Three-dimensional Visualization Study Of The Relationship Between Tubules And Afferent Arterioles After Macula Densa In Kidney

Objective:Tubuloglomerular feedback is one of the mechanisms by which nephrons self-regulate renal blood flow and glomerular filtration rate.The macula densa senses the NaCl concentration of the end of the thick ascending loop,which located at the the glomerular pole.Transport of NaCl increased ATP consumption and adenosine production,inhibited renin secretion and promoted contraction of afferent arteriole,thereby regulating renal blood flow and glomerular filtration rate to normal levels.The thick ascending loop travels at different distances after macula densa,and transforms into distal convoluted tubule,and then transforms into connecting tubule and connects to collecting duct.Recent studies have shown that there is another physical contact between tubules and afferent arterioles after macula densa.In vitro perfusion tests of separated connecting tubules and adjacent afferent arterioles show that the increased concentration of Na⁺in tubular fluid dilated the afferent arteriole,which was defined as Crosstalk.Epithelium sodium channel（ENaC）plays a major role in Crosstalk that aldosterone promotes it.Pendrin,Cl^-/HCO₃^-exchanger,locates on the apical membrane of type B and non-A-non-B type intercalated cells,promotes NaCl reabsorption with ENaC by aldosterone.Due to the lack of direct morphological evidence,the mechanism of Crosstalk is still under debate.Many questions need to be solved urgently,such as the type of nephron exists in Crosstalk,the contact rules of tubules and afferent arteriole of different nephron types,whether Pendrin of intercalated cells participates in Crosstalk,and so on.Exploring the adjacent relationship between tubules and afferent arteriole will provide important morphological information for further study of the mechanism of Crosstalk.Methods:Three C57/BL/6J mice were fixed by perfusion,and embedded in Epon 812.Tissue blocks were cut perpendicular to the longitudinal axis of the kidney.And a total of2000,2.5-μm-thick consecutive sections were obtained from renal capsule to the papillary tip.Toluidine blue staining was performed to take photos and images.After image recording and alignment,137 nephrons and their afferent arterioles were traced by the computer assisted digital tracing and three-dimensional visualization programs.On the basis of three-dimensional reconstruction,the target sections with tubule-afferent arteriole contact after macula densa were selected,and semi-thin sections of 0.5-?m-thick were prepared.The modified immunoperoxidase staining method was used to detect the functional protein called Pendrin on intercalated cells.Three-?m-thick paraffin sections were selected for Pendrin staining and photographed by Nikon 90i for further analysis.The tubules were in contact with the afferent arterioles and Pendrin staining at the contact site were found on the slice.Results:1.The distal convoluted tubules commenced downstream from the macula densa at an average length of 40¹80μm.The cell of distal convoluted tubules turned abruptly from simple cuboidal to high columnar,and the cell nuclei located near the apical membrane of the cell.The distal convoluted tubules went surround their parent glomerulus locating at cortical labyrinth.Virtually,the distal convoluted tubule rarely intermingled with the proximal tubule and occupied its own separate territory in the cortex.As the depth of the cortex increases,the number of convolutions of the distal convoluted tubule increases.Usually,the distal convoluted tubules of superficial nephron returned and made coils on the way to touch the capsule just for one time.The length of distal convoluted tubule is 500⁹00μm.2.Each collecting duct received connecting tubules from 5⁷nephrons.The connecting tubules from superficial nephron joined collecting ducts directly,while the connecting tubules from middle cortical and juxtamedullary nephron joined each other to form an arcade,and the latter drained into collecting ducts at superficial cortex.The connecting tubule was often accompanied by interlobular veins and was in intimate contact with the afferent arteriole of their own glomeruli.The length of the connecting tubules of the superficial nephron and the mid-cortical nephron was 150⁶50μm,and the length of the connecting tubule derived from the juxtamedullary nephron is 400⁹00μm.3.The afferent arteriole was branched by the interlobular artery in the cortical labyrinth,extended to the glomeruli with the length of 15²80μm.The afferent arteriole in superficial cortex that originated from the interlobular artery ran towards the capsule,but those in cortico-medullary junction ran in the opposite direction.Those located in the middle cortex also ran in horizontal direction besides above two directions.4.The contact after the macula densa founded between the distal convoluted tubules and（or）the connecting tubules and the afferent arterioles were diversified.Crosstalk existed in most superficial and middle cortical nephrons that have such contact,but there is no such contact existed in the juxtamedullary nephrons that ran close to the cortico-medullary junction.5.Crosstalk occurred in the distal convoluted tubules and（or）the connecting tubules.The morphological basis was the non-A-non-B type of intercalated cells of the tubule,and the contact site in which was Pendrin-positive cell.Conclusion:The course of the renal tubules after macula densa and afferent arterioles determines that the Crosstalk mainly exists in the superficial and mid-cortical nephrons.Pendrin-positive non-A-non-B intercalated cells at the end of distal convoluted tubules and/or connecting tubules form close contact with the afferent arterioles,providing structural basis for Crosstalk.The morphological intersection of tubules after macula densa with the afferent arterioles may be another important regulatory site of the renin-angiotensin-aldosterone system.