Renal Expression Of The Ammonia Transporters, Rhbg And Rhcg, In Response To Low Protein Diet

Background Acid-base homeostasis depends on renal ammonia metabolism.The generation of new bicarbonate is necessary to replace the alkali consumed in the buffering of endogenous and exogenous acids.Under normal conditions 60-70%of the new bicarbonate formed by the kidney is due to renal ammonia metabolism. Recent studies have yielded important new insights into the mechanisms of renal ammonia transport.In particular,the theory that ammonia transport occurs almost exclusively through nonionic NH₃ diffusion has been replaced by the observation that a variety of proteins specifically transport NH₃ and NH₄⁺ and that this transport is critical for normal ammonia metabolism.Renal ammonia metabolism and transport involve integrated responses of multiple portions of the kidney,including specific transport mechanisms in the proximal tubule, thick ascending limb of the loop of Henle,and the collecting duct.Proximal tubule segments metabolize the amino acid,glutamine,producing equal amounts of NH₄⁺ and bicarbonate molecules.The proximal tubule secretes NH₄⁺ into the luminal fluid primarily through the action of the apical sodium/hydrogen ion exchanger,NHE-3.In the thick ascending limb of the loop of Henle,the apical Na⁺-K^-/2Cl^- cotransporter NKCC2 predominantly reabsorbs the majority of luminal ammonia into the renal interstitium that result in renal interstitial ammonia accumulation.And then,the collecting duct secrets ammonia of the renal interstitium into the luminal fluid. Approximately,70-80%of total urinary ammonia is secreted by the collecting duct, indicating the important role of understanding the metabolism of collecting duct ammonia transport.An important evidence suggests ammonium transporter(Amt)/ mythylamine permease(MEP)/human Rhesus(Rh) glycoprotein protein family functions as vital role in ammonia metabolism and transport.Plant,bacterial Amt and MEP are subject to the nitrogen control and the expression is increased under poor nitrogen supply. Furthermore,MEP may function both to ammonia transport and to component of nitrogen sensor.In human,the Rh family of protein,both erythroid(Rhag) and nonerythroid(Rhbg and Rhcg),also show conservation with the Amt and MEP family throughout their consequence.We hypothesis that it might have the similar regulation in the condition of low protein diet in animal;and then to illuminate the Rh glycoprotein's role in the low protein diet;to explore the mechanism of ammonia regulation in the kidney;to deepen the recognition to the regulation of Acid-base of kidney.Objective To explore the effect of the ammonia transporters,Rhbg and Rhcg,in response to low protein diet.Methods1.To explore the BUN,Creatinine,K⁺,Na⁺,Cl^- of the serum between low protein diet and control;to explore the ammonia,Creatinine,K⁺,Na⁺,Cl^- of the urine between low protein diet and control.2.Detect the protein expression of Rhbg and Rhcg in cortex,outer medullar,inner medulla between low protein diet and normal control by Western blot.3.Detect the mRNA expression of Rhbg and Rhcg in cortex,outer medullar,inner medulla between low protein diet and control by real-time PCR4.Using single labeling,double labling and quantitative immunohistochemistry to detect the expression of Rhbg and Rhcg in principal cells and intercalated cells in CCD,OMCD,IMCD.Results1.Physiological'date.Serum:Rats fed with low protein diet showed significantly lower in BUN(19.7±3.9 VS 13.3±1.5 mg/dl) and CO2(25.2±1.3 VS 22.9±2.2 mmol/L) values,but not difference in Creatinine,K⁺,Na⁺,Cl^-.Urine:Rats fed with low protein diet showed significantly lower in Na⁺ (192.83±2408.97 VS 45±1319.6),FE_Na+(0.80±0.06 VS 0.18±0.03) and had no difference in urea,creatinine,K⁺,Na⁺,Cl^-,FE_K+,FE_Cl-,and FE_Urea. 2.Rhbg protein expression:Low protein diet significantly increased Rhbg in high molecular weigh(trimeric) expression and no change in outer medulla,inner medulla (P＜0.05,n = 6 in each group).3.Rhcg protein expression:No difference in Rhcg immunoreactivity was observed in proteins isolated from the cortex,outer medulla,or inner medulla.4.Rhbg mRNA quantification:We next quantified Rhbg mRNA expressions.No significant differences in Rhbg mRNA expression was observed in any of these regions.5.Rhcg mRNA quantification:We quantified Rhcg mRNA expressions.No significant differences in Rhcg mRNA expression was observed in any of these regions.6.Rhbg immunolocalization:In the cortex of the kidney from low protein diet rats, more intensity of immunolabel was observed than from control,but no difference was demonstrated between outer medulla and inner medulla.7.Rhcg immunolocalization.In kidneys from low protein diet rat,there were no detectable differences in Rhcg immunoreactivity in the cortex,outer medulla,or the inner medulla.8.Morphometric and quantitative immunohistochemistry analysis.The Rhbg immunoreactivity increased significantly between low protein diet and control both in the principal cells and intercalated cells of cortical collecting duct.Conclusion Low protein diet increases Rhbg expression in the cortex,especially in trimeric state and this increase is consistence with more distinct difference in basolateral Rhbg immunoreactivity especially in the principal cells and intercalated cells in CCD.It is possible that Rhcg-mediated ammonia transport is not regulated in response to low protein diet.