The Effect Of Renal Lymph Circulation Disorder On Renal Function And Structure In Rats

Background and objective:Recent surveys have revealed that the prevalence of chronic kidney disease (CKD) is surprisingly high in the general population. Most patients with CKD develop end-stage renal failure, which requires costly treatment and unaffordable renal replacement therapy. Therefore, to find out the cause of chronic renal damage and to develop the effective treatment seemed to be emergency. Renent year, the study of lymph ultrastructure in some organs and their circlulation disorders have been done, especially in brian, liver, and stomach intestine. However, there is very few about renal lymph ultrastructure and the effect of its disorder on renal function and structure. The knowledge on renal lymphatics is very limited and their importance is almost always neglected. The role of lymph in keeping the balance between reabsorption of fluid from the tubules and uptake of reabsorbed fluid by the peritubular capillaries is highlighted. Early papers demonstrated that acute ligation of hilar lymphatic ducts has a significant effect on renal function, such as changes of sodium excretion, urine volume. As long as the time goes by, it appears diffuse lesion in renal interstitium. Therefore, what is the relationship between renal lymph circlulation and renal structure, what is the exact mechanism to interfere with? There is nobody to study.In our pre-study, we demonstrated that rats developed significant and progressive proteinuria and elevating serum creatinine, when renal hilar lymph ducts was ligated. These kidneys were characterized by tubular cell detachment, tubular cell necrosis, tubular atrophy or dilatation and deposition of ECM. Thus,we suggest that renal lymph disorder induces renal fibrosis and renal cell apoptosis. As we know, tubulointerstitial fibrosis is critical determinants of renal function, and is the common final way of end stage of renal disease. The renal epithelial -mesenchymal transition(EMT) is one of the important mechanism of tubulointerstitial fibrosis. It is well known that TGF-β1 plays a significant role in the progression of EMT. Smad proteins have been identified recently as important components of the TGF-β₁ signaling pathway. Apoptosis is so called programmed cell death, and rencent study revealed that Bcl-2 family members are significant to cell apoptosis. The expression and regulation of Bcl-2 family proteins play a key role in apoptotic signal transduction pathway. Proapoptotic Bax and antiapoptotic Bcl-2 are the typical genes in Bcl-2 family, and furthermore, bax is the chief active regulating factor of bcl-2. In this study, we established the rat model of renal lymph circlulation disorder to show the time course of renal lesions caused by lymphatic ligation ; and to reveal the exact mechanism of renal lesion through detect the molecule involved in TGF-β₁/ SMAD and Bcl-2 /bax pathway.Methods:1. The study of the distribution and morphologic feature of the intrarenal lymphatics1.1 There are 20 female and male Wistar rats were involved in this study and nephridial tissue were obtained from each animal. Double immunofluorescence was applied to detect the lymphatic distribution by using Podoplanin and CD34. Using Leica fluorescence microscope , green light was detect under 543nm and red light under 633nm wavelength of light.1.2 Observe of renal lymphatic ultrastructure :renal tissues were obtained from the cortex, medulla and junctional zone , and put in 3% glutaric dialdehyde stationary liquid for transmission electron microscope to observe the fine distribution and morphologic feature of the intrarenal lymphatics.2. The study of rats renal function and structure2.1 Animals were divided into two groups: control group and model group (30 rats in each group) .Establish the rat model of renal lymph circlulation disorder by renal lymph ligation. Six rats in each group were sacrifice at 7,14,28,56d after operation. Urine aliquot and blood preparation were obtained. Proteinuria and serum creatinine value were examed by automatic analyser .Creatinine clearance (Ccr) formula : urine creatinine×urine volume per minute/serum creatinine, corrected by body weight.2.2 The renal tissue was examined by PAS and Masson staining after fixation, dehydration, paraffin imbedding. The assessment of tubulus injury index , TIFI and GSI was performed with the use of semiquantitative scores .2. 3 Obtained 1mm³ of renal cortex for fixation, embedding sectioning. The ultrastructure of renal tissue was detected by electron microscope.3. The study of renal interstitial fibrosis induced by renal lymph circulation disorderRats in each group were sacrifice at 7,14,28,56d after operation. Nephridial tissue were obtained for extracting protein , mRNA and paraffin section. Real-time PCR, immunostaining and western blot techniques were applied to detect the expression of COLI , TGF-β₁, Smad2/3, P-smad2/3 in renal tissue.4. The study of renal cell apoptosis induced by renal lymph circlulation disorderRats in each group were sacrifice at 7,14,28,56d after operation. Nephridial tissue were obtained for extracting protein , mRNA and paraffin section. Real-time PCR, immunostaining and western blot techniques were applied to detect the expression of Bax/Bcl-2, Caspase3 in renal tissue. Apoptotic nuclei were labeled using the terminal deoxynucleotidyltransferase dUTP nick-end-label (TUNEL) technique.Results:1. The study of the distribution and morphologic feature of the intrarenal lymphatics1.1 Under immunofluorescence double staining, blood capillary showed schillerization, and lymphatic capillary showed green color. lymphatic capillary appeared anomalism, large lumina , thin wall and located in connective tissue region. Cortilymph obviously exceed medulla lymph in rats.1.2 Under electron microscope, the basal lamina of lymphatic was discontinuity.A lot of lysosome and vesicles existed in the endothelial of capillary lymphatics. Many of the vesicles liberated in cytoplasm. The intercellular junctions of the renal lymphatics are held in close apposition.2. The study of rats renal function and structure2.1 Model group rats developed severe proteinuria and elevated serum creatinine and reduced creatinine clearance were observed.2.2 Histomorphological changes appeared in ligated kidneys, characterized by tubular damage, tubulointerstitial fibrosis, and expansion of the mesangium. The index of tubular damage and tubulointerstitial fibrosis were remarkably aggravated in model group.2.3 Under electron microscope, renal tubular epithelial cell andpodocyte showed edema; the number of vacuolus increased in intracytoplasm; mitochondrial crista disappeared; foot process got fusion; mesenterium got proliferation and insertion;ground substance became electron-dense3. The study of renal interstitial fibrosis induced by renal lymph circlulation disorder 3.1 Immunohistochemical staining indicated that Overexpression of transforming growth factor-β₁ (TGF-β₁) , Smad2/3, COL I genes and proteins were detected in model group rats. These proteins were mainly expressed in renal tubulointerstitium .3.2 Western blot showed protein expression of TGF-β₁ , Smad2/3 were increased in model group rats. The expression of p-Smad2/3 increased from week 1 and keep high level after then.3.3 Real-time PCR revealed that the expression of Col I, TGF-β₁, Smad2, Smad 3 mRNA were up-regulation from week 1, as 4.5,1.6,1.7,1.8 folds as control group.4. The study of renal cell apoptosis induced by renal lymph circlulation disorder4.1 TUNEL exam showed that many positive staining cells in model group rats. cell apoptosis mainly occured in dilate and emarcide renal tubule. Apoptotic index elevated from day 1 in model group , peaked at day 14.4.2 Immunohistochemical staining indicated that overexpression of Bax and caspase-3 in model group rats, mainly in renal tubulointerstitium. Oppositely, Bcl-2 was weakly.4.3 Western blot showed protein expression of Bax, caspase-3 increased in model group rats, peaked at day 14. The active unit of caspase-3 (17 KD) was also elevated in model group. Icreasing bax to bcl-2 ratio were also detected in model group rats.4. 4 Real-time PCR show that the expression of bax and caspase-3 were up-regulation in all time points. However, bcl-2 mRNA decreased in model group. Icreasing bax to bcl-2 ratio were also detected in model group rats. Conclusions:1. The intercellular junctions of the renal lymphatics are held in close apposition by adhesion devices and a lot of lysosome and vesicles existed in the endothelial of capillary lymphatics, which are the base for renal to transport big molecular and fluids.2. Renal lymph disorder took an obviorsly effect on renal function and structure, which induced severe chronic renal lesion and chronic renal failure. The lesion was aggravated by the time of the disorder..3. Renal lymph disorder induces tubulointerstitial fibrosis, and enhanced activation of the TGF-β₁/Smad singanling played a key role in the progress of the fibrosis and epithelial -mesenchymal transition .4. Renal lymph disorder enhanced renal cells apoptosis. Bax/Bcl-2 pathway mediates apoptosis in this desease.