Mechanisms Of NaHCO₃ And TMAO Regulate Renal Afferent Arteriolar Tone And Glomerular Filtration Rate In Hypertension

Circulating sodium bicarbonate and trimethylamine N-oxide?TMAO?play an important role in the development of many diseases,such as hypertension and chronic kidney disease?CKD?.Knockout of one of the Na⁺/HCO₃^-cotransporters?NBCn1?impairs acetylcholine-induced vasodilatation of mesenteric artery via reducing nitric oxide?NO?generation.Aging and CKD increase circulating TMAO level and lead to excessive production of reactive oxygen species?ROS?,inhibit the activity of endothelial nitric oxide synthase?e NOS?,reduce NO generation,and ultimately attenuate the relaxation of mesenteric arteries induced by acetylcholine.Since renal afferent arterioles?Afs?are the major resistance vessels to the whole body,the vasoconstriction and vasodilatation regulate the resistance of Af and thereby affect blood flow and glomerular filtration rate?GFR?of single nephron,which affects the systemic blood volume and then blood pressure.However,whether sodium bicarbonate and TMAO affect the resistance of Af or not?Can the changed resistance of Afs affects the GFR and blood pressure in mice?If so,what mechanisms does it work in?Therefore,isolated and microperfused mouse renal Af was used to explore the effects and mechanisms of NaHCO₃and TMAO on the Af tone and GFR in hypertensive mice.Which provides new ideas for clinical treatment of CKD and hypertension and other related diseases.Part one:The mechanism of sodium bicarbonate regulates renal afferent arteriolar tone and glomerular filtration rateAims:The amount of ultrafiltrate generated by both kidneys per unit of time is called glomerular filtration rate?GFR?.The resistance of afferent arteriole?Af?influences the glomerular capillary filtration pressure,which affects the renal GFR.In patients with chronic kidney disease?CKD?,GFR gradually decreases with the development of disease.Diabetes patients will have an increase in GFR in the early stage.The increased GFR is a risk factor for diabetic nephropathy.Improving the tension of the Af reverses renal GFR and then protects renal function.Oral NaHCO₃ preserves the decline of GFR by reducing metabolic acidosis in CKD patients,thus having a protective effect on kidney function.However,it is not clear that the mechanisms of renal function protection from NaHCO₃.Knockout of one of the Na⁺/HCO₃^-cotransporters?NBCn1?impairs acetylcholine-induced vasodilatation of mesenteric artery.We hypothesis that NaHCO₃ dilates Af by stimulating nitric oxide?NO?release mediated by upregulated of the Na⁺/HCO₃^-cotransporters?NBCs?contributing to the elevation in GFR.Methods:1.C57Bl/6 mice were injected with FITC-sinistrin by intravenous,which dissolved in saline or 44 m M NaHCO₃ solution.Renal GFR was measured by the plasma FITCsinistrin clearance.The effect of intravenous injection NaHCO₃ solution on mice blood pressure and plasma pH were measured by catheterizing carotid artery and a calibrated pH electrode.2.The Af with attached glomerulus was microdissected under a stereomicroscope and then transferred to a temperature-controlled chamber on the stage of a confocal microscope.After a 15-minute equilibration period,Af was given different bath solution according to the different research purpose,and then recorded the diameter of Af.3.The expression of Slc4 bicarbonate transporter family in Af and mouse primary cultured renal vascular endothelial cell?EC?and vascular smooth muscle cell?VSMC?were measured by reverse transcription–polymerase chain reaction?RT-PCR?,quantitative polymerase chain reaction?q PCR?,immunoblotting and immunofluorescence.4.Microperfused Af were loaded with 4,5-diaminofluorescein diacetate?DAF-2 DA?and 2?,7?-bis-?2-Carboxyethyl?-5-?and-6?carboxyfluorescein,acetoxymethyl ester?BCECF-AM?to examine the alteration of nitric oxide?NO?and intracellular pH.Results:1.NaHCO₃ enhances the renal GFR in mice,however,administration of NaHCO₃ and Na Cl did not significantly change the mean arterial pressure or plasma pH in mice.NaHCO₃ can dilate renal Af,but the physiological Na Cl solution can't.2.NBCe1 and NBCn1 were detected in Af and primary cultured mouse renal vascular EC and VSMC.S0859,an inhibitor of the Na⁺/HCO₃-cotransporter?NBC?family,which inhibited the NaHCO₃ induced vasodilation.3.NaHCO₃ increases the intracellular pH,which can be blocked by S0859.Meanwhile,increasing the pH of bath solution dilates renal Af.4.NaHCO₃ and increasing the pH of bath solution enhance the NO generation of Af.Endothelial nitric oxide synthase?e NOS?inhibitor L-NG-Nitroarginine Methyl Ester?L-NAME?blocked NaHCO₃ and increasing the pH of bath solution induced NO generation and vasodilation.Conclusion:We conclude that NBCn1 and NBCe1 are expressed in Af.HCO₃-is transported into Af via NBCe1 and NBCn1,thereby increasing pHi and activating e NOS that enhances NO generation.The increased NO dilates renal Af and consequently contributes to an increase in GFR.Part two: The action and mechanism of trimethylamine Noxide in regulating angiotensin ?-induced hypertensionAims:Over the past decades,increasing evidence has linked the activity and composition of the gut microbiota with human health and disease.Trimethylamine N-oxide?TMAO?is a product of gut microbiota,which plays an important role in the progress of many diseases.Previous studies suggest that TMAO is implicated in the pathogenesis of many diseases such as chronic kidney disease?CKD?and atherosclerosis.However,whether and how TMAO affects the progression of hypertension have not been investigated.Since Afs are the major resistance vessels to the whole body,the vasoconstriction and vasodilatation regulate the resistance of Af and thereby regulate blood flow and GFR of single nephron,which affects the systemic blood volume and then blood pressure.Therefore,we hypothesis that TMAO affects the resistance of Af,thereby regulating blood pressure and GFR,which plays an important role in hypertension.Methods:1.Using Isolated and microperfused Af system and a wire myograph system to test whether TMAO directly contracts Af and mesenteric artery?MR?.Isolated and microperfused Af was used to investigate whether and how 200 ?M TMAO affects angiotensin ??Ang ??induced vasoconstriction.Some relevant inhibitors,including ROS scavenger 4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl?Tempol?,calcium/calmodulin-dependent kinase ??Ca MK??inhibitor KN-93 and phospholipase C?PLC?inhibitor U73122),were used to study the mechanisms of TMAO in regulating Ang ? induced vasoconstriction.Microperfused Af were loaded with fluorescence probe Fluo-4 to examine the intracellular Ca²⁺ release.2.Mice injected with TMAO or TMAO combined with Ang ? to examine whether acute administration of TMAO influences the basal blood pressure or Ang ? induced blood pressure elevation.The acute variation of blood pressure was measured by catheterizing carotid artery.3.Mice implanted with osmotic pumps to test the effect of TMAO on chronic Ang ? infusion induced hypertension.To examine whether inhibition of TMAO generation affects the chronic Ang ? infusion induced hypertension,antibiotic was administered through drinking water in the Ang ? infused mice.Therefore,the mice were randomly divided into six groups: control group,TMAO group,Ang ? group,TMAO + Ang ? group,antibiotic group and antibiotic + Ang ? group.Tail-cuff was used to record the chronic blood pressure.The plasma TMAO and Ang ? concentration were measured by enzyme linked immunosorbent assay?Elisa?kit in the six groups.Isolated Af and MR from the six groups were used to study its constrictor response to vasoconstrictors,such as Ang ?,phenylephrine?PE?and endothelin-1.4.Cultured mouse aortic smooth muscle cells?MOVAS?were treated with different concentrations of TMAO for 24 hours or a fixed concentration of TMAO?100 ?M?for different time periods and then the relative protein expression was detected by Western blot.The generation of reactive oxygen species?ROS?were detected by relative fluorescence probes,including Mito SOX,Dihydroethidium and JC-1.Results:1.Low concentrations of TMAO have no effect on Af and mesenteric arterial vascular tone,however,high concentrations of TMAO directly contracts Af and MR,and Af are more sensitive to TMAO than MR.Meanwhile,similar effects on blood pressure were observed when mice acute administration of TMAO.2.Isolated and microperfused Af pretreatment with pathophysiological concentrations of TMAO enhances Ang ? induced vasoconstriction.Tempol,KN-93 and U73122 suppress the effect of TMAO on Ang ? induced vasoconstriction.Mice were injected with TMAO enhances pressor response to acute administration of Ang ?.Af pretreatment with TMAO augments intracellular calcium elevation triggered by Ang ?.3.TMAO or antibiotic treatment had marginal effects on blood pressure.Importantly,in the Ang ?–infused mice,hypertension was significantly aggravated by TMAO treatment but alleviated by antibiotics treatment.Antibiotics reduce plasma TMAO concentration,however,mice infused with Ang ? increases plasma TMAO level.At the same time,Af and MR from TMAO group mice showed a stronger response to Ang ?,which was blocked by U73122.4.TMAO enhances the ROS generation and thereby activates Ca MK?/PLC ?3/Ca²⁺ signaling pathway in MOVAS.Conclusion:High concentrations of TMAO contracts Af and MR and then increases blood pressure,however,low concentrations of TMAO has no effective.Low concentrations of TMAO enhances Ang ? induced vasoconstriction and acute pressor response via activating ROS/Ca MK?/PLC ?3/Ca²⁺ signaling pathway.Orally administered TMAO aggravates Ang ? induced hypertension.On the other hand,antibiotic inhibits gut microbiota and leads to reduce the TMAO generation and consequently alleviates Ang ? induced hypertension.