Effects Of IAMC On Electrolyte Metabolism And Renal Antioxidant Indexes In Transport Stressed Rats

Animals suffer from shortness of breath,sweating and incontinence during transport stress,resulting in varying degrees of dehydration and electrolyte disturbances.The kidney is an important site of electrolyte metabolism in the animal body and plays an important role in maintaining its electrolyte balance.When animals are subjected to transport stress,the kidney regulates ion transport-related factors in renal tissues through the Renin-Angiotensin-Aldosterone System（RAAS）,which regulates the uptake and excretion of Na^+,Cl^-and K⁺ions to maintain the body’s electrolyte balance.IAMC is an alkaline water-soluble ionophore additive that has been found to regulate serum Na⁺concentrations in cows and alleviate metabolic acidosis in beef cattle.However,the effect of IAMC on electrolyte disturbances in animals due to transport stress is not known.Therefore,rats were selected as research objects in this study to explore the effects of Balpiril on electrolyte metabolism and renal antioxidant indexes in transport stressed rats.There were five time points in this experiment:three days before transportation（D-3）,before transportation（D0）,after transportation（D0+）,on the third day after transportation（D3）and on the tenth day after transportation（D10）.Two hundred and forty 25-day-old male SD rats were randomly divided into six groups:blank control group（I,60 rats,tested at time points D-3,D0,D0+,D3,D10）,transport stress group（II,36 rats,tested at time points D0+,D3,D10）,IAMC pre-transport addition group（III,48 rats,tested at time points D0,D0+,D3,D10）,vitamin C（Ⅳ,48 rats,the same time points as group III）,IAMC post-transport（V,24rats,the time points were D3 and D10）and vitamin C post-transport（VI,24 rats,the same time points as group V）.Rats in group I were not treated with transport,while rats in groups II,III,IV,V and VI were treated with transport stress for 3.5 h on the day of transport（D0+）（road transport,average speed 42 km/h,total distance 116 km,temperature 26°C,sunny on that day）.According to the experimental design,12 rats were randomly selected and sampled from different test groups at each time point.The following tests were conducted:Experiment 1:Effect of IAMC on indicators related to renal function and histomorphology in transport-stressed rats:The contents of BUN,Cre and CysC in serum of rats were detected by automatic biochemical analyzer,and the morphology of kidney tissue was observed by HE staining.Results（1）Morphology of renal tissue:No obvious pathological changes were observed in D0,Ⅰ,ⅢandⅣgroups.In D0+,compared with rats in groupⅠ,the kidney of rats in groupsⅡ,ⅢandⅣshowed different degrees of renal interstitial small vessel congestion,and a small number of epithelial cells were shed in the renal tubules of rats in groupⅡ.In D3,Ⅱ-Ⅵrats,the interstitial small vessel congestion was obvious,and a few renal tubular epithelial cell granules and vacuoles were denaturated inⅢandⅤrats.The morphology of kidney tissue in D10 ratsⅡ-Ⅵwas restored to normal.（2）Related indexes of renal function:in D0,compared with groupⅠ,serum BUN of groupⅢshowed an increasing trend and renal index showed a decreasing trend.In D0+,compared with groupⅠ,the kidney index of rats in groupⅡshowed an increased trend,and the contents of serum Cre,BUN and CysC had no significant difference.In D3,compared with groupⅠ,the contents of serum Cre,BUN and CysC in groupⅡwere significantly decreased,but the renal index had no significant difference.Compared with groupⅡ,the contents of serum Cre,BUN,CysC and renal index of rats in groupⅢ-Ⅵhad no significant differences.In D10,compared with groupⅠ,the contents of serum Cre,BUN,CysC and renal index of rats in groupⅡhad basically recovered.Experiment 2:Effect of IAMC on the antioxidant capacity of the kidney in transport-stressed rats:The levels of ROS in rat kidney tissues were detected by DHE staining and the antioxidant indexes in rat kidney tissues were detected by colorimetric method.Results（1）Compared with groupⅠ,ROS of rats in D0,SOD and CAT of rats inⅢshowed an increasing trend,while MDA showed a decreasing trend.Kidney GSH-PX in groupⅣwas significantly increased.（2）In D0+,compared with groupⅠ,ROS,GSH-PX and MDA in groupⅡwere significantly increased,while SOD was decreased.Compared with groupⅡ,SOD,GSH-PX and T-AOC in kidney of ratsⅢshowed an increasing trend,while MDA showed a decreasing trend.（3）In D3,compared with groupⅠ,ROS,GSH,T-AOC,SOD and GSH-Px were significantly increased in groupⅡ,while CAT and MDA were increased.Compared with groupⅡ,kidney GSH and Gsh-Px in groupsⅢ-Ⅵwere significantly increased,while ROS was significantly decreased;The kidney T-AOC and SOD of groupⅢwere increased significantly.In groupⅤ,kidney T-AOC increased significantly,SOD and CAT increased.（4）In D10,compared with groupⅠ,kidney GSH in groupⅡwas significantly increased,while other indexes had no significant changes.Compared with groupⅡ,kidney ROS of ratsⅤdecreased significantly,kidney T-AOC,GSH,CAT increased significantly,and SOD increased.The above results indicate that the supplementation of balpiril before and after transport reduces the ROS level of the kidney,improves the activity of antioxidant enzymes in the kidney,and is beneficial to alleviate the oxidative stress of the kidney caused by transport stress.Experiment 3:Effects of IAMC on serum ion levels and hormones in rats under transport stress:The serum ion levels of rats were detected by automatic biochemical instrument,and the serum renin,AngⅡand ALD levels were determined by ELISA.Results（1）Serum ion levels:（1）In D0,compared with groupⅠ,serum Na⁺level of ratsⅢwas significantly increased;（2）In D0+,compared with groupⅠ,serum Na⁺and Cl^-levels of ratsⅡwere significantly increased,Ca²⁺level was significantly decreased,and K+level showed a downward trend.Compared with groupⅡ,the serum Na+and Ca²⁺levels of ratsⅢwere significantly increased,the serum K⁺level showed an increasing trend,and the serum Cl^-level showed a decreasing trend.（3）In D3,compared with groupⅠ,the level of Cl^-and Mg²⁺in groupⅡwas significantly increased,while the level of Na⁺and K⁺was significantly decreased.Compared with groupⅡ,the serum Mg²⁺level of ratsⅢwas significantly increased,the serum K⁺level was increased,and the serum Na⁺level was decreased.The serum Cl^-level of rats in groupⅤwas significantly decreased,and the K⁺level showed a downward trend.（4）In D10,compared with groupⅠ,serum K⁺and Cl^-levels of rats in groupⅡwere significantly increased,and the levels of Na⁺,Mg²⁺and Ca²⁺had basically recovered.Compared with groupⅡ,the serum Na⁺level of rats in groupsⅢ-Ⅵwas significantly increased,the Cl^-level was significantly decreased,and the levels of Mg²⁺and Ca²⁺were not significantly different.The serum K⁺level of rats in groupsⅢandⅤwas significantly decreased.（2）Serum hormone levels:（1）In D0,compared with groupⅠ,serum AngⅡand ALD levels of ratsⅢshowed an increasing trend;（2）In D0+,compared with groupⅠ,serum renin level of ratsⅡwas significantly increased,and AngⅡand ALD levels showed an increasing trend.Compared with groupⅡ,the serum renin level of rats in groupⅢwas significantly decreased,AngⅡlevel was increased,and ALD level was decreased.（3）Compared with groupⅠ,the serum levels of renin,AngⅡand ALD in groupⅡshowed an increasing trend in D3.Compared with groupⅡ,the serum renin level of rats in groupⅢwas decreased,and that in groupⅤwas significantly decreased.The serum AngⅡlevel in groupsⅢandⅤshowed a decreasing trend;The serum ALD level of rats in groupⅢ-Ⅵshowed a decreasing trend.（4）In D10,compared with groupⅠ,the serum hormone level of rats in groupⅡhad basically recovered.Compared with groupⅡ,the serum hormone level of groupⅢhad no significant difference.The serum AngⅡlevel of rats in groupⅤshowed an increasing trend,while the serum ALD level showed a decreasing trend.The above results indicated that adding balpiril before and after transportation could reduce the serum ion level caused by transportation stress in rats to some extent,and affect the changes of serum renin,AngⅡand ALD levels in rats.Experiment 4:Effect of IAMC on renal ion transport function in transport-stressed rats:The mRNA and protein expression levels of renal ion transport proteins（α-ENAC,AQP1,AQP2,ROMK,NCC）were detected by RT-q PCR with WB method,and NKA enzyme activity in rat kidney was detected by colorimetric method.Results（1）Compared with groupⅠ,ROMK protein level in groupⅢand NCC mRNA and protein level in groupⅢwere significantly increased in D0.（2）In D0+,compared with groupⅠ,the levels ofα-ENAC mRNA and protein in the kidney of rats in groupⅡwere increased significantly,the levels of AQP2 protein were increased,and the activity of NKA was decreased significantly.Compared with groupⅡ,NKA activity,NCC mRNA and protein levels were significantly increased in groupⅢ,whileα-ENAC mRNA and protein levels were decreased.（3）In D3,compared with groupⅠ,NKA activity in kidney of rats in groupⅡwas significantly increased,mRNA and protein levels ofα-ENAC and NCC were increased,while ROMK protein levels were decreased.Compared with groupⅡ,the protein expressions of AQP2,α-ENAC and ROMK in kidney of groupⅢwere significantly increased,while the activity of NKA was significantly decreased.In groupⅤ,NKA activity,AQP2,NCC mRNA and protein expressions were significantly increased,whileα-ENAC,ROMK mRNA and protein expressions were increased.（4）In D10,compared with groupⅠ,mRNA and protein expressions of AQP2,α-ENAC,ROMK and NCC in groupⅡshowed no significant differences,and NKA activity showed an upward trend.Compared with groupⅡ,the expression of NCC mRNA and protein in kidney of groupⅢwas significantly increased,the expression ofα-ENAC protein was significantly decreased,and the activity of NKA was decreased.The mRNA and protein expressions of AQP2 in groupⅤwere significantly increased.These results suggest that Balpiril may regulate the expression of ion transporters to relieve the electrolyte disorder caused by transport stress.In conclusion,transport stress leads to ROS accumulation,antioxidant enzyme activity fluctuations,tissue morphological changes,serum electrolyte disturbance,and interference with protein transport-related protein levels in rat kidneys.Adding IAMC before and after transportation alleviated the changes of the above indexes to a certain extent,suggesting that IAMC can alleviate the serum electrolyte disturbance and renal oxidative stress caused by transportation stress in rats to a certain extent.