Activation of renal vascular smooth muscle TRPV4 channels by 5-hydroxytryptamine impairs kidney function in neonatal pigs.

Control of microvascular reactivity by 5-hydroxytryptamine (5-HT; serotonin) is complex and may depend on vascular bed type and 5-HT receptors. 5-HT receptors consist of seven families (5-HT1-5-HT7), with 5-HT2 predominantly mediating renal vasoconstriction. Cyclooxygenase (COX) and smooth muscle intracellular Ca2+ levels ([Ca2+]i) have been implicated in 5-HT-induced vascular reactivity. Although 5-HT receptor expression and circulating 5-HT levels are known to be dependent on postnatal age, control of neonatal renal microvascular function by 5-HT is unclear. In the present study, we demonstrate that 5-HT stimulated human TRPV4 transiently expressed in Chinese hamster ovary cells. 5-HT2A is the predominant 5-HT2 receptor subtype in freshly isolated neonatal pig renal microvascular smooth muscle cells (SMCs). HC-067047 (HC), a selective TRPV4 blocker, attenuated cation currents induced by 5-HT in the SMCs. HC also inhibited the 5-HT-induced increase in renal microvascular [Ca2+]i and constriction. Intrarenal artery infusion of 5-HT had minimal effects on systemic hemodynamics but reduced renal blood flow (RBF) and increased renal vascular resistance (RVR) in the pigs. Transdermal measurement of glomerular filtration rate (GFR) indicated that kidney infusion of 5-HT reduced GFR. HC and 5-HT2 receptor antagonist ritanserin attenuated 5-HT effects on RBF, RVR, and GFR. Moreover, the serum and urinary COX-1 and COX-2 levels in 5-HT-treated piglets were unchanged compared with the control. These data suggest that activation of renal microvascular SMC TRPV4 channels by 5-HT impairs kidney function in neonatal pigs independently of COX production.

KV7.1 channel blockade inhibits neonatal renal autoregulation triggered by a step decrease in arterial pressure.

KV7 channels, the voltage-gated K+ channels encoded by KCNQ genes, mediate heterogeneous vascular responses in rodents. Postnatal changes in the functional expression of KV7 channels have been reported in rodent saphenous arteries, but their physiological function in the neonatal renal vascular bed is unclear. Here, we report that, unlike adult pigs, only KCNQ1 (KV7.1) out of the five members of KCNQ genes was detected in neonatal pig renal microvessels. KCNQ1 is present in fetal pig kidneys as early as day 50 of gestation, and the level of expression remains the same up to postnatal day 21. Activation of renal vascular smooth muscle cell (SMC) KV7.1 stimulated whole cell currents, inhibited by HMR1556 (HMR), a selective KV7.1 blocker. HMR did not change the steady-state diameter of isolated renal microvessels. Similarly, intrarenal artery infusion of HMR did not alter mean arterial pressure, renal blood flow, and renal vascular resistance in the pigs. An ∼20 mmHg reduction in mean arterial pressure evoked effective autoregulation of renal blood flow, which HMR inhibited. We conclude that 1) the expression of KCNQ isoforms in porcine renal microvessels is dependent on kidney maturation, 2) KV7.1 is functionally expressed in neonatal pig renal vascular SMCs, 3) a decrease in arterial pressure up to 20 mmHg induces renal autoregulation in neonatal pigs, and 4) SMC KV7.1 does not control basal renal vascular tone but contributes to neonatal renal autoregulation triggered by a step decrease in arterial pressure.NEW & NOTEWORTHY KV7.1 is present in fetal pig kidneys as early as day 50 of gestation, and the level of expression remains the same up to postnatal day 21. KV7.1 is functionally expressed in neonatal pig renal vascular smooth muscle cells (SMCs). A decrease in arterial pressure up to 20 mmHg induces renal autoregulation in neonatal pigs. Although SMC KV7.1 does not control basal renal vascular resistance, its inhibition blunts neonatal renal autoregulation engendered by a step decrease in arterial pressure.

Interleukin 1 beta-induced calcium signaling via TRPA1 channels promotes mitogen-activated protein kinase-dependent mesangial cell proliferation.

Glomerular mesangial cell (GMC)-derived pleiotropic cytokine, interleukin-1 (IL-1), contributes to hypercellularity in human and experimental proliferative glomerulonephritis. IL-1 promotes mesangial proliferation and may stimulate extracellular matrix accumulation, mechanisms of which are unclear. The present study shows that the beta isoform of IL-1 (IL-1ß) is a potent inducer of IL-1 type I receptor-dependent Ca2+ entry in mouse GMCs. We also demonstrate that the transient receptor potential ankyrin 1 (TRPA1) is an intracellular store-independent diacylglycerol-sensitive Ca2+ channel in the cells. IL-1ß-induced Ca2+ and Ba2+ influxes in the cells were negated by pharmacological inhibition and siRNA-mediated knockdown of TRPA1 channels. IL-1ß did not stimulate fibronectin production in cultured mouse GMCs and glomerular explants but promoted Ca2+ -dependent cell proliferation. IL-1ß also stimulated TRPA1-dependent ERK mitogen-activated protein kinase (MAPK) phosphorylation in the cells. Concomitantly, IL-1ß-induced GMC proliferation was attenuated by TRPA1 and RAF1/ MEK/ERK inhibitors. These findings suggest that IL-1ß-induced Ca2+ entry via TRPA1 channels engenders MAPK-dependent mesangial cell proliferation. Hence, TRPA1-mediated Ca2+ signaling could be of pathological significance in proliferative glomerulonephritis.

Doxorubicin-Induced Fetal Mesangial Cell Death Occurs Independently of TRPC6 Channel Upregulation but Involves Mitochondrial Generation of Reactive Oxygen Species.

Doxorubicin (DOX), a category D pregnancy drug, is a chemotherapeutic agent that has been shown in animal studies to induce fetal toxicity, including renal abnormalities. Upregulation of the transient receptor potential cation (TRPC) 6 channel is involved in DOX-induced podocyte apoptosis. We have previously reported that TRPC6-mediated Ca2+ signaling promotes neonatal glomerular mesangial cell (GMC) death. However, it is unknown whether DOX alters mesangial TRPC expression or viability in the fetus. In this study, cell growth was tracked in control and DOX-treated primary GMCs derived from fetal pigs. Live-cell imaging demonstrated that exposure to DOX inhibited the proliferation of fetal pig GMCs and induced cell death. DOX did not alter the TRPC3 expression levels. By contrast, TRPC6 protein expression in the cells was markedly reduced by DOX. DOX treatment also attenuated the TRPC6-mediated intracellular Ca2+ elevation. DOX stimulated mitochondrial reactive oxygen species (mtROS) generation and mitophagy by the GMCs. The DOX-induced mtROS generation and apoptosis were reversed by the mitochondria-targeted antioxidant mitoquinone. These data suggest that DOX-induced fetal pig GMC apoptosis is independent of TRPC6 channel upregulation but requires mtROS production. The mtROS-dependent GMC death may contribute to DOX-induced fetal nephrotoxicity when administered prenatally.

γ-secretase inhibitor DAPT mitigates cisplatin-induced acute kidney injury by suppressing Notch1 signaling.

Organ toxicity, including kidney injury, limits the use of cisplatin for the treatment of multiple human cancers. Hence, interventions to alleviate cisplatin-induced nephropathy are of benefit to cancer patients. Recent studies have demonstrated that pharmacological inhibition of the Notch signaling pathway enhances cisplatin efficacy against several cancer cells. However, whether augmentation of the anti-cancer effect of cisplatin by Notch inhibition comes at the cost of increased kidney injury is unclear. We show here that treatment of mice with cisplatin resulted in a significant increase in Notch ligand Delta-like 1 (Dll1) and Notch1 intracellular domain (N1ICD) protein expression levels in the kidneys. N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a γ-secretase inhibitor reversed cisplatin-induced increase in renal N1ICD expression and plasma or urinary levels of predictive biomarkers of acute kidney injury (AKI). DAPT also mitigated cisplatin-induced tubular injury and reduction in glomerular filtration rate. Real-time multiphoton microscopy revealed marked necrosis and peritubular vascular dysfunction in the kidneys of cisplatin-treated mice which were abrogated by DAPT. Cisplatin-induced Dll1/Notch1 signaling was recapitulated in a human proximal tubule epithelial cell line (HK-2). siRNA-mediated Dll1 knockdown and DAPT attenuated cisplatin-induced Notch1 cleavage and cytotoxicity in HK-2 cells. These data suggest that Dll1-mediated Notch1 signaling contributes to cisplatin-induced AKI. Hence, the Notch signaling pathway could be a potential therapeutic target to alleviate renal complications associated with cisplatin chemotherapy.

Acute hydroxyurea treatment reduces tubular damage following bilateral ischemia-reperfusion injury in a mouse model of sickle cell disease.

Ischemic injury is a primary contributor to the initiation of renal tubular epithelial cell damage in sickle cell disease (SCD). In this study, we investigated the effects of bilateral ischemia-reperfusion injury, which is a common type of acute kidney injury (AKI), in male and female genetic mouse model of SCD. Bilateral occlusion of both renal hila for 21 min led to a significantly higher detection of established serum markers of AKI (creatinine, KIM-1 and NGAL) compared to sham-operated male SCD mice. Severe damage to the outer medullary tubules was determined in the ischemia-reperfision injury (IRI)-treated SCD male mice. In female SCD mice with a longer ischemic time (23 min), the serum markers of AKI were not as highly elevated compared to their male counterparts, and the extent of outer medullary tubular injury was less severe. To assess the potential benefit in the use of hydroxyurea (50 mg/kg IP) following bilateral renal IRI, we observed that the serum markers of AKI and the outer medullary tubular damage were markedly improved compared to male SCD mice that were not treated with hydroxyurea. In this study, we confirmed that male SCD mice were more susceptible to increased tubular damage and a loss in renal function compared to female SCD mice, and that hydroxyurea may partially prevent the extent of tubular injury following severe ischemia-reperfusion injury in SCD.

Pharmacological inhibition of TRPV4 channels protects against ischemia-reperfusion-induced renal insufficiency in neonatal pigs.

Renal vasoconstriction, an early manifestation of ischemic acute kidney injury (AKI), results in renal hypoperfusion and a rapid decline in kidney function. The pathophysiological mechanisms that underlie ischemia-reperfusion (IR)-induced renal insufficiency are poorly understood, but possibilities include alterations in ion channel-dependent renal vasoregulation. In the present study, we show that pharmacological activation of TRPV4 channels constricted preglomerular microvessels and elicited renal hypoperfusion in neonatal pigs. Bilateral renal ischemia followed by short-term reperfusion increased TRPV4 protein expression in resistance size renal vessels and TRPV4-dependent cation currents in renal vascular smooth muscle cells (SMCs). Selective TRPV4 channel blockers attenuated IR-induced reduction in total renal blood flow (RBF), cortical perfusion, and glomerular filtration rate (GFR). TRPV4 inhibition also diminished renal IR-induced increase in AKI biomarkers. Furthermore, the level of angiotensin II (Ang II) was higher in the urine of IR- compared with sham-operated neonatal pigs. IR did not alter renal vascular expression of Ang II type 1 (AT1) receptors. However, losartan, a selective AT1 receptor antagonist, ameliorated IR-induced renal insufficiency in the pigs. Blockade of TRPV4 channels attenuated Ang II-evoked receptor-operated Ca2+ entry and constriction in preglomerular microvessels. TRPV4 inhibition also blunted Ang II-induced increase in renal vascular resistance (RVR) and hypoperfusion in the pigs. Together, our data suggest that SMC TRPV4-mediated renal vasoconstriction and the ensuing increase in RVR contribute to early hypoperfusion and renal insufficiency elicited by renal IR in neonatal pigs. We propose that multimodal signaling by renal vascular SMC TRPV4 channels controls neonatal renal microcirculation in health and disease.

Oxidant-induced increase in norepinephrine secretion from PC12 cells is dependent on TRPM8 channel-mediated intracellular calcium elevation.

Reactive oxygen species (ROS) modulate neuronal function, including plasticity and neurotransmitter biosynthesis and release. The cellular mechanisms that underlie redox modulation of neurotransmission are not fully resolved, but potential pathways include ROS-induced alterations in Ca2+ signaling in nerve terminals. In this study, we show that cold-sensitive receptor TRPM8 is activated by pro-oxidant tert-butyl hydroperoxide (tBHP). Polymerase chain reaction, Western immunoblotting, and immunofluorescence indicated that TRPM8 channels are expressed in rat pheochromocytoma 12 (PC12) cells, a phenotypic model of sympathetic neurosecretion when differentiated with nerve growth factor. WS-12, a selective TRPM8 channel agonist, and tBHP increased intracellular Ca2+ concentration in differentiated PC12 cells; an effect attenuated by AMTB, a selective TRPM8 channel blocker, and siRNA-mediated TRPM8 knockdown. Blockade of TRPM8 channels also reduced WS-12- and tBHP-evoked norepinephrine secretion from the cells. These data suggest that TRPM8 channels contribute to oxidant-induced neurotransmission in PC12 cells.

Cisplatin-induced oxidative stress stimulates renal Fas ligand shedding.

Acute kidney injury (AKI), a significant complication of cisplatin chemotherapy is associated with reactive oxygen species (ROS)-dependent renal cell death, but the cellular targets of ROS in cisplatin nephrotoxicity are not fully resolved. Here, we investigated cisplatin-induced oxidative renal damage and tested the hypothesis that ROS-dependent shedding of death activator Fas ligand (FasL) occurs in cisplatin nephropathy. We show that intraperitoneal injection of sulfobutyl ether-ß-cyclodextrin (Captisol™)-solubilized cisplatin elevated the level of lipid peroxidation product malondialdehyde in mouse kidneys and urinary concentration of oxidative DNA damage biomarker 8-hydroxy-2'-deoxyguanosine. Cisplatin increased mouse kidney-to-body weight ratio and the plasma or urinary levels of predictive biomarkers of AKI, including creatinine, blood urea nitrogen, microalbumin, neutrophil gelatinase-associated lipocalin, and cystatin C. Histological analysis and dUTP nick end labeling of kidney sections indicated tubular injury and renal apoptosis, respectively in cisplatin-treated mice. Whereas the plasma concentration of soluble FasL (sFasL) was unaltered, urinary sFasL was increased ∼4-fold in cisplatin-treated mice. Real-time quantitative live-cell imaging and lactate dehydrogenase assay showed that cisplatin stimulated caspase 3/7 activation and cytotoxicity in a human proximal tubule epithelial cell line which were attenuated by inhibitors of the FasL/Fas system and poly [ADP-ribose] polymerase-1. Moreover, TEMPOL, an intracellular free radical scavenger mitigated cisplatin-induced renal oxidative stress and injury, AKI biomarker and urinary sFasL elevation, and proximal tubule cell death. Our findings indicate that cisplatin-induced oxidative stress triggers the shedding of membrane-bound FasL to sFasL in the kidney. We demonstrate that cisplatin elicits nephrotoxicity by promoting FasL/Fas-dependent oxidative renal tubular cell death.

TRPV4 channels contribute to renal myogenic autoregulation in neonatal pigs.

Myogenic response, a phenomenon in which resistance size arteries and arterioles swiftly constrict or dilate in response to an acute elevation or reduction, respectively, in intravascular pressure is a key component of renal autoregulation mechanisms. Although it is well established that the renal system is functionally immature in neonates, mechanisms that regulate neonatal renal blood flow (RBF) remain poorly understood. In this study, we investigated the hypothesis that members of the transient receptor potential vanilloid (TRPV) channels are molecular components of renal myogenic constriction in newborns. We show that unlike TRPV1-3, TRPV4 channels are predominantly expressed in neonatal pig preglomerular vascular smooth muscle cells (SMCs). Intracellular Ca2+ concentration ([Ca2+]i) elevation induced by osmotic cell swelling was attenuated by TRPV4, L-type Ca2+, and stretch-activated Ca2+ channel blockers but not phospholipase A2 inhibitor. Blockade of TRPV4 channels reversed steady-state myogenic tone and inhibited pressure-induced membrane depolarization, [Ca2+]i elevation, and constriction in distal interlobular arteries. A step increase in arterial pressure induced efficient autoregulation of renal cortical perfusion and total RBF in anesthetized and mechanically ventilated neonatal pigs. Moreover, intrarenal arterial infusion of the TRPV4 channel blockers HC 067047 and RN 1734 attenuated renal autoregulation in the pigs. These data suggest that renal myogenic autoregulation is functional in neonates. Our findings also indicate that TRPV4 channels are mechanosensors in neonatal pig preglomerular vascular SMCs and contribute to renal myogenic autoregulation.

Adenosine A1 receptor-operated calcium entry in renal afferent arterioles is dependent on postnatal maturation of TRPC3 channels.

Adenosine, a regulator of cardiovascular development and renal function, constricts renal afferent arterioles by inducing intracellular Ca2+ concentration ([Ca2+]i) elevation in smooth muscle cells (SMCs) via activation of its cognate A1 receptors (A1Rs). Mechanisms that underlie A1R-dependent [Ca2+]i elevation in renal vascular SMCs are not fully resolved. Whether A1R expression and function in preglomerular microvessels are dependent on postnatal kidney maturation is also unclear. In this study, we show that selective activation of A1Rs by 2-chloro-N6-cyclopentyladenosine (CCPA) does not stimulate store-operated Ca2+ entry in afferent arterioles isolated from neonatal pigs. However, CCPA-induced [Ca2+]i elevation is dependent on phospholipase C and transient receptor potential cation channel, subfamily C, member 3 (TRPC3). Basal [Ca2+]i was unchanged in afferent arterioles isolated from newborn (0-day-old) pigs compared with their 20-day-old counterparts. By contrast, CCPA treatment resulted in significantly larger [Ca2+]i in afferent arterioles from 20-day-old pigs. A1R protein expression levels in the kidneys and afferent arterioles were unaltered in 0- vs. 20-day-old pigs. However, the TRPC3 channel protein expression level was ~92 and 78% higher in 20-day-old pig kidneys and afferent arterioles, respectively. These data suggest that activation of A1Rs elicits receptor-operated Ca2+ entry in porcine afferent arterioles, the level of which is dependent on postnatal maturation of TRPC3 channels. We propose that TRPC3 channels may contribute to the physiology and pathophysiology of A1Rs.

Early septic insult in neonatal pigs increases serum and urinary soluble Fas ligand and decreases kidney function without inducing significant renal apoptosis.

Apoptosis of renal tubular and glomerular cells during kidney disease involves activation of Fas ligand (FasL)-dependent death pathway. The significance of FasL in neonates with septic acute kidney injury (AKI) is unresolved, but an increase in renal FasL production, and/or infiltration of circulating FasL into the kidneys may occur following initial septic insult. Here, we examined whether soluble Fas ligand (sFasL) levels are altered during early phase of septic AKI in neonates. Six hours of polymicrobial sepsis elicited by cecal ligation and puncture (CLP) elevated serum C-reactive protein (CRP) (a bacteremia and sepsis marker) concentration in anesthetized and mechanically ventilated neonatal pigs. Serum creatinine and urea nitrogen concentrations were increased by ∼39% and 46%, respectively, following 6 h of CLP in the pigs. The urinary level of NGAL, an early marker of AKI was also elevated by ∼71% in the septic pigs. The basal concentration of sFasL in the serum and urine of neonatal pigs was similar. Six hours of CLP significantly increased serum and urine sFasL levels in the pigs by ∼24% and 68%, respectively. However, there was no evidence of caspase activation to suggest an induction of cellular apoptotic process in the kidneys of the septic pigs. These findings suggest that an increase in circulating and urinary sFasL during early septic AKI in neonatal pigs is not associated with renal apoptosis.

Lipid rafts are required for signal transduction by angiotensin II receptor type 1 in neonatal glomerular mesangial cells.

Angiotensin II (ANG-II) receptors (AGTRs) contribute to renal physiology and pathophysiology, but the underlying mechanisms that regulate AGTR function in glomerular mesangium are poorly understood. Here, we show that AGTR1 is the functional AGTR subtype expressed in neonatal pig glomerular mesangial cells (GMCs). Cyclodextrin (CDX)-mediated cholesterol depletion attenuated cell surface AGTR1 protein expression and ANG-II-induced intracellular Ca(2+) ([Ca(2+)]i) elevation in the cells. The COOH-terminus of porcine AGTR1 contains a caveolin (CAV)-binding motif. However, neonatal GMCs express CAV-1, but not CAV-2 and CAV-3. Colocalization and in situ proximity ligation assay detected an association between endogenous AGTR1 and CAV-1 in the cells. A synthetic peptide corresponding to the CAV-1 scaffolding domain (CSD) sequence also reduced ANG-II-induced [Ca(2+)]i elevation in the cells. Real-time imaging of cell growth revealed that ANG-II stimulates neonatal GMC proliferation. ANG-II-induced GMC growth was attenuated by EMD 66684, an AGTR1 antagonist; BAPTA, a [Ca(2+)]i chelator; KN-93, a Ca(2+)/calmodulin-dependent protein kinase II inhibitor; CDX; and a CSD peptide, but not PD 123319, a selective AGTR2 antagonist. Collectively, our data demonstrate [Ca(2+)]i-dependent proliferative effect of ANG-II and highlight a critical role for lipid raft microdomains in AGTR1-mediated signal transduction in neonatal GMCs.

Postnatal kidney maturation regulates renal artery myogenic constriction.

AIM: Intravascular pressure-induced vasoconstriction (myogenic constriction) is central to renal blood flow autoregulation. At term, kidney maturation is functionally incomplete. Premature neonates are at risk of kidney dysfunction. However, it is unclear whether renal artery myogenic constriction is altered after preterm birth. Here, we compared renal artery myogenic constriction in full-term and preterm pigs during the first week of life. METHODS: We investigated myogenic constriction in small interlobular arteries isolated from the kidneys of pigs delivered at term and at 91% of term (with and without 96 h of neonatal intensive care). RESULTS: Cross-sectional area, media/lumen ratio, and luminal diameter measured under passive conditions were similar in arteries from full-term and preterm pig kidneys. An acute elevation in intravascular pressure from 20 to 100 mm Hg increased arterial wall tension and induced steady-state constriction of the arteries. However, arteries isolated from newly born preterm pigs (within 24 h) developed greater myogenic tone and lower active wall tension compared with arteries from full-term and 4-day-old preterm neonates. Pressure-induced elevation in intracellular Ca2+ was also larger in arteries from newly born preterm pigs compared with full-term and 4-day-old preterm pigs. CONCLUSION: Myogenic constriction is elevated in newly born preterm pigs. Our data also suggests that postnatal kidney maturation may modulate renal blood flow autoregulation.

Urotensin II system in chronic kidney disease.

Chronic kidney disease (CKD) is a progressive and long-term condition marked by a gradual decline in kidney function. CKD is prevalent among those with conditions such as diabetes mellitus, hypertension, and glomerulonephritis. Affecting over 10% of the global population, CKD stands as a significant cause of morbidity and mortality. Despite substantial advances in understanding CKD pathophysiology and management, there is still a need to explore novel mechanisms and potential therapeutic targets. Urotensin II (UII), a potent vasoactive peptide, has garnered attention for its possible role in the development and progression of CKD. The UII system consists of endogenous ligands UII and UII-related peptide (URP) and their receptor, UT. URP pathophysiology is understudied, but alterations in tissue expression levels of UII and UT and blood or urinary UII concentrations have been linked to cardiovascular and kidney dysfunctions, including systemic hypertension, chronic heart failure, glomerulonephritis, and diabetes. UII gene polymorphisms are associated with increased risk of diabetes. Pharmacological inhibition or genetic ablation of UT mitigated kidney and cardiovascular disease in rodents, making the UII system a potential target for slowing CKD progression. However, a deeper understanding of the UII system's cellular mechanisms in renal and extrarenal organs is essential for comprehending its role in CKD pathophysiology. This review explores the evolving connections between the UII system and CKD, addressing potential mechanisms, therapeutic implications, controversies, and unexplored concepts.

Post-injury Inhibition of Endothelin-1 Dependent Renal Vasoregulation Mitigates Rhabdomyolysis-Induced Acute Kidney Injury.

In patients with rhabdomyolysis, the overwhelming release of myoglobin into the 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, post-injury inhibition of ET-1-mediated renal vasoregulation may provide therapeutic targets for rhabdomyolysis-induced AKI.

Loss of urotensin II receptor diminishes hyperglycemia and kidney injury in streptozotocin-treated mice.

Beyond the CNS, urotensin II (UII) and its receptor (UT) are functionally expressed in peripheral tissues of the endocrine, cardiovascular, and renal systems. The expression levels of UII and UT in the kidney and circulating UII levels are increased in diabetes. UII also promotes mesangial proliferation and matrix accumulation in vitro. Here, we evaluate the effect of UT deletion on the development of hyperglycemia and diabetic kidney disease (DKD) in streptozotocin (STZ)-treated mice. Ten-week-old WT and UT knockout (KO) mice were injected with STZ for 5 days to induce diabetes. Blood glucose levels were measured weekly, and necropsy was performed 12 weeks after STZ injection. UT ablation slowed hyperglycemia and glucosuria in STZ-treated mice. UT KO also ameliorated STZ-induced increase in HbA1c, but not STZ-induced decrease in plasma insulin levels. However, STZ-induced increases in plasma glucagon concentration and immunohistochemical staining for glucagon in pancreatic islets were lessened in UT KO mice. UT ablation also protected against STZ-induced kidney derangements, including albuminuria, mesangial expansion, glomerular lesions, and glomerular endoplasmic reticulum stress. UT is expressed in a cultured pancreatic alpha cell line, and its activation by UII triggered membrane depolarization, T- and L-type voltage-gated Ca2+channel-dependent Ca2+influx, and glucagon secretion. These findings suggest that apart from direct action on the kidneys to cause injury, UT activation by UII may result in DKD by promoting hyperglycemia via induction of glucagon secretion by pancreatic alpha cells.

Induction of reactive oxygen species by mechanical stretch drives endothelin production in neonatal pig renal epithelial cells.

Vasoactive endothelin (ET) is generated by ET converting enzyme (ECE)-induced proteolytic processing of pro-molecule big ET to biologically active peptides. H2O2 has been shown to increase the expression of ECE1 via transactivation of its promoter. The present study demonstrates that H2O2 triggered ECE1-dependent ET1-3 production in neonatal pig proximal tubule (PT) epithelial cells. A uniaxial stretch of PT cells decreased catalase, increased NADPH oxidase (NOX)2 and NOX4, and increased H2O2 levels. Stretch also increased cellular ECE1, an effect reversed by EUK-134 (a synthetic superoxide dismutase/catalase mimetic), NOX inhibitor apocynin, and siRNA-mediated knockdown of NOX2 and NOX4. Short-term unilateral ureteral obstruction (UUO), an inducer of renal tubular cell stretch and oxidative stress, increased renal ET1-3 generation and vascular resistance (RVR) in neonatal pigs. Despite removing the obstruction, UUO-induced increase in RVR persisted, resulting in early acute kidney injury (AKI). ET receptor (ETR)-operated Ca2+ entry in renal microvascular smooth muscle (SM) via transient receptor potential channel 3 (TRPC3) channels reduced renal blood flow and increased RVR. Although acute reversible UUO (rUUO) did not change protein expression levels of ETR and TRPC3 in renal microvessels, inhibition of ECE1, ETR, and TRPC3 protected against renal hypoperfusion, RVR increase, and early AKI. These data suggest that mechanical stretch-driven oxyradical generation stimulates ET production in neonatal pig renal epithelial cells. ET activates renal microvascular SM TRPC3, leading to persistent vasoconstriction and reduction in renal blood flow. These mechanisms may underlie rUUO-induced renal insufficiency in infants.

Beneficial effects of carbon monoxide-releasing molecule-2 (CORM-2) on acute doxorubicin cardiotoxicity in mice: role of oxidative stress and apoptosis.

Doxorubicin (DXR) has been used in variety of human malignancies for decades. Despite its efficacy in cancer, clinical usage is limited because of its cardiotoxicity, which has been associated with oxidative stress and apoptosis. Carbon monoxide-releasing molecules (CORMs) have been shown to reduce the oxidative damage and apoptosis. The present study investigated the effects of CORM-2, a fast CO-releaser, against DXR-induced cardiotoxicity in mice using biochemical, histopathological and gene expression approaches. CORM-2 (3, 10 and 30 mg/kg/day) was administered intraperitoneally (i.p.) for 10 days and terminated the study on day 11. DXR (20 mg/kg, i.p.) was injected before 72 h of termination. Mice treated with DXR showed cardiotoxicity as evidenced by elevation of serum creatine kinase (CK) and lactate dehydrogenase (LDH), tissue malondialdehyde (MDA), caspase-3 and decrease the level of total antioxidant status (TAS) in heart tissues. Pre- and post-treatment with CORM-2 (30 mg/kg, i.p.) elicited significant improvement in CK, LDH, MDA, caspase-3 and TAS levels. Histopathological studies showed that cardiac damage with DXR has been reversed with CORM-2+DXR treatment. There was dramatic decrease in hematological count in DXR-treated mice, which has been improved with CORM-2. Furthermore, there was also elevation of mRNA expression of heme oxygenase-1, hypoxia inducible factor-1 alpha, vascular endothelial growth factor and decrease in inducible-nitric oxide synthase expression upon treatment with CORM-2 that might be linked to cardioprotection. These data suggest that CORM-2 treatment provides cardioprotection against acute doxorubicin-induced cardiotoxicity in mice and this effect may be attributed to CORM-2-mediated antioxidant and anti-apoptotic properties.