The Functional Differentiation Of Nephron Distal Parts And Collecting Duct During Development Of Mouse

Objective: The nephron is the basic structural and functional unit of the kidney to filter the blood and eliminate the various metabolic waste products.Each nephron consists of the renal corpuscle and the renal tubule including the proximal tubule,the thin limbs and the distal tubule.The proximal tubules is responsible for the reabsorption most ultrafiltrate of renal corpuscle.Although the fine regulation of ultrafiltrate occurs in the distal tubule,which is crucial for the maintenance of body water volume and control acid-base balance.The realization of function depends on membrane transporters distributed distal tubule and collecting duct,which absorb sodium,potassium,calcium and water and secrete acid,base and bicarbonate.Structurally the distal convoluted tubule(DCT)connects to collecting duct(CD)by connecting tubule(CNT).Which continue the regulation and elimination of nephron on ultrafiltrate.The CNT is a transitional region between the DCT and CD.In addition to the CNT cells which are characteristic of this segment,there are intermingling cells from neighbouring segments,i.e,DCT cells,intercalated cells and principal cells.As a result,main functions of the CNT with respect not only DCT and CD but also CNT cells.There are specificity membrane transporters in CNTs: Natrium chlorinum cotransporter(NCC)is expressed DCT cells;aquaporin2(AQP2)is distributed in principal cells;H-ATPase is distributed in intercalated cells;calbindin,kallikrein and epithelial sodium channel(ENa C)are distributed in CNT cells.It follows that the CNT possesses numerous physiological function.So how take place of CNT?Kidney morphogenesis originates from a series of signal inductive interactions between the epithelial ureteric bud and metanephric mesenchyme.The former branches repeatedly,giving rise to the urinary collecting system and all of the CDs.Simultaneously,the latter is induced to undergo cell proliferation,migration,differentiation to become nephrons.The nephron proceeds through a series of renal vesicle,comma shape,S-shape,capillary loop and mature stage.The ureteric bud vesicle connected with nephron in the early development.The extension of connected part differentiated and developed the adult CNT.Recently,high flux genetic testing technology reveals that the brisk proliferation of distal cells and invasion into the epithelia of ureteric bud is the initial stage of two structures connected.However,how this occurs remains incompletely understood.Over recent years,cellular origin of connecting tubule(CNT),a segment between two embryonically different structures,collecting duct originating from ureteric bud(UB),and nephron derived from cap mesenchyme,has been genetically characterized,however,intuitional visualization of the cell activities at the initial connection of the two structures is limited.In this study,all our works adopt computer image processing and target architecture program trace techniques as well as three dimensional reconstruction of the nephron distal part including DCT and CNT.And on the foundation shows the precise location of membrane transproters in DCT,CNT and CD combing with immunohisto-chemisty technology.The aims are to explore the time and space of occurs and functional differentiation of the nephron distal part,and provide histocytology basis of research in etiology and therapeutics.Methods: The pup kidneys were preserved by in vivo perfusion fixation through the left ventricle of the heart.The kidneys were dehydrated with graded ethanol and cleared with xylene,and then embedded in paraffin wax.Subsequently,5-?m-thick sections were obtained from the different developing days.In addition,kidneys were post-fixed for 1 h in 1% Os O4,dehydrated in graded ethanol and acetone,and embedded in Epon 812.A total of 700 2.5-?m-thick epoxy sections were obtained from E17.5 kidneys for computer-assisted tubular tracing.Based on the tracing,sections containing emerging nephron-CD connections were re-embedded in Epon 812,and 50-nm-thick ultrathin sections were cut for ultrastructural analysis.Micrographs of paraffin sections were obtained with a light-field microscope equipped with a Nikon DS digital camera.Electron micrographs were obtained,at magnifications of ×3,000,×8,000,×15,000,and ×30,000 using electron microscopes.For each of serial epoxy sections from E17.5,four partly overlapping digital images were recorded and combined into one 24-bit color image using analy SIS.The digitized images were aligned using a custom-made computer program running under Linux,as previously described in detail.The spatial course of the renal tubules was traced with another custom-made computer program running on the Linux computer as previously described in detail Briefly,the aligned image stack was interactively viewed on a computer screen and the x-,y-,and z-coordinates along the courses of the renal tubules were demarcated with a computer mouse and recorded in a data file.Subsequently,this data was displayed in 3D to visualize the relationship between the nephrons and the UBs.At the same time,the target antigens were retrieved by high temperature and high pressure,soon after the expression of transproters was tested by immunohistochemical method.The positive tubule were traced with series of computer programs written in C on the linux-based PC.At last,immunohistochemistry technology combined with stereology analysis method were used to explore the expression of membrane transporters on renal sections of 12.5-,14.5-,16.5-,18.5-day-old fetuses and 1-,3-,5-,7-,14-,21-,28-and 40-day-old pups.Results: 1.The expression of membrane transporters in DCT and CD during development.Three membrane transporters appeared first at E14.5 d.NCC and AQP2 was mainly located on the free surface of cell.H+-ATPase was mainly located on the free surface or the cytoplasm or the basal surface of cell that depends on the types of cell(Type A intercalated cells have free surface H-ATPase.Type B intercalated cells have basal surface and cytoplasmic H+-ATPase.Non A-non B intercalated cells have free surface and or cytoplasmic H+-ATPase.)2.The expression of NCC in developing DCT.NCC-positive cells faintly appeared first in the developing DCT at E14.5 d.Now the cells of DCT show features of immature cells: Larger nucleus,cytoplasm is less.From 16.5 d of gestation,NCC-positive cells were observed not only the developing DCT but also mature DCT.With the development of nephron,NCC-positive DCT tends to mature DCT.Nucleus closes to luminal surface of cytoplasm,and basal longitudinal striations present below the nucleus.3.The expression of AQP2 in developing CD.AQP2-positive cells appeared in the CD connected with mature nephron at every time of development.In ureteric bud connected with early development nephron,there were no expression of membrane transporters,and very faint labeling of cells were observed.With the development of mudulla,AQP2-positive cells were present through-out the medullary CD.AQP2-positive cells distributed the whole lumen in inner medul-lary CD.AQP2-positive cells dispersed distributed in CD located in the subcapsule and medullary ray.4.H+-ATPase was mainly located in distal part of the DCT and CD.The distribution of H+-ATPase and AQP2 in the CD is complementary.Both alternated expressed in relatively mature CD.Therefore,H+-ATPase-positive cells were mainly present in cortex CD,and very faint labeling cells were observed in medullary CD.In addition,type A intercalated cells distributed in the distal part of DCT and CD;Type B intercalated cells distributed in cortex CD;Non A-non B intercalated cells distributed in the initial CD.5.In the CD,the Numerical Area Density(NA)of AQP2 and H-ATPase gradually increased during the development of the kidney.Howere,H+-ATPase mainly increased in cortex;NCC positive cells mainly increased in medulla.6.The development of CNT and the distribution of membrane transporters.The present study demonstrated on electron micrographs that,in mice,the initial connection was composed of a group of cells,arising from renal vesicle(RV),the youngest nephron,and plaque-like attaching at the basal epithelium of the terminal UB tip.The identification of the two structures was based on tubular tracing on serial 2.5-?m-thick epoxy sections.The cells at the initial connection was characterized by highly irregular shape with apical cytoplasmic processes,finger-like or edge ruffle or even lamellipodia;electron dense nuclei;abundant inter-and intra-cellular spaces,and extensive cell-cell focal contacts equipped with various cell membrane junctions and occasional discrete fusion of opposing membranes in addition to numerous mitochondria,densely packed rosette-like polyribosome,widespread branching r ER and microtubules between the nucleus and cell membrane.Moreover,a terminal UB tip was traced to often connect to two nephrons at different developing stages.The tip and initial connection associated RV and S-shaped body were further revealed to not express NCC,H+-ATPase,and AQP2,the expression of which were however shown in immature CNT at capillary-loop nephron stage.Conclusion: 1.Three types of membrane transporters appeared first at E14.5 d.The morphologyical structure of membrane transporters-positive cells is development stages,which the ratio of nucleoplasm is big,which suggests the function of DCT and CD has been initially formed,but the structure has not yet mature.2.With the development of medulla,transporters of CD are segmental distribution.The intercalated cells are mainly located in cortical CD.However,the principal cells are mainly located in medullary CD.Which hints there are regional differences between two types of cells in regulation of water and acid base balance.3.The cell features at the tip and the initial connection reflect a high demand in protein and energy production and cell-cell communications for probably further cell division and invasion to tip,rather than for regulating the balance of water-salt,acid-bass of the body fluid.The membrane transporters appeared late in CNT.The membrane transporters positive CNT is short.Which point out the function of transporters in CNT is crucial with the development of CNT length increases.