Piezo1 Mediates Vasodilation Induced by Acute Hyperglycemia in Mouse Renal Arteries and Microvessels.

BACKGROUND: Acute hyperglycemia is a risk factor for developing acute kidney injury and poor renal outcome in critically ill patients, whereby the role of renal vasculature remains unclear. We hypothesize that hyperglycemia-associated hyperosmolarity facilitates vasodilation through Piezo1-mediated eNOS (endothelial NO synthase) activation. METHODS: Vasoreactivity was analyzed using wire myography in isolated mouse mesenteric arteries and renal interlobar, and using microvascular perfusion in renal afferent arterioles and efferent arterioles, and vasa recta. Immunofluorescence and Western blot were used for molecular analyses of isolated mouse blood vessels and human umbilical vein endothelial cells. RESULTS: Pretreatment with hyperglycemia (44 mmol/L glucose; 4 hours) increased acetylcholine-induced relaxation in interlobar arteries and mesenteric arteries, which was prevented by eNOS inhibition using Nω-nitro-L-arginine methylester hydrochloride. Hyperosmotic mannitol solution had a similar effect. Hyperglycemia induced an immediate, Nω-nitro-L-arginine methylester hydrochloride-inhibitable dilation in afferent arterioles, efferent arterioles, and vasa recta, whereby stronger dilation in afferent arterioles compared to efferent arterioles. Hyperglycemia also increased glomerular filtration rate in mice. In human umbilical vein endothelial cells, hyperglycemia, and the Piezo1 activator Yoda-1 increased levels of Piezo1 protein, p-CaMKII (phosphorylated Ca2+/Calmodulin-dependent protein kinase type II), Akt (protein kinase B), and p-eNOS (phosphorylated eNOS). The hyperglycemia effect could be prevented by inhibiting Piezo1 using GsMTx4 (Grammostola spatulata mechanotoxin 4) and CaMKII using KN93 (N-[2-[[[3-(4-Chlorophenyl)-2-propenyl]-methylamino]-methyl]-phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide). Furthermore, in arteries and microvessels, inhibition of Piezo1 using GsMTx4 prevented the hyperglycemia -effect, while Yoda-1 caused relaxation and dilation, respectively. CONCLUSIONS: Results reveal that Piezo1 mediates renal vasodilation induced by hyperosmolarity in acute hyperglycemia. This mechanism may contribute to the pathogenesis of renal damage by acute hyperglycemia.

Nitric Oxide Signalling in Descending Vasa Recta after Hypoxia/Re-Oxygenation.

Reduced renal medullary oxygen supply is a key factor in the pathogenesis of acute kidney injury (AKI). As the medulla exclusively receives blood through descending vasa recta (DVR), dilating these microvessels after AKI may help in renoprotection by restoring renal medullary blood flow. We stimulated the NO-sGC-cGMP signalling pathway in DVR at three different levels before and after hypoxia/re-oxygenation (H/R). Rat DVR were isolated and perfused under isobaric conditions. The phosphodiesterase 5 (PDE5) inhibitor sildenafil (10-6 mol/L) impaired cGMP degradation and dilated DVR pre-constricted with angiotensin II (Ang II, 10-6 mol/L). Dilations by the soluble guanylyl cyclase (sGC) activator BAY 60-2770 as well as the nitric oxide donor sodium nitroprusside (SNP, 10-3 mol/L) were equally effective. Hypoxia (0.1% O2) augmented DVR constriction by Ang II, thus potentially aggravating tissue hypoxia. H/R left DVR unresponsive to sildenafil, yet sGC activation by BAY 60-2770 effectively dilated DVR. Dilation to SNP under H/R is delayed. In conclusion, H/R renders PDE5 inhibition ineffective in dilating the crucial vessels supplying the area at risk for hypoxic damage. Stimulating sGC appears to be the most effective in restoring renal medullary blood flow after H/R and may prove to be the best target for maintaining oxygenation to this vulnerable area of the kidney.

Age Impairs Soluble Guanylyl Cyclase Function in Mouse Mesenteric Arteries.

Endothelial dysfunction (ED) comes with age, even without overt vessel damage such as that which occurs in atherosclerosis and diabetic vasculopathy. We hypothesized that aging would affect the downstream signalling of the endothelial nitric oxide (NO) system in the vascular smooth muscle (VSM). With this in mind, resistance mesenteric arteries were isolated from 13-week (juvenile) and 40-week-old (aged) mice and tested under isometric conditions using wire myography. Acetylcholine (ACh)-induced relaxation was reduced in aged as compared to juvenile vessels. Pretreatment with L-NAME, which inhibits nitrix oxide synthases (NOS), decreased ACh-mediated vasorelaxation, whereby differences in vasorelaxation between groups disappeared. Endothelium-independent vasorelaxation by the NO donor sodium nitroprusside (SNP) was similar in both groups; however, SNP bolus application (10-6 mol L-1) as well as soluble guanylyl cyclase (sGC) activation by runcaciguat (10-6 mol L-1) caused faster responses in juvenile vessels. This was accompanied by higher cGMP concentrations and a stronger response to the PDE5 inhibitor sildenafil in juvenile vessels. Mesenteric arteries and aortas did not reveal apparent histological differences between groups (van Gieson staining). The mRNA expression of the α1 and α2 subunits of sGC was lower in aged animals, as was PDE5 mRNA expression. In conclusion, vasorelaxation is compromised at an early age in mice even in the absence of histopathological alterations. Vascular smooth muscle sGC is a key element in aged vessel dysfunction.

Protective Effect of Tempol on Acute Kidney Injury Through PI3K/Akt/Nrf2 Signaling Pathway.

BACKGROUND/AIMS: Tempol is a protective antioxidant against ischemic injury in many animal models. The molecular mechanisms are not well understood. Nuclear factor erythroid 2-related factor (Nrf2) is a master transcription factor during oxidative stress, which is enhanced by activation of protein kinase C (PKC) pathway. Another factor, tubular epithelial apoptosis, is mediated by activation of phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB, Akt) signaling pathway during renal ischemic injury. We tested the hypothesis that tempol activates PKC or PI3K/Akt/Nrf2 pathways to transcribe many genes that coordinate endogenous antioxidant defense. METHODS: The right renal pedicle was clamped for 45 minutes and the left kidney was removed to study renal ischemia/reperfusion (I/R) injury in C57BL/6 mice. The response was assessed from serum parameters, renal morphology and renal expression of PKC, phosphorylated-PKC (p-PKC), Nrf2, heme oxygenase-1 (HO-1), Akt, phosphorylated-Akt (p-Akt), pro-caspase-3 and cleaved caspase-3 in groups of sham and I/R mice given vehicle, or tempol (50 or 100 mg/kg, intraperitoneal injection). RESULTS: The serum malondialdehyde (MDA, marker of reactive oxygen species) doubled and the BUN and creatinine increased 5- to 10-fold after I/R injury. Tempol (50 or 100 mg/kg) prevented the increases in MDA but only tempol (50 mg/kg) lessened the increases in BUN and creatinine and moderated the acute tubular necrosis. I/R did not change expression of PKC or p-PKC but reduced renal expression of Nrf2, p-Akt, HO-1 and pro-caspase-3 and increased cleaved caspase-3. Tempol (50 mg/kg) prevented these changes produced by I/R whereas tempol (100 mg/kg) had lesser or inconsistent effects. CONCLUSION: Tempol (50 mg/kg) prevents lipid peroxidation and attenuates renal damage after I/R injury. The beneficial pathway apparently is not dependent on upregulation or phosphorylation of PKC, at lower tempol doses, does implicate upregulation of Akt with expression of Nrf2 that could account for the increase in the antioxidant gene HO-1 and a reduction in the cleavage of the cellular damage marker pro-caspase-3.