猪肾小球内皮细胞与系膜细胞的发育过程及相互关系

目的:明确中国实验用小型猪肾小球内皮细胞与系膜细胞的发育过程及相互关系。方法:采集不同时间点(胚胎28～112d及出生后1d、7d、14d、21d)中国实验用小型猪肾组织,应用免疫荧光技术检测胚肾发育不同阶段(帽状间充质、肾小囊体、逗号形体、"S"形体、毛细血管袢期肾小球及成熟肾小球)内皮细胞标志物CD31与系膜细胞标志物α-SMA的表达情况。结果:内皮细胞标志物CD31在胚肾早期呈散在性分布,继而围绕肾小囊体和逗号形体呈"环抱"状分布,然后进入"S"形体下端的血管裂隙内聚集形成无管腔的"前毛细血管束",最后表达于成熟肾小球毛细血管内皮细胞。系膜细胞标志物α-SMA在早期胚肾、肾小囊体和逗号形体阶段均无表达;"S"形体早期分布于"S"形体周围,后期进入"S"形体下端的血管裂隙;毛细血管袢期聚集在肾小球血管极根部;随着肾小球发育逐渐向肾小球内延伸,最终表达于成熟肾小球系膜区。CD31与α-SMA双重染色的结果显示,在毛细血管袢期CD31阳性的内皮细胞聚集形成无管腔的前毛细血管束,而α-SMA阳性的系膜细胞聚集在肾小球血管极的根部;随着肾小球发育,α-SMA阳性的系膜细胞逐渐由血管极根部向肾小球内迁移,同时CD31阳性的内皮细胞逐渐形成带有管腔的毛细血管丛。结论:中国实验用小型猪肾小球内皮细胞的发育开始于后肾间充质阶段,系膜细胞的发育开始于"S"形体阶段,即肾小球系膜细胞发育在内皮细胞之后;在肾小球血管丛形成过程中,内皮细胞与系膜细胞间可能存在重要的相互作用。     

Reconstruction of glomerular tufts--observation of mesangiolysis induced by anti-thymocyte serum]

The reconstruction process of the glomerular capillary structure following mesangiolysis induced in rats by anti-thymocyte serum (ATS) was investigated for three months. Cystic ballooning change of the glomerular tufts was remarkable four days after the administration of ATS, which was followed by inflammatory cell infiltration, and hypercellularity in mesangial area was observed one week later. Glomerular tufts were gradually reconstructed thereafter, leaving focal and segmental sclerotic or adhesive lesions. In the periphery of the ballooning area, a large vascular lumen was subdivided into smaller capillary lumina by endothelial and mesangial bridging, which resembled to the process in the fetal glomerulogenesis. The endothelial cells in mesangiolysis connected one another by junctional complexes and formed new capillary structure. The mesangial cells got in touch with the endothelial cells by mesangial bridging. In the proliferative lesion, endothelium-like cells could be recognized by immunostaining and had fenestrae with diaphragmatoid structure without mature basement membrane in the initial stage on electron microscopic observation. Interstitial-typed collagen fibers were found in the proliferative lesion, and they still existed in the focal segmental sclerotic lesion in the late stage of the experiment. The glomerular basement membrane (GBM) was seen to be reformed by fusing the original GBM with the newly synthesized one covering subendothelial space, where mesangial cells were interposed. Endothelial cells, endothelial-mesangial interaction and extracellular matrix formation seemed to play an important role in reconstruction of the glomerular tufts after mesangiolysis induced by ATS.     

Development of the glomerular mesangium

The development of the glomerular mesangium was studied in fetal and newborn rat kidneys by using a widefield electron microscope which can cover a whole glomerulus within one low-power viewfield. A three-dimensional observation of the immature glomeruli was done by performing ultrathin serial sectionings of the specimen for electron microscopy. Scanning electron microscopic observation of the developing glomeruli was also performed. The developmental distribution of contractile protein (actin) in mesangial cells and the main intrinsic component of the extracellular matrix protein (type IV collagen) of the mesangium were examined by immunohistological techniques. The widefield electron micrograph revealed a precise relationship between the mesangium and other components of the glomerulus. The results confirmed that the capillary extends into the S-shaped body from the sur-rounding vascular system at the initiation of nephronogenesis. The mesangial cells are always continuous to the vascular pericyte-smooth muscle cell system during the whole course of glomerular development and they participate in the sub-division of the capillary network during glomeru-logenesis. Morphological findings and the changing distribution of intra- and extracellular proteins of the mesangium during development suggest that the mesangial cell differentiates from the primitive pericyte of the immature capillary.     

Abnormal development of glomerular endothelial and mesangial cells in mice with targeted disruption of the lama3 gene

Mice with targeted disruption of the lama3 gene, which encodes the alpha3 chain of laminin-5 (alpha3beta3gamma2, 332), develop a blistering skin disease similar to junctional epidermolysis bullosa in humans. These animals also develop abnormalities in glomerulogenesis. In both wild-type and mutant animals (lama3(-/-)), podocytes secrete glomerular basement membrane and develop foot processes. Endothelial cells migrate into this scaffolding and secrete a layer of basement membrane that fuses with the one formed by the podocyte. In lama3(-/-) animals, glomerular maturation arrests at this stage. Endothelial cells do not attenuate, develop fenestrae, or form typical lumens, and mesangial cells (MCs) were not identified. LN alpha3 subunit (LAMA3) protein was identified in the basement membrane adjacent to glomerular endothelial cells (GEnCs) in normal rats and mice. In developing rat glomeruli, the LAMA3 subunit was first detectable in the early capillary loop stage, which corresponds to the stage at which maturation arrest was observed in the mutant mice. Lama3 mRNA and protein were identified in isolated rat and mouse glomeruli and cultured rat GEnCs, but not MC. These data document expression of LAMA3 in glomeruli and support a critical role for it in GEnC differentiation. Furthermore, LAMA3 chain expression and/or another product of endothelial cells are required for MC migration into the developing glomerulus.     

Endothelial cell apoptosis during glomerular capillary lumen formation in vivo

Transforming growth factor-beta (TGF-beta) stimulates endothelial cell apoptosis in vitro, and inhibition of TGF-beta1 leads to retention of undifferentiated endothelial cells in developing glomerular capillaries and reduced lumen formation in vivo. This study explored the question whether glomerular capillary lumen formation in vivo may involve TGF-beta1-dependent endothelial cell apoptosis. Neutralizing anti-TGF-beta1 or non-immune IgY were infused into the renal arteries of 3-d-old rats, and the kidneys were examined 2 d later. By transmission electron microscopy, endocapillary apoptotic cells were observed at a frequency of 0.10/loop in immature glomeruli of 3-d-old rat pups. In 5-d-old rat pups given neutralizing TGF-beta1 antibody or control IgY, the frequency of endocapillary apoptotic cells was 0.03 and 0.09/loop, respectively (P < 0.001, chi(2)). Dual labeling with TUNEL and anti-von Willebrand factor (vWF) antibody showed that apoptotic cells in immature glomeruli of 5-d-old rat pups are endothelial cells. Quantitative analysis showed significantly fewer TUNEL/vWF-labeled cells in glomeruli after anti-TGF-beta1 antibody infusion than in controls. No endocapillary apoptotic cells were observed in any group in C-shaped or S-shaped bodies, and the TUNEL assay revealed no glomerular apoptotic cells in kidneys from mature rats. These findings suggest that superfluous endothelial cells are cleared from immature glomerular capillaries by apoptosis, a process regulated by TGF-beta1. Taken together with the previous finding, that TGF-beta1 blockade blunts glomerular capillary lumen formation in vivo, it is proposed that TGF-beta1-dependent apoptosis serves to open capillary lumens in this vascular bed during glomerular development.     

Neutralizing TGF-beta1 antibody infusion in neonatal rat delays in vivo glomerular capillary formation 1.

BACKGROUND: The interruption of transforming growth factor-beta (TGF-beta) signaling with dominant negative type II TGF-beta receptors in bovine glomerular endothelial cells abrogates capillary morphogenesis in vitro, and genetic defects in the TGF-beta1 signaling cascade in mice and humans result in abnormalities of blood vessel morphology. This study sought to determine whether TGF-beta1 participates in renal glomerular capillary development in vivo. METHODS: To inhibit TGF-beta1 action, neutralizing anti-TGF-beta1 IgG was infused intra-arterially into the suprarenal aorta of three-day-old rats, and the glomerular endothelial cell appearance was evaluated two days later by immunohistochemical detection of the endothelium-specific von Willebrand factor, in situ analysis of vascular endothelial growth factor receptor binding, and morphometric study of developing glomerular structures by transmission electron microscopy. RESULTS: The infusion of neutralizing the TGF-beta1 antibody markedly reduced the invasion of comma- and S-shaped bodies by endothelial cells, and inhibited organization of endothelial cells into capillaries in these structures. In addition, capillary lumen formation and endothelial cell fenestration in developing cortical, but not in deep, already mature glomeruli were inhibited by neutralizing TGF-beta1 antibody. Seven days after TGF-beta1 antibody infusion, glomeruli appeared normal, and no reduction in glomerular number was observed. CONCLUSIONS: These findings suggest that TGF-beta1 plays a critical role in the formation of glomerular capillaries during renal development in the rat, and that flattening and fenestration of glomerular capillaries require the action of TGF-beta1.     

Glomerular endothelial cells and podocytes jointly synthesize laminin-1 and -11 chains

BACKGROUND: The glomerular basement membrane (GBM) originates in development from fusion of subendothelial and subepithelial matrices. Subsequently, newly synthesized subepithelial matrix is added as glomerular capillary loops expand. During GBM assembly, the laminin-1 heterotrimer (alpha 1, beta 1, and gamma 1 chains), initially expressed in vascular clefts of comma- and S-shaped bodies, is eventually replaced by laminin-11 (alpha 5, beta 2, and gamma 1 chains), which persists into maturation. The cellular source(s) of these laminins is not known and prompted this study. METHODS: To determine which cells synthesize the various laminin chains, postfixation immunoelectron microscopy of developing mouse kidney was performed using monoclonal and polyclonal antibodies that specifically recognized laminin alpha 1, beta 1, alpha 5, or beta 2 chains. RESULTS: Intracellular labeling for laminin alpha 1, beta 1 (laminin-1), and alpha 5 and beta 2 (laminin-11) chains was observed in developing glomerular endothelial cells and podocytes of comma- and S-shaped nephric figures. Laminin-1 was also seen in unfused GBMs at this stage, whereas laminin-11 was only found intracellularly. In capillary loop stage GBMs, laminin alpha 1 chain was completely absent, whereas labeling for laminin alpha 5 was intense, indicating rapid substitution between alpha chains. In contrast, laminin beta 1 chain labeling remained strong both intracellularly and in GBMs of capillary loop stage glomeruli, and beta 2 was up-regulated as well. In maturing stage glomeruli, beta 1 labeling declined, and alpha 5 and beta 2 remained at high levels intracellularly in both endothelial cells and podocytes and in GBMs. CONCLUSIONS: Our results show that both endothelial cells and podocytes synthesize laminin-1 and -11 chains throughout glomerular development. The sustained and comparatively high level of laminin synthesis by endothelial cells was unexpected, suggesting that the endothelium may be an important source of GBM proteins in glomerular disease.     

Differential expression of the intermediate filament protein nestin during renal development and its localization in adult podocytes

Nestin, an intermediate filament protein, is widely used as stem cell marker. Nestin has been shown to interact with other cytoskeleton proteins, suggesting a role in regulating cellular cytoskeletal structure. These studies examined renal nestin localization and developmental expression in mice. In developing kidney, anti-nestin antibody revealed strong immunoreactivity in vascular cleft of the S-shaped body and vascular tuft of capillary loop-stage glomerulus. The nestin-positive structures also were labeled by endothelial cell markers FLK1 and CD31 in immature glomeruli. Nestin was not detected in epithelial cells of immature glomeruli. In contrast, in mature glomerular, nestin immunoreactivity was observed only outside laminin-positive glomerular basement membrane, and co-localized with nephrin, consistent with podocyte nestin expression. In adult kidney, podocytes were the only cells that exhibited persistent nestin expression. Nestin was not detected in ureteric bud and its derivatives throughout renal development. Cell lineage studies, using a nestin promoter-driven Cre mouse and a ROSA26 reporter mouse, showed a strong beta-galactosidase activity in intermediate mesoderm in an embryonic day 10 embryo and all of the structures except those that were derived from ureteric bud in embryonic kidney through adult kidney. These studies show that nestin is expressed in progenitors of glomerular endothelial cells and renal progenitors that are derived from metanephric mesenchyme. In the adult kidney, nestin expression is restricted to differentiated podocytes, suggesting that nestin could play an important role in maintaining the structural integrity of the podocytes.     

Impaired glomerular maturation and lack of VEGF165b in Denys-Drash syndrome

Individuals with Denys-Drash syndrome (DDS) develop diffuse mesangial sclerosis, ultimately leading to renal failure. The disease is caused by mutations that affect the zinc finger structure of the Wilms' tumor protein (WT1), but the mechanisms whereby these mutations result in glomerulosclerosis remain largely obscure. How WT1 regulates genes is likely to be complex, because it has multiple splice forms, binds both DNA and RNA, and associates with spliceosomes. Herein is described that in DDS podocytes, the ratio of both WT1 +KTS isoforms C to D differs considerably from that of normal child and adult control podocytes and more closely resembles fetal profiles. Aside from the delay in podocyte maturation, DDS glomeruli show swollen endothelial cells, reminiscent of endotheliosis, together with incompletely fused capillary basement membranes; a dramatic decrease in collagen alpha4(IV) and laminin beta2 chains; and the presence of immature or activated mesangial cells that express alpha-smooth muscle actin. Because appropriate vascular endothelial growth factor A (VEGF-A) expression is known to be essential for the development and maintenance of glomerular architecture and function, this article addresses the question of whether VEGF-A expression is deregulated in DDS. The data presented here show that DDS podocytes express high levels of the proangiogenic isoform VEGF165, but completely lack the inhibitory isoform VEGF165b. The VEGF165/VEGF165b ratio in DDS resembles that of fetal S-shaped bodies, rather than that of normal child or adult control subjects. The alteration in VEGF-A expression presented here may provide a mechanistic insight into the pathogenesis of DDS.     

Involvement of endothelial cell adhesion molecules in the development of anti-Thy-1 nephritis

To study an involvement of glomerular endothelial cells in the development of anti-Thy-1 nephritis, we examined the expression of endothelial cell adhesion molecules during the course of this model. Ribonuclease protection assay elucidated that expression of mRNA for intercellular adhesion molecule-1 (ICAM-1) was markedly enhanced in the glomeruli with a peak at 2 h (6.5-fold, p < 0.05) after the anti-Thy-1 antibody injection when mesangial cell lysis was recognized and IL-1beta mRNA expression was induced in the glomeruli. The glomerular ICAM-1 was predominantly localized in the endothelial cells and was intensely immunostained at day 1 in the glomerular endothelial cells. In contrast, platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial-cadherin mRNA expression increased gradually with a peak at day 6 (2.6-fold (p < 0.05) and 4.2-fold (p < 0.05), respectively) in the glomeruli with mesangial proliferative lesion. PECAM-1 was also immunolocalized in the glomerular endothelial cells and the immunoreactivity was greatly enhanced at day 6. Glomerular expression of vascular cell adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 (E-selectin) was unchanged at a low level during the course of anti-Thy-1 nephritis. Blocking of ICAM-1 by administration of anti-ICAM-1 antibody showed significant decrease in the number of polymorphonuclear leukocytes accumulating in the glomeruli by 45.7% (9.4 +/- 0.2 vs. 5.1 +/- 0.1 per glomerular cross section, p < 0.01) at 2 h. These results suggest a significant involvement of glomerular endothelial cells in the development and repair of anti-Thy-1 nephritis via direct or indirect intercellular interactions between mesangial cells and glomerular endothelial cells.     

Maturation of the developing renal glomerulus with respect to basement membrane proteoglycans

Maturation of extracellular matrices in relation to heparan sulfate proteoglycans was investigated during glomerular development. Antibodies directed against the core-protein (Mr = 18,000) of the basement membrane heparan sulfate-proteoglycan were utilized. The IgG fraction of the antibodies was conjugated with either fluorescein or rhodamine or 125I-iodine. The fluoresceinated and radioiodinated antibodies were given intravenously to three-day-old rats; their kidneys were obtained 6, 48, and 120 hours later and processed for immunohistochemical and tissue autoradiographic studies. At six hours (day 0), matrices of all the stages were labeled. The labeling was lowest in the vesicle stage and highest in the capillary stage. The autoradiographic grain-densities of vesicle, S-shaped, precapillary, and capillary stages were 10.62 +/- 0.54, 7.44 +/- 0.65, 11.37 +/- 1.03, and 18.35 +/- 1.97, respectively. After 48 hours (day 5), the grain-density for S-shaped body decreased from 7.44 +/- 0.65 to 4.38 +/- 0.61, while no appreciable change in the precapillary and capillary stages was observed. At this time interval, the fluorescein-labeled glomeruli were seen in the deeper cortex only. After 120 hours (day 8), the grain-density for the capillary stage decreased from 18.35 +/- 1.97 to 2.82 +/- 0.92. The double-labeling experiments included a second injection of rhodaminated antibody at 48 hours (day 5). In these experiments, newly developed glomeruli of varying stages were seen in the superficial cortex. With the use of double filters, the immature glomeruli in the superficial cortex and mature ones in the deeper cortex were visualized.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelial localization of receptor tyrosine phosphatase, ECRTP/DEP-1, in developing and mature renal vasculature.

Developmental assembly of the renal microvasculature requires spatially and temporally coordinated migration, assembly, differentiation, and maturation of endothelial cells in the context of adjacent epithelial and mesangial cells. In this study, endothelial expression and distribution of the receptor tyrosine phosphatase ECRTP/DEP-1 were evaluated during and after developmental assembly of the renal microvasculature. Monoclonal antibodies against ECRTP/DEP-1 ectodomain epitopes localize its expression to membrane surfaces of endothelial cells in glomerular, peritubular capillary, and arterial renal sites of mature human and murine kidney. During kidney development, ECRTP/DEP-1 immunostaining is evident on a subpopulation of metanephric mesenchymal cells and on putative progenitors of glomerular capillary endothelial cells early in their recruitment to developing glomeruli. ECRTP/DEP-1 is prominently displayed on luminal membrane surfaces with punctate accumulations at inter-endothelial contacts that overlap with vascular endothelial-cadherin staining. ECRTP/DEP-1 is recruited to inter-endothelial contacts in confluent cultured human renal and dermal microvascular endothelial cells, yet experimental dissociation of vascular endothelial-cadherin from endothelial junctional complexes fails to redistribute ECRTP/DEP-1. These findings indicate that ECRTP/DEP-1 is expressed in anticipation of glomerular capillary endothelial recruitment during development, and suggest that ECRTP/DEP-1 ectodomain interacts with endothelial surface ligands that are engaged by cell-cell contact.     

Developmental patterns of PDGF B-chain, PDGF-receptor, and alpha-actin expression in human glomerulogenesis.

Expression of PDGF B-chain and the PDGF receptor beta-subunit (PDGFR beta) is detected immunocytochemically during the development of glomeruli in human kidneys of 54 to 105 days gestational age. During the early stages (vesicular, comma-shape and S-shape) of glomerulogenesis, PDGF B-chain is localized to differentiating epithelium of the glomerular vesicle, while PDGFR beta is expressed in the undifferentiated metanephric blastema, vascular structures, and interstitial cells. During this stage PDGF may be acting as a paracrine growth factor and as a chemoattractant acting to recruit mesangial progenitor cells into the developing glomerulus. As the glomerular tuft forms, both PDGF B-chain and PDGFR beta can be detected in an arboreal pattern radiating from the hilus of the glomerular tuft. Immunocytochemical studies using markers specific to endothelium (Ulex europaeus I lectin, Factor VIII related antigen), and smooth muscle (alpha-smooth muscle actin), indicate that the PDGF B-chain and PDGFR beta are both expressed primarily by mesangial cells. During this stage, PDGF may be acting primarily to provide an autocrine factor to mediate further mesangial cell proliferation. Glomerular expression of alpha-smooth muscle actin is limited to later stages of glomerulogenesis; at these stages the pattern of expression is similar to that of PDGF-B chain and PDGFR beta. The upregulation of mesangial PDGF, PDGFR beta, and alpha-smooth muscle actin expression that has been identified in some disease states in both humans and experimental animals appears to represent a recapitulation of this normal developmental process.     

Hypoxia regulates PDGF-B interactions between glomerular capillary endothelial and mesangial cells

BACKGROUND: Platelet-derived growth factor (PDGF)-B regulates mesangial cell and vessel development during embryogenesis, and contributes to the pathogenesis of adult renal and vascular diseases. Endothelial cell PDGF-B exerts paracrine effects on mesangial cells, but its regulation is not well defined. We examined the impact of hypoxia on PDGF-B-mediated interactions between glomerular endothelial and mesangial cells, a condition of potential relevance in developing, and diseased adult, kidneys. METHODS: Glomerular endothelial or mesangial cells were subjected to hypoxia and responses compared to normoxic cells. Endothelial PDGF-B was studied by Northern and Western analysis. Mesangial proliferative responses to PDGF-B were assessed by (3)H-thymidine incorporation, and migration by a modified Boyden chamber assay. Hypoxia-induced changes in receptor specific binding capacity were studied by saturation binding assays. RESULTS: Hypoxia stimulated increases in endothelial PDGF-B mRNA and protein. In normoxic mesangial cells, PDGF-B stimulated dose-dependent proliferation, but the proliferative response of hypoxic cells was two to three times greater. Exogenous PDGF-B also caused prompter migration in hypoxic mesangial cells. Mesangial cells were treated with endothelial cell-conditioned medium. More cells migrated when hypoxic cells were stimulated with hypoxic conditioned medium, than when normoxic cells were stimulated with normoxic conditioned medium. Preincubating conditioned medium with PDGF-B neutralizing antibody greatly decreased the chemoattractant activity. Binding studies demonstrated increased specific binding capacity in hypoxic cells. CONCLUSION: Hypoxia enhances PDGF-B paracrine interactions between glomerular endothelial and mesangial cells. These hypoxia-regulated interactions may be important during glomerulogenesis in fetal life and during the pathogenesis of adult glomerular disease.     

Bone marrow is a reservoir of repopulating mesangial cells during glomerular remodeling.

The renal glomerulus, whose cellular components are developmentally derived from the mesenchyme, plays a pivotal role in filtratating plasma. Irretrievable changes of glomerular components are responsible for the initiation and progression of impaired renal function. Recently, it has been shown that functional stem cells exist in the bone marrow of adult bodies and that they can reconstitute damaged tissues of the mesenchymal origin. To examine whether the bone marrow provides stem cells to damaged glomeruli, transgenic rats carrying enhanced green fluorescence protein (EGFP rat) were established in a systemic and constitutive manner. After transplanting the bone marrow of EGFP rats into wild-type rats, the progeny of the transplanted marrow cells were tracked with a tag of EGFP. Recruitment of bone marrow-derived cells into glomeruli was dramatically facilitated in response to mesangiolysis evoked in anti-Thy1 antibody-mediated glomerulonephritis. In the restored glomeruli, 11% to 12% of glomerular cells were derived from the transplanted bone marrow. The number of bone marrow-derived CD45(+) cells transiently increased during the disease process, and CD45-negative cells constantly accounted for more than half of the bone marrow-derived population in glomeruli. Bone marrow-derived Thy1(+) cells kept increasing in number until the remodeling ceased and finally made up 7% to 8% of glomerular cells. Laser scanning microscopy displayed that the bone marrow-derived Thy1(+) cells provide structural support for glomerular capillaries, which indicates that they are mesangial cells. Although CD45(-)Thy1(-) bone marrow-derived cells exist during the remodeling of glomeruli, none of them expressed endothelial markers such as Factor VIII and RECA1 as long as they were tested. The results indicate that the bone marrow can give rise to mesangial cells in vivo.     

Expression of transforming growth factor-β type II receptor mRNA in embryonic and adult rat kidney

Summary: The transforming growth factor-β (TGF-β) family of growth factors regulates cell proliferation, differentiation, extracellular matrix synthesis and angiogenesis in many developing tissues. Transforming growth factor-β1 was recently shown to affect the branching of ureteric epithelium and nephron formation in cultured rat metanephroi. As the TGF-β type II receptor is specific for the TGF-β family, the present study used in situ hybridization to localize mRNA for this receptor in metanephroi from Sprague-Dawley rat embryos. Transforming growth factor-β type II receptor mRNA was located in ureteric duct epithelium, undifferentiated mesenchymal cells in the nephrogenic zone, vesicles, comma-shaped bodies and S-shaped bodies. In some S-shaped bodies, TGF-β type II receptor mRNA was not expressed in the lower limb, which subsequently forms the renal corpuscle. Expression was not observed in capillary loop stage glomeruli and maturing glomeruli, or in proximal tubules and interstitial cells. In adult rat kidney, TGF-β type II receptor mRNA was expressed in cortical collecting ducts and distal tubules but not in glomeruli or proximal tubules. These findings demonstrate that the prominent expression of TGF-β type II receptor mRNA decreases as glomeruli and tubules develop. Expression then remains undetectable in adult glomeruli and proximal tubules. the developmentally-regulated expression of this receptor suggests a key role in glomerular and nephron development.     

Culture of endothelial cells from baboon and human glomeruli.

Whereas mesangial and epithelial cells from glomeruli are commonly grown in vitro, there has been a failure to isolate and propagate human glomerular capillary endothelial cells. This study defines the conditions for the reproducible isolation and growth of homogeneous monolayers of primate (baboon and human) glomerular capillary endothelial cells. Using selective media and growth factors, the following criteria were identified to optimize the isolation and proliferation of glomerular endothelial cells: (1) collagenase treatment of isolated glomeruli; (2) requirement for 20% serum, endothelial cell growth factor and heparin; (3) requirement of fibronectin as surface matrix; and (4) isolation from donors less than 60 years old, as premature senescence was directly proportional to the age of the human kidney donor. Under these conditions, primary cultures with an endothelial cell composition greater than 70% were reproducibly obtained. Homogeneous endothelial monolayers were developed from 20 of 23 human kidneys, and maintained for 5 to 10 passages, depending on the age of the kidney donor. Purification to homogeneity was achieved by patch cloning or by fluorescence-activated cell sorting. Glomerular capillary endothelial cells exhibited a cobblestone morphology at confluence, expressed factor VIII-related antigen, angiotensin converting enzyme activity, and endocytosed acetylated low-density lipoproteins. Electron microscopy revealed the presence of intracellular Weibel-Palade bodies and caveolae and microvillous projections on the luminal surface. Glomerular cells also stained positive for Ulex europaeus, a lectin characteristic of human endothelial cells. In addition, preliminary results indicate that human glomerular endothelial cells increase intracellular cyGMP in response to alpha-human 5 to 28 atrial natriuretic peptide and intracellular free calcium in response to thrombin.     

Intussusceptive capillary growth is required for glomerular repair in rat Thy-1.1 nephritis

BACKGROUND: Injection of anti-Thy-1.1 antibodies to rats causes mesangiolysis, with subsequent capillary loss. This dramatic event is followed by almost complete recovery of glomerular architecture. However, the precise cellular mechanisms of revascularization are not fully understood. METHODS: Glomerulonephritis was induced by the injection of monoclonal anti-Thy-1.1 antibody to rats. Structural changes in the glomerular vasculature, with special emphasis on the repair phase, were studied with corrosion casting technique, light microscopy (LM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). RESULTS: Specifically during the extensive revascularization period, numerous tiny holes of about 1.5 microm in diameter were found on the glomerular cast. The presence of transluminal tissue pillar was confirmed with LM and SEM. These findings indicate the involvement of intussusceptive capillary growth. TEM study demonstrated that some tissue pillars were composed of endothelial cells only and others had cytoplasmic process of mesangial cells in their cores sandwiched both sides by endothelial walls. CONCLUSION: Intussusceptive capillary growth, or nonsprouting angiogenesis is involved in the postinjury angiogenesis in the glomeruli, in which process mesangial cells as well as endothelial cells may play important roles.