| PrefaceIn some mammalian species,such as human,rabbit,rat,mice,etc.,renal vasculature has been previously described in details.For example,in adult mouse kidney,the renal artery divides into several arteries before entering the renal hilum.These vessels,whose several divisions run along the corticomedullary boundary and is called the arcuate artery.From the arcuate arteries,the interlobular arteries branch more or less sharply,most divide several times as they extend towards the renal capsule.Afferent arterioles leading to the glomeruli arise from the smaller branches of interlobular arteries.Glomerulus is connected to the peritubular capillaries by the way of the efferent arterioles.The efferent arterioles arising from the superficial and midcortical glomeruli form the peritubular capillary networks distributing around the renal tubules,whereas the efferent arteriole of juxta-medullary glomeruli accompanied with Henle's loop descending deep into the medulla and give rise to vasa recta supplying the medulla.In the present study,the arcuate,interlobular arteries and afferent arterioles are referred as the vascular tree.In addition,the glomeruli and cortical peritubular capillary in the cortex,as well as vascular bundle and interbundle capillary networks in the medulla,constitute the renal microcirculation.The majority of renal blood flow is conducted directly to the glomerular capillary where the metabolic waste is excreted.When the ultrafiltrate flows through the renal tubules,most of the water and solutes in it is reabsorbed and returned back to the systemic circulation through the surrounding peritubular capillaries.Moreover,the vasa recta participate in the countercurrent exchange and play an important role in the control of blood pressure and the maintenance of a hypertonic gradient.Thus,the proper and timely assembly of the renal vasculature with the various parts of renal tubules in cortex and medulla is not only necessary for kidney's physiological function,but also is a crucial morphogenetic event formed through the precisely and timely renal development in the long term evolution.Like other organs,the formation of renal blood vessels includes two basic processes: vasculogenesis and angiogenesis.Vasculogenesis is endothelial cell precursors also called angioblasts differentiate in suit and assemble primary vascular plexus.Angiogenesis is the process that vascular endothelial cells form new blood vessels by budding,nesting and bridging on the basis of existing blood vessels.Both of these two events happen in a coordinated manner and consist of the efficient and complex renal vasculature network in the future.However,their contribution in the embryonic development of the renal vasculature still remains to be determined.Based on the interspecies grafting experiments,it has been proved that extra renal arteries are involved in the formation of renal blood vessels.Using specific antibodies Flk-1,a receptor for vascular endothelial growth factor,researchers found that endothelial progenitors capable of forming the glomerular and peritubular capillaries are already present in the metanephric mesenchyme before vessels can be identified.It has been further detected that except for the intrinsic progenitor cells within the renal stroma,there are some extra-renal progenitors migrate into the kidney through renal artery.Thus,the formation of renal artery and its branches is considered as angiogenesis,whereas the formation of glomerular and peritubualr capillaries is derived from vasculogensis.Based on gene knockout and transgenic animal model experiments,some genes and factors which participate in the other organogenesis,such as vascular endothelial growth factor,angiopoietin-1,renin-angiotensin system,etc.,are also involved in the regulation of renal blood vessels.Due to the presence of the cortex and medulla,the vascular pattern in these two distinct regions may be regulated by different signaling factors.The growth of peritubular capillaries in the cortex or medulla has been demonstrated to be regulated by the angiogenic factors released by the developing renal tubules and ureteric bud.In addition,the mesenchyme cells in the medulla may also be involved in the formation of vasa recta.Recent studies have found that ureteral bud epithelial cells participated in the formation of medullary vascular network through Wnt7 b signaling pathway.It is generally accepted that renal vascularization is synchronized with nephrogenesis.Nephrogenesis dynamically results from the reciprocal inductive interaction between the ureteric bud and metanephric mesenchyme.Metanephric mesenchyme induces the ureteric bud to continue to grow into the kidney,while ureteric bud induces metanephric mesenchyme condense around the tip and develop into vesicles,comma-shaped bodies,S-shaped bodies,and finally into mature nephrons.At E11.5,the branches of dorsal artery(equivalent to the adult renal artery)companied with the ureteric bud invading into the kidney,meanwhile the angioblasts appear to initiate vasculogenesis.Nephrogenesis is a precisely process that gene knockout experiments have shown that gene mutations or lack of key vascular angiogenic factors occur during the development particularly in the new born period,often result in arterial and arteriolar abnormalities,reduced glomeruli,followed by the deterioration of kidney structure and function.In 1980 s,Brenner et al.observed a link between the low number of human glomeruli(Nglom)and increasing risk of hypertension.Due to the close proximity of peritubular capillariesto tubules,the progression of chronic kidney diseases caused by hypertension,diabetes,and atherosclerosis is associated with the progressive vascular changes includes thickening of lamina intima,reduction of peritubular capillary,and nephrosclerosis with podocyte depletion.Thus,a systemic knowledge about the basic principles of renal vasculature morphogenesis is essential not only for understanding the aetiology of congenital and acquired kidney diseases,but also for finding new strategies for vascular target therapy to decelerate the progression of chronic kidney disease.The present study investigated mainly the morphological formation of renal vasculature with nephrogenesis based on serial histological sections and computer-assisted tubular tracing,furthermore the study estimated the volume densities of the glomeruli and peritubular capillaries in cortex and medulla,which hopefully to better understand the signal dependent morphogenic processes,and provides a reference for studying the renal microvasculature of developing,adult kidneys,as well as injured kidneys.Methods1.Renal tissue preparationPrenatal kidneys were obtained from E14.5,E16.5,E17.5,and E18.5 fetuses,while postnatal kidneys were obtained from P1,P3,P5,and P7 pups,as well as from P21,P28,P40,and 8-week-old mice.Five-?m-thick coronary sections and 2.5-?m-thick serial epoxy sections were obtained from the entire kidneys or tissue blocks using a microtome.2.Image recording and alignmentImages of serial sections from developing kidneys were obtained using a TDI linear scanning system.All images were aligned into image stacks by a series of custom-made computer programs.3.Digital tracing and 3-D representationThe spatial courses of the renal tubules and vasculature were traced with a series of custom-made computer programs running on a Linux-based PC.The tracing program generated automatically a data file containing x-,y-,and z-coordinates of the traced path.The spatial course of the path was visualized in 3D using the free Plot MTV visualization program.4.CD34 immunohistochemical staining for the vascular systemSections containing the cortex and medulla from each kidney were immunostained for CD34,a marker of vascular endothelium,for estimating volume densities of the glomeruli and capillary beds.5.The measurement of microvascular volume density in the developing kidneyThree to five sections randomly sampled from three to five kidneys at each developing day.The whole cortex was randomly sampled from serial sections.On each micrograph,volume densities in cortex and medulla were estimated using point counting with a stereological grid test system installed in Photoshop CS6.Results1.3D representation of blood vessels in the developing kidneysThe renal vasculature tree consisted of six main arteries running between the cortex and the medulla.Dozens of lateral branches sprouted from the main trunk and further divided in 1 to 2 times to form even smaller branches connecting to the earliest formed immature glomeruli.All components of the renal arterial tree extended beneath the nephrogenic zone whereas veins accompanied with them elongating within the region.With rapidly increasing number of nephrons,a dense branched network of renal vasculature was formed at E17.5 with tremendous lateral and bifid branching.As well as the renal papillae began to develop and elongate,the efferent arterioles of the earlier born glomeruli accompanying with Henle's ascending limb extended into the deep medulla and divided into descending vasa recta,which composed the further typical vascular bundle with ascending vasa recta.Soon after,renal vasculature tree at P5 showed a burst increasing both in number and in length to adapt the expansion of renal volume.In addition,interlobular arteries arising from the arcuate artery mainly as a main trunk divided into four times before extending beneath the renal capsule,which was same as the adult kidney.Thus,the basic renal vascular network within the cortex fully formed until P5.After that,with the expansion of especially the medullary volume,the renal vasculature remodeled into the typical spatial arrangement,including main,arcuate,and interlobular arteries,as well as afferent and efferent arteriolesAt E14.5,the diameter of arcuate,interlobular artery and afferent arteriole was 20 to 30?m,10 to 20?m,and 5 to 10?m,respectively.At E17.5,the diameter of arcuate,interlobular artery and afferent arteriole was 20 to 45?m,15 to 30?m,and10?m,respectively.At P5,the diameter of arcuate,interlobular artery and afferent arteriole was 40 to 65?m,15 to 45?m,and 10 to 15?m,respectively.2.General morphology of microvasculature in developing kidneysAssisted with immunostaining for CD34,a marker of endothelial cells,the morphology formation of renal vasculature was observed.Early at E14.5,many tiny cross sections of CD34 positive vessels scattered between the stromal or mesenchymal cells in the nephrogenic zone,while others closely associated with the developing renal tubules.The vascularized glomeruli were observed to be simple loop connecting to afferent and efferent arteriole,at the lower cleft of S-shaped nephrons,whereas the renal vesicle and comma-shaped nephrons were negative for CD34.In addition,the ladder-like CD34 negative mesenchyme was arranged perpendicular to the developing tubules in the medulla.Noticeably,the efferent arterioles arising from early(juxtamedullary)glomeruli consistently ran in close contact with their own thick ascending limbs.No loop bends between the descending and ascending vasa recta were found.During the kidney development,the thickness of the nephrogenic zone gradually decreased with time and vanished completely after P5.As more and more tubular loops extended with a convoluted course in the cortical labyrinth and a straight course from the medullary rays into the medulla,the mesenchymal cells became less and less visible,meanwhile the distribution of the cortical and medullary microvasculature increased rapidly.The typical vascular bundles and interbundle regions in the inner stripe of outer medulla did not appear until P7.3.Estimations of volume densities of glomeruli and peritubular capillairesThe volume density of glomeruli varied slightly through the kidney development,4.61 ± 0.47% at E14.5 to 6.07 ± 0.2% at P7 to 4.19 ± 0.47% at P40.The volume density of cortical and medullary peritubular capillary increased 3.3-and 2.6-fold respectively,from(2.34 ± 0.13% and 7.03 ± 0.09%)at E14.5 to(7.71 ± 0.44% and 18.27 ± 1.17%)at P40.The increase in volume density of medullary peritubular capillary of the entire medulla was paralleled by a growth of that in different medullary zones.Conclusion1.The formation of renal vascular pattern consisted of iterative bifid branching and lateral sprouting.These two angiogenic modes alternately and synchronously occurred in the morphogenesis of renal vasculature through the development.For example,during the development of renal vasculature,the main arteries were formed by bifid branching with the same diameter of vascular lumen.Whereas the main interlobular arteries sprouted from the arcuate artery through a smaller lateral branching and extended towards the cortex,as well as the afferent arteriole.In addition,lateral sprouting typically extend toward the renal capsule,it may be suggest that the growth and regulated signal pathway of renal blood vessels of the cortex may be different from that of the medulla.2.The development of renal vasculature is associated with nephrogenesis.There are no new interlobular arteries and afferent arterioles arise from the bigger arteries since P5.With the increasing number of juxta-medullary nephron,more and more vasa recta companied with the Henle's loop descending deep into the medulla,as well as the density of peritubular capillary surrounding the medullary tubules increased.These results reflect that the establishment of urine concentration mechanism related to the formation of medullary osmotic gradient which is mainly depending on the vascular-tubular relations after birth.3.The volume density of glomeruli changed slightly during the kidney development,suggesting that the establishment of renal filtration not only correspond to functional development of the tubules,but also adapt to the demands of metabolism. |
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