| Neonatal kidney injury is one of common diseases in PICU,also is the leading cause of death in PICU. Neonatal acute kidney injury is very common, but the studies were relatively few. Death caused by acute kidney injury is very common, and in later period can cause renal insufficiency easier, especially in very low birth weight,and neonate with other diseases. In recent years, the research on neonatal acute kidney injury was increasing. In the case of low blood volume, a variety of pathological conditions such as shock, infection, hypothermia can easy case kidney damage, also can cause neonatal renal insufficiency. The mortality of acute renal failure caused by endotoxin accounts for about 70%. At present, the pathological mechanism of endotoxin induced acute renal failure was poorly understood.Lipopolysaccharide(LPS) frequently used as endotoxin, widely used in the animal during the preparation of endotoxin damage models. Endotoxin of kidney damage can lead renal hemodynamic changes, make the body appear ischemia anoxic; Endotoxin of kidney damage can also lead the oxidative metabolism of the cells destroyed.Studies have shown that hypoxia-inducible factor, as the regulation gene in the anoxic condition, can produce nucleoprotein under the condition of oxygen nucleoprotein, combine with its target genes and bring about a change in adaptation to hypoxia environment condition, thus cause HIF-1a expression increased. HIF-1α can promote the release of inflammatory cytokines through NF-k B signal pathways, thus worsen kidney damage.Other studies suggested that glutamine(Gln) as substrates ofrenal dysplasia sugar, and as fuel of renal tubular cell oxidative metabolism.Endotoxemia occurs, Gln can inhibit the apoptosis of renal tubular epithelial cells and can slow down the inhibition degree of mitochondrial respiratory function. PHC is a new anticholinergic agent widely usede in clinical anesthesia.Studies have shown that PHC has plays a key role of multiple organs in improving microcirculation,can be considered as ideal vasoactive agent for correct the microcirculation cramps when endotoxic shock.Upon this, through a large number of clinical cases we found that PHC has a protective function for acute kidney injury. Therefore, this study aims to observe the effects of preconditioning with penehyclidine hydrochloride on HIF-1αand Gln that expressed in LPS-induced kidney injury in neonatal rats, to further discuss the specific mechanism.ObjectiveTo explore the expression of HIF-1α and Gln in LPS-induced kidney injury in neonatal rats, and the effects of pretreatment with PHC on HIF-1α、Gln and other relative cytokins expression, and looking forward to find new treatment targets for clinical neonatal kidney injury.Materials and Methods1 Experimental AnimalsSeventy-two SD rats, SPF, 7 days old, healthy, supplied by animal center of Henan province, male and female unlimited, weighting from 15.0 to 18.0g.2 Experimental Methods2.1 Experimental animal groupsBased on the research content, experimental animals were randomly divided into two parts: 48 and 24.The first one, forty-eight rats were divided into 3 groups by random number table, 16 for each group. Group C: control group; Group K :Endotoxin kidney injurymodel group and Group P :Penehyclidine hydrochloride pretreatment group. This section of experimental animals were aim to observe the behavioral science and the change of mental state, and calculate the mortality of 24 h after building.The second part, twenty-four rats were also stochasticly divided into 3 groups use the way of part 1, 8 for each group. We detection the expression of HIF-1α,Gln and relation factors, kidney determination of the wet/dry weight ratio, and then observed the histopathlogy converts in kidney tissues.2.2 Experimental model preparationGroup C: Intraperitoneal injection of PBS 10ml/kg;Group P: Firstly, intraperitoneal injection of penehyclidine hydrochloride2mg/kg,diluteding the concentration of 0.10mg/ml, then, intraperitoneal injection of LPS after 30 minutes.Group K: Intraperitoneal injection of LPS 5 mg/kg, diluteding the concentration of 1.0 mg/ml;Injection site requirements are consistent, withdrawing without blood and gas.After molding, toe clipping mark clear,then back into the cage and mothers feeding together. Keep feeding environment balanced. The second part of the mice in building 6 hours after anesthesia, materials, frozen storage, set aside.2.3 Kidney tissue specimen collectionIntraperitoneal injection of chloral hydrate 3ml/kg, then connected with animal anesthesia machine. Fixed the rats on the operating table. Opened the chest, touching the heart beats, collected blood by heart puncture with 5 needles. Let stand for 1hour, then 1200 rpm centrifuged 10 min under room temperature, gathered the supernate, and saved at-80℃ for more forward a single step of inspection. Removed the left and the right kidney after laparotomy. Took upper half on the right kidney,homogenated for the determining of HIF-1α and Gln; Took the other half on the right kidney, extracted RNA,used for RT-PCR analysis of HIF-1α m RNA expression in kidney tissue. Took upper half on the left kidney, measuring the wet weight and dry weight of the kidney tissue; Took the other half on the left kidney, HE dyed,observing the histopathological change under optical microscope.2.4 Test indexes2.4.1 Numeration of renal tissues wet/dry weight ratioMade use of the filter paper dried the kidney tissue and weighted the wet weight by electronic scales,recorded it, and then, threw them into 70℃ oven for 2 days,arided in, weighed it, and recorded it. At last,we numerated the ratio.2.4.2 Histopathological observation of kidney tissuesMade the lower half on the left kidney tissue into 10% paraformaldehyde(the volume of paraformaldehyde is three times greater than the kidney tissues) 48 h for fixing fully. Paraffin embedded, HE stained. Observing the histopathological change under optical microscope.2.4.3 Measurements of TNF-α, IL-6, HIF-1α, Gln in kidney tissue in serum by ELISATo weigh the upper half on the right kidney kidney tissues, PBS buffer fluid,using slurry preparation tissue homogenate. Under the condition of 4000 r/min,centrifuged 10 minutes, collected supernatant,- 80 refrigerator save to use. ELISA kit to use manual detection of TNF-α, IL- 6, the expression of HIF-1α and Gln.2.4.4 RT-PCR analyzes the expressing levels of HIF-1α m RNAGot the lower half on the right kidney tissue, flushed by DEPC water, then throw in-80 refrigerator waiting for inspection.Used Trizol extracting of total RNA,through the following steps: inverse transcription, amplification and agarose gel electrophoresis,etc.We determinated the expression of the HIF-1α m RNA levels in the kidney tissue.Using SPSS17.0 statistics software package analyzed the datas. Statistical analysis used the method of One-Way ANOVA and Least Significant Difference test(LSD-t). 0.05 was setted as the inspection standard. When the consequence <0.05 proves the difference is statistical significance.Results1. Living condition observation After administration of LPS, 1 hour later, the newborn mice of group K began to shake, shortness of breath. About after 3 hours temperature is reduced, skin turn grey, depression, unresponsive, and breathing activity intensity is reduced.About 6 hours later, the symptoms and signs of the rats were worsen, even severe depletion of oxygen. Apparently, At the same time compared to group K, the symptoms and signs of group P were substantial improvement. The symptoms and signs of all rats in group C were normal. The mortality rate of 24 h in Group C, P and K group were 0%, 12.4% and 50%respectively.2. The kidney wet/dry weight ratio results: the groups K and P compared with group C were significantly higher(P<0.05). But compared with group K, group P was significantly reduced(P < 0.05).3. Renal histopathological results showed: no significant changes in renal tissue morphology in group C. In group K, we saw renal tubular epithelial cells markedly swollen, lumen narrowed, congestion, part of the renal tubular epithelial cell cavity deformation or transparent degeneration. Exfoliated cells and red blood cells could be saw in the lumen, interstitial angiectasis hyperemia, and a large number of inflammatory cells infiltration.Compare to group K, the signs and symptoms were substantial improvement.4. The assay of IL-6,TNF-α, and HIF-1α in kidney tissue were significantly higher in both group K and P that compared with group C(P<0.05), but the content of Gln was lower in both group K and P(P<0.05); compared to group K, the content of TNF-α, IL-6, and HIF-1α were significantly lower,but the content of Gln was higher(P<0.05) in group P.5. The results of kidney HIF- 1 alpha m RNA expression: both K and P group of the mass were increased compared with group C(P < 0.05). And group P decreased(P < 0.05) compared with group K.ConclusionPreconditioning with PHC can obviously alleviate renal tissue inflammation in LPS induced acute kidney injury of neonatal rats, and improve its histopathlogy. The mechanisms of the protective effects might be that preconditioning with PHC may down-regulate the expression of HIF-1α in kidney tissue, alleviate inflammation;Besides, may also by protecting kidney glutamine metabolic pathways under acute kidney injury, reduce the consumption of glutamine. |
PDF Full Text Request