the kidneys. Rhabdomyolysis‐induced AKI was not sex‐dependent. At 24 h, rhabdomyolysis also increased renal reactive oxygen species (ROS), ECE‐1, and ET‐1 levels. Furthermore, ET‐1 activated rat renal vascular smooth muscle cell TRPC3 leading to a reduction in RBF and increased RVR. Protein expression levels of the ET receptors (ETA and ETB) and TRPC3 channels in renal microvessels were unaltered. However, postinjury inhibition of ECE1, ET Receptors, and TRPC3 mitigated rhabdomyolysis‐induced GFR impairment, renal hypoperfusion, AKI biomarker elevation, and morphological kidney damage. To further explore the role of TRPC3 ion channels in rhabdomyolysis‐induced AKI, we subjected TRPC3 knock‐out (KO) rats to rhabdomyolysis. Compared to their wild‐type counterparts, KO rats show protection against rhabdomyolysis‐induced AKI. Our findings suggest that ECE‐driven proteolytic ET‐1 production contributes to rhabdomyolysis‐induced AKI. Downstream, ET‐derived DAG activates renal vascular smooth muscle cell TRPC3 channels leading to extracellular calcium entry, vasoconstriction, increased RVR, and reduced GFR.

Foremost among the ion channels that control vascular tone is the transient receptor potential (TRP) cation channels. The TRPM subfamily of the TRP channels includes eight members, expressed in various excitable and non-excitable cells and involved in diverse functions, such as sensing membrane potential, cold, taste, redox, osmolarity, and cell survival. Previous analysis of TRPM gene expression showed the highest expression of TRPM8 in intact blood vessels compared with other isoforms (TRPM1-7). Studies have also revealed that activating TRPM8 by menthol and icilin modulates endothelium-dependent and independent vascular reactivity. However, the non-selectivity of pharmacological TRPM8 modulators constitutes a significant limitation in the prior studies aimed at delineating TRPM8-dependent vasoregulation. In this study, we demonstrate that acetylcholine (ACH)- and sodium nitroprusside (SNP)-induced reduction in arterial pressure was unaltered in mice lacking TRPM8. We generated endothelial-specific knockout (KO) of TRPM8 (ecTRPM8-/-) by breeding TRPM8 floxed (TRPM8fl/fl) with Cdh5-Cre (VE-Cadherin-Cre) mice. Unlike global TRPM8 KO, ecTRPM8-/- mice were not resistant to cold temperatures. Phenylephrine-induced contraction of renal and mesenteric arteries isolated from TRPM8fl/fl and ecTRPM8-/- mice were similar. Also, ACH- and SNP-induced relaxation of phenylephrine-precontracted renal and mesenteric arteries from TRPM8fl/fl and ecTRPM8-/- mice were comparable. These findings suggest that TRPM8 channels do not regulate endothelium-dependent and -independent vasorelaxation in mice. Disclosure of Funding: NHLBI: R01 HL151735-01 and R01HL151735-S 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.

Acute kidney injury (AKI) is the most severe and life-threatening complication of rhabdomyolysis, with 10-40% of patients developing some form of kidney insufficiency within days. Experimental evidence indicates that renal vasoconstriction plays a significant role in the pathophysiology of rhabdomyolysis-induced AKI. Rodents subjected to glycerol-induced rhabdomyolysis exhibited increased plasma concentrations of endothelin-1 (ET-1). Treating glomerular mesangial cells with myoglobin has also been shown to increase the cellular production of ET-1. The downstream effectors of ET-1-induced vasoconstriction are smooth muscle Ca2+ permeable transient receptor potential canonical (TRPC) channels. There are seven members of the TRPC gene family (TRPC1-7). Whereas TRPC2 and 7 are absent, TRPC1, 3, 4, 5, and 6 are expressed in renal vessels, with TRPC3 predominant. TRPC3, TRPC6, and TRPC7 are highly homologous. Although the ET system is known to be associated with rhabdomyolysis-induced AKI, the function of renal vascular ion channels in the disease is unclear. Here, we investigated the involvement of TRPC3 and TRPC6 in glycerol-induced rhabdomyolysis in rats. ET-1 production is increased in myoglobin-treated epithelial cells, the primary source of renal ET. Rhabdomyolysis also increased renal ET-1 production, decreased renal blood flow (RBF), and increased renal vascular resistance, effects attenuated by ET receptor and TRPC channel blocker Pyr3. Furthermore, we used TRPC3 and TRPC6 knockout (KO) rats to support the pharmacological approaches. Basal day and night arterial pressure, heart rate, GFR, plasma creatinine, and BUN were unchanged in WT vs. KO rats. Twenty-four h rhabdomyolysis led to comparable increases in urinary ET-1 production in WT and KO rats. However, rhabdomyolysis-induced decreases in GFR, elevations in BUN, and plasma creatinine were all attenuated in the TRPC3 but not TRPC6 KO rats. Similarly, morphological kidney damage (tubular necrosis, casts, and dilatation) was mitigated in the TRPC3 KO with no protection offered by the KO of TRPC6 channels. Together, our data suggest that myoglobin-driven ET-1 production and downstream activation of TRPC3- but not TRPC6-dependent vasoconstriction contributes to rhabdomyolysis-induced AKI. American Heart Association Predoctoral Fellowship 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.

Individuals with sickle cell disease (SCD) are at greater risk for developing rhabdomyolysis, a potentially life-threatening syndrome resulting from the breakdown of skeletal muscle fibers. Acute kidney injury (AKI) is one of the most severe complications of rhabdomyolysis. Chronic kidney and cardiovascular disease, which account for SCD mortality, are long-term consequences of AKI. Although SCD elevates the risks of rhabdomyolysis-induced sudden death, the mechanisms that underlie rhabdomyolysis-induced AKI in SCD are unclear. Here, we examined the effects of glycerol-induced rhabdomyolysis on kidney health in transgenic humanized mice that express human sickle hemoglobin (HbSS-Townes model). Unlike the control mice (AA), homozygous SCD mice (SS) exhibited 100% mortality 8-24 h after intramuscular glycerol injection. Five hours of glycerol-induced rhabdomyolysis caused a more significant increase in myoglobinuria and plasma creatine kinase levels in SS compared to AA mice. Since SS mice experience chronic hemolysis, releasing free heme and iron into the bloodstream, and the induction of rhabdomyolysis releases myoglobin, we examined heme and renal iron parameters in these mice. Basal plasma heme, iron accumulation, and kidney tissue ferric iron levels were significantly higher in SS than in AA control mice. In contrast to AA, rhabdomyolysis aggravated these parameters in SS mice. Rhabdomyolysis also amplified renal oxidative stress in SS compared to AA mice. SS mice exhibited worse renal function, exemplified by a significant decrease in GFR, increased plasma and urinary biomarkers of early AKI, and more significant renal tubular injury. The free radical scavenger TEMPOL ameliorated rhabdomyolysis-induced AKI in the SS mice. These findings suggest that rhabdomyolysis and subsequent AKI are heightened in SCD mice. It can also be inferred that oxidative stress driven by renal iron accumulation may underlie rhabdomyolysis-induced early AKI in SCD. NHLBI: R01 HL151735-01 and R01HL151735-S 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.