Development Of Metanephric Cap Mesenchyme Cells In Pigs And The Effect On Renal Tubular Epithelial-Mesenchymal Transition

Objective: To explore the development of metanephric cap mesenchyme cells (MCMCs)and glomeruli in pigs and the effect on renal tubular epithelial-mesenchymal transition(EMT)Methods:1. The development of MCMCs and glomeruli in pigs: periodic acid-Schiffstaining (PAS) was used to identify the morphological changes of MCMCs andglomerular development at different points, and immunofluorescence staining wasapplied to examine the expression and morphological changes of markers forglomerular podocytes, endothelial cells and mesangial cells at different developmentalstages in Chinese experimental miniature pig.2. The effect of MCMCs on renal tubular EMT: The model of EMT in LLC-PK1cells (a porcine, proximal tubule cell line) in vitro was established by treating the cellswith10ng/ml transforming growth factor β1(TGF-β1) for24h,48h and72h. Primarypig MCMCs were co-cultured with LLC-PK1cells in Transwell system to investigatethe effect on TGF-β1-induced EMT.Results:1. The development of MCMCs and glomeruli in pigs:(1) The morphologicalchanges of glomerular development at different points: PAS staining revealed thatmetanephric development in pigs began at embryonic day28(E28d), when the uretericbud (UB) formed a T-shaped bifurcation that was surrounded by a population ofmetanephric mesenchyme cells, called the cap mesenchyme; a renal vesicle,comma-shaped body and S-shaped body were formed. Glomeruli were formed at E35d,including the immature glomeruli (glomeruli at the capillary loop stage), which werelocated below the nephrogenic zone, and the mature glomeruli (indicated byerythrocytes in the capillary lumens), which were located juxtamedullary. A pig wasborn at E112d-E114d. Nephrogenesis continued after birth because the nephrogeniczone and the developing cap mesenchyme, renal vesicle, comma-shaped body and S-shaped body were detected at postnatal days1,7and14(P1d, P7d and P14d).Glomerulogenesis was completed by P21d when the nephrogenic zone disappeared.(2)The development of MCMCs at different points: Immunofluorescence stainingindicated that Six2, a marker for MCMCs was expressed in the porcine MCMCsdiffusely, the level of which was highest at E28d. As gestation advanced, thenephrogenic zone length decreased, and the expression of Six2was reduced. Thenephrogenic zone disappeared at P21d when the expression of Six2was lost. Six2andWT1(a podocyte marker) both expressed in the MCMCs.(3) The expression andmorphological changes of markers for glomerular inherent cells at differentdevelopmental stages: immunofluorescence staining showed diffuse WT1expressionwas observed in metanephric cap mesenchyme cells and then, in succession, in the renalvesicle, the comma-shaped body, the tail of the comma-shaped body, the lower aspect ofthe S-shaped body and the glomerular podocytes. CD31, a marker for endothelial cells,was scattered throughout the fetal kidneys and then surrounded the developing renalvesicle and comma-shaped body. At the S-shaped stage, CD31-positive cells migratedinto the vascular cleft to form precapillary cords in the immature glomeruli and finallylocalized in the endothelium of mature glomeruli. The changes in α-SMA (a marker formesangial cells) staining were as follows: expression was not present in the early fetalkidney, renal vesicle or comma-shaped body, but expression appeared near theperiphery of the S-shaped body at the early stage of the S-shaped body, streamed intothe vascular cleft by the late S-shaped body stage, aggregated at the root of thedeveloping glomerulus, migrated to the periphery of the glomerular tuft, and finallylocalized to the mesenterium in the mature glomerulus.2. The inhibition of MCMCs on renal tubular EMT: Treatment with10ng/ml TGF-β1for24h,48h and72h could trigger EMT in LLC-PK1cells which acquired afibroblast-like shape, and the Length/breadth ratios were increased. The expression ofthe epithelial marker E-cadherin was decreased. The level of mesenchymal proteinsα-SMA and vimentin were increased. The effect was most apparent after induction for72h. MCMCs could repressTGF-β1-induced EMT, including the inhibition of the downregulation of E-cadherin and the upregulation of α-SMA and vimentin，which wereaccompanied by decreased expression of EMT signal molecules β-catenin and ZEB1.Conclusion: Metanephric development in pigs begins at E28d. Glomeruli are formed atE35d and complete by P14-21d. Kidney development in pigs follows a pattern similarto that in humans; the podocytes arise from metanephric cap mesenchyme cells whichare Six2-positive. Molecular cross-talk between cells in the glomerular compartmentsmay be important during the development of the tuft. Six2is required to maintain anephron progenitor population of MCMCs, and primary pig MCMCs can inhibitTGF-β1-induced EMT in LLC-PK1cells.