Protective Effect And Mechanism Of TRPA1 In Contrast-induced Nephropathy

Background:Contrast-induced nephropathy（CIN）is defined as acute renal insufficiency that appears within a short period of time after contrast agent injection under the premise of excluding the effects of other diseases.The continuous progress of coronary intervention surgery and the rapid upgrading of imaging hardware facilities provide strong support for the diagnosis and treatment of cardiovascular and cerebrovascular system diseases.At the same time,the massive use of contrast agent and the increase of combined underlying diseases make CIN a common complication after cardiac catheterization and coronary intervention.CIN has become one of the main causes of hospital-acquired impaired renal function.The diagnostic criteria for CIN are not fully uniform,and according to the latest recommendations of KDIGO,CIN is defined as:an increase in creatinine levels≥0.3 mg/d L（26.5μmol/L）from baseline levels within48 h after contrast agent use,or an increase of more than 1.5 times baseline values within 7 days.Compared with the normal population,the incidence of CIN is significantly increased in people with underlying diseases such as chronic kidney disease（CKD）,diabetes and hypertension.For the diagnosed CIN,there is a lack of effective means to reverse the disease progression of CIN in clinical practice.Therefore,how to effectively prevent or improve acute renal impairment caused by contrast agent use has been a hot issue in the medical field.At present,it is generally accepted that the pathogenesis of contrast-induced nephropathy is closely related to its direct toxic effects,renal blood perfusion changes,oxidative stress,inflammatory response,and apoptosis.Among them,oxidative stress has been paid more attention.Due to its unique high viscosity and high permeability,contrast agent can prolong its retention time in the kidney compared with other drugs,causing toxic effects on cells,resulting in massive necrosis of tubular epithelial cells and decreased glomerular filtration rate.At the same time,it can affect the normal physiological function of vascular endothelial cells,reduce the production of substances with vasodilator effect,lead to continuous contraction of renal vessels,and aggravate renal tissue ischemia and hypoxia.Each of the above stages can produce excessive reactive oxygen species（ROS）,which inhibit the function of nitric oxide synthetase（NOS）and prostacyclin lyase,resulting in reduced nitric oxide（NO）and prostaglandin（PG）production and further aggravating kidney injury.It has been found that reducing renal ROS levels attenuates the damage of CIN.Transient receptor potential channel A1（TRPA1）,a member of the TRP subfamily of mammalian cell membrane-anchored proteins,is a non-selective cation channel protein with good permeability to Ca²⁺.This channel can be activated by byproducts of a variety of reactive oxygen species,which can sense changes in the level of oxidative stress in the body.It can also function as a cell membrane receptor,and TRPA1 can be activated by cold stimulation（<17℃）,mustard oil,cinnamaldehyde,and allicin.It was previously believed that TRPA1 is mainly expressed in the heart,blood vessels,gastrointestinal tract,pancreas and other tissues.TRPA1 can sense noxious stimuli such as heat,cold and itching,and pain.At the same time,TRPA1 is also involved in the regulation of vasodilatation and inflammatory response.Recent studies have found that TRPA1 is still expressed in renal tissue,and is up-regulated in the renal tubules of AKI patients,which is considered to be closely related to the progression of AKI to CKD.TRPA1 may therefore be closely related to the development of renal disease,but the role of TRPA1 in contrast-induced nephropathy is currently unknown.Therefore,in this study,mice were used as experimental subjects,and after the construction of contrast-induced nephropathy models in line with clinicopathological characteristics,TRPA1-specific agonist allyl isothiocyanate（AITC）and inhibitor HC030031 were given for early intervention,and the changes in serum creatinine and urea nitrogen,renal pathological changes,renal oxidative stress levels,and changes in the expression levels of TRPA1 and apoptosis-related proteins in renal tissues of mice in each experimental group were observed by H&E staining,TUNEL fluorescence staining,Western blot,and enzyme-linked immunosorbent assay（Elisa）techniques.To investigate the specific role and mechanism of TRPA1 in contrast-induced renal injury in order to provide some theoretical reference for the prevention and treatment of contrast-induced renal injury in clinical practice.Purposes:1.C57BL/6J mice were used to construct a mouse model of contrast-induced renal injury that met the clinical characteristics by intraperitoneal injection of indomethacin（Indomethacin）,L-NAME,followed by tail vein injection of iodixanol after water deprivation treatment.2.TRPA1 agonist AITC and inhibitor HC030031 were used to intervene in advance,and the role and mechanism of TRPA1 in CIN were preliminarily explored by observing the changes of kidney morphology and function.Methods:1.20 SPF male C57BL/6J mice were randomly divided into blank control group（control）,contrast agent group（CM）,renal injury group（Indo+L-NAME）,and model group（Indo+L-NAME+CM）,with 5 mice in each group.Indomethacin and nitroso-L-arginine methyl ester（L-NAME）were injected intraperitoneally in advance,followed by liquid iodixanol from the tail vein of mice,and blood and kidney samples were collected 24 hours after administration to analyze renal function and morphological changes.2.After verifying that the model was successfully constructed,25 SPF grade male C57BL/6J mice were randomly divided into blank control group（control）,contrast-induced nephropathy group（CIN）,vehicle control group（DMSO+CIN）,agonist group（AITC+CIN）,and inhibitor group（HC030031+CIN）,with 5 mice in each group.The construction method of contrast-induced nephropathy model was the same as above.DMSO+CIN was treated with the same amount of solvent before intervention;AITC+CIN was intraperitoneally injected 30 minutes before modeling;HC030031+CIN was intraperitoneally injected 30 minutes before modeling.Blood and kidney samples were taken 24 hours after intervention to observe serum creatinine,urea nitrogen,renal pathological changes,TRPA1 expression,oxidative stress levels and apoptosis,and mitochondrial morphology in each group of mice.Result:1.Compared with the CM group,the serum creatinine and urea nitrogen levels of mice in the Indo+L-NAME+CM group increased significantly（P<0.01）,and the increased creatinine level reached the diagnostic criteria of contrast-induced nephropathy.There was no statistically significant difference in creatinine and urea nitrogen levels between the control,CM,and Indo+L-NAME groups（P>0.05）.H&E staining of kidney tissue revealed that in the control group,the tubular epithelial cells were arranged regularly,the cells were full,and there was no significant injury.In the CM group,individual tubular epithelial cells showed similar vacuole-like changes,and the overall injury was not significant.The renal tissue in the Indo+L-NAME group was arranged regularly and did not show significant damage.In the Indo+L-NAME+CM group,a large range of tubular epithelial cell detachment and vacuolar degeneration were observed,the brushing edge injury was significant,some basement membranes were exposed,and the degree of tissue injury was significantly aggravated compared with the CM group,and the difference was statistically significant（P<0.01）.DHE staining of kidney tissue revealed that ROS levels in kidney tissue were significantly increased in Indo+L-NAME+CM compared with CM group,and the difference was statistically significant（P<0.01）.TUNEL staining of kidney tissue suggested that compared with the control group and CM group,Indo+L-NAME+CM kidney tissue showed more significant tubular epithelial cell apoptosis,and the difference was statistically significant（P<0.01）.2.Immunofluorescence staining revealed that TRPA1 was normally expressed in a certain amount of renal tissue.Compared with the control group,the expression of TRPA1 in the CIN group was increased,and the difference was statistically significant（P<0.01）;the expression of TRPA1 was significantly up-regulated in the AITC+CIN group（P<0.01）,while the expression of TRPA1 was significantly decreased in the HC030031+CIN group（P<0.01）.In terms of renal function,serum creatinine levels and serum urea nitrogen levels were significantly decreased in the AITC+CIN group compared with the CIN group（P<0.01）,and serum creatinine levels and serum urea nitrogen levels were significantly increased in the HC030031+CIN group（P<0.01）.In terms of pathological injury,H&E and TUNEL staining suggested that the degree of tubular injury and the level of tubular epithelial cell apoptosis were significantly reduced in the AITC+CIN group compared with the CIN group（P<0.01）,and the degree of injury and apoptosis was significantly increased in the HC030031+CIN group（P<0.01）.DHE staining suggested that ROS levels were significantly decreased in the AITC+CIN group compared with the CIN group（P<0.01）,while ROS levels were increased in the HC030031+CIN group（P<0.01）.The results of SOD and MDA in kidney tissue suggested that compared with CIN group,SOD activity was increased（P<0.01）and MDA content was significantly decreased（P<0.01）in AITC+CIN group,while SOD activity was significantly decreased（P<0.01）and MDA content was significantly increased（P<0.01）in HC030031+CIN group.Compared with CIN group,the levels of TRPA1 and Bcl-2 m RNA were increased（P<0.01）,and the levels of Bax and caspase-3 m RNA were decreased（P<0.01）;compared with HC030031+CIN group,the levels of TRPA1 and Bcl-2 m RNA were decreased（P<0.01）,and the levels of Bax and caspase-3 m RNA were increased（P<0.01）.Compared with CIN group,the expression of TRPA1 and Bcl-2 protein was increased（P<0.05）,while the expression of Bax and caspase-3 protein was relatively decreased（P<0.01）in AITC+CIN group;the expression of Bcl-2 protein was decreased（P<0.01）,and the expression of Bax and caspase-3 protein was relatively increased（P<0.05,P<0.01）in HC030031+CIN group.Electron microscopy showed that mitochondria in CIN DMSO+CIN group showed excessive division,mitochondrial structure was destroyed,and nuclei were shrunken.The degree of mitochondrial damage was alleviated in the AITC+CIN group compared with the CIN group,while the number of mitochondria was reduced,the mitochondrial structure was destroyed,autophagosome formation was observed,and nuclear shrinkage was evident in the HC030031+CIN group.Conclusion:1.The mice were treated with water deprivation for 24 h before administration of contrast agent,and intraperitoneal injection of vasoconstrictor drugs Indomethacin and L-NAME,followed by intravenous injection of iodixanol injection through tail vein.The serum creatinine level in mice was increased by more than 0.3 mg/d L（26.5μmol/L）compared with the baseline value,reaching the diagnostic criteria of contrast-induced nephropathy,and the mouse model of contrast-induced nephropathy was successfully established.2.Pharmacological agonists of TRPA1 can prevent renal pathological damage and renal function injury in CIN by decreasing renal ROS levels,increasing antioxidant capacity,reducing tubular epithelial cell apoptosis,and improving mitochondrial injury.