NKCC1-mediated Renal Lymphatic Dysfunction In Puromycin Aminonucleoside Rats

BackgroundNephrotic syndrome(NS),with major clinical symptom of proteinuria,hypoalbuminemia,severe edema and hyperlipidemia,is one of the most common kidney diseases.Proteinuria is one of the risk factors for a variety of cardiovascular diseases,as well as one of the independent factors for the progression of chronic kidney disease(CKD).NS patients are often suffered from water and sodium retention.So far,there are two theories to explain this phenomenon:hypofilling theory and overfilling theory.Hypofilling theory,which has been generally accepted,challenged by many researchers in recent years.For example,the vascular volume of NS patients varies greatly among individuals,and hypoproteinemia does not necessarily cause edema.Therefore,the theory of hyperfilling is gradually being valued.However,the mechanism of primary retention of water and sodium in the kidney is still unclear.Under physiological conditions,the lymphatic vessels,renal tubules,and renal blood vessels work together to maintain the balance in the kidney.So,any changes in the function of any of them will cause insufficient kidney clearance.Previous studies have focused on renal tubules and renal blood vessels,and there are only few studies on renal lymph vessels.Na+-K+-2Cl-cotransporter 1(NKCC1),whose expression and function is regulated by sodium ion concentration,also exists on renal lymphatic vessels.Therefore,we speculated that elevated sodium ion concentration in the kidney affects the contraction and pumping function of lymphatic vessels by changing the expression or function of NKCC1 on the lymphatic vessels.This research aims to provide new directions for future treatment of patients with NS.ObjectiveTo find out the role of NKCC1 transporter on lymphatic vessels in nephrotic syndrome.Methods(1)Puromycin amino nucleoside was used to build nephrotic syndrome rat model(PAN),and magnetic resonance imaging(MRI)was used to detect sodium ion content in the kidneys of normal and PAN model rats.(2)Collected rat kidney lymph fluid under a microscope,and colorimetric method was used to measure sodium concentration in rat lymph fluid and serum.Normal rats were used as control group.(3)Separated the renal lymphatic vessels of normal rats and PAN rats under a microscope,and digital image capture system was used with the microvascular perfusion chamber to detect the contraction and pumping function of the lymphatic vessels under normal sodium and high sodium environment.(4)The renal lymphatics of PAN model rats and normal rats were extracted under a microscope.The expression of NKCC1 on the lymphatic vessels was detected by PCR and immunohistochemistry.(5)Lymphatic endothelial cells(LECs)were cultured with normal and highsodium medium to simulate the in vivo environment of normal rats and PAN rats.PCR and western blot were used separately to detect the expression and phosphorylation of NKCC1 in LECs.Results(1)Magnetic resonance technique showed that sodium retention existed in the kidney of PAN rats.The sodium concentration in the kidneys of PAN model rats were significantly higher than those of normal rats(PAN vs.normal:22.91±9.12 mmol/L mmol/L vs.84.10±10.71 mmol/L,P<0.05).(2)The colorimetric results showed that sodium concentration in the renal lymph of PAN rats was significantly increased compared with that of normal rats(P=0.011),while there was no significant difference existed between two groups if compared serum sodium concentration(P=0.078).(3)PCR results and immunohistochemistry results showed that the expression of NKCC1 in the lymphatic vessels of PAN rats was significantly higher than that of normal rats(P=0.011).(4)The PCR method showed that the expression of NKCC1 in the high-sodium culture medium was significantly higher than that in the normal medium(P=0.0001).Western blot showed that compared with the medium environment of normal sodium ion concentration,the function of NKCC1 in LEC was significantly reduced in a high sodium environment,and the decrease of NKCC1 function was independent of the increase of osmotic pressure(P=0.0099).The use of the NKCC1 inhibitor furosemide reduced the contraction amplitude and pumping fraction of lymphatic vessels.The downstream eNOS and functions of NKCC1 are inhibited by the high sodium environment.(5)Isolated rat lymphatic vessels and in vitro experiments showed:compared with baseline,normal rats’ lymphatic vessels had increased contraction frequency,decreased contraction amplitude and pumping fraction in a high-sodium environment.However,the contractility of lymphatic vessels in PAN rats did not change significantly from baseline in high sodium environment(P<0.05).ConclusionsThere exists sodium retention in the kidney of NS rats.Although the expression of NKCC1 on the renal lymphatics increases with sodium concentration,the function of this cotransporter decreases.Lower phosphorate NKCC1 affects the contraction function and pumping fraction of lymphatic vessels,which leads to aggravation of retention in the kidneys as well as progression of renal function.