TGF-β type II receptor in rat renal vascular development: Localization to juxtaglomerular cells

TGF-γ type II receptor in rat renal vascular development: Localization to juxtaglomerular cells. To further define the role of transforming growth factor-beta (TGF-γ) receptors in renal vascular development, detailed immunohistochemical studies of TGF-γ receptor expression were performed from gestational day 15 through adulthood. On gestational day 15, TGF-γ type II receptor immunoreactivity was restricted to perirenal stromal and vascular cells. On gestational day 17 TGF-γ type II receptor immunoreactive stromal cells were observed within the kidney, with the same distribution as stromal α-smooth muscle actin and renin immunoreactive cells, and intense stromal TGF-γ type II receptor immunoreactivity continued through postnatal day 5. As vascular development progressed, TGF-γ type II receptor, α-smooth muscle actin and renin immunoreactivity became progressively restricted to small renal arteries and arterioles. Expression of TGF-γ type II receptors and renin was very intense in afferent glomerular arterioles during postnatal days 5 to 15, and then became progressively restricted only to juxtaglomerular cells in the mature kidney. TGF-γ type I receptor (ALK-5, ALK-1 and ALK-2) immunoreactivity was not detected in stromal or vascular elements during development or in the mature kidney. Intense TGF-γ type II receptor expression in renal stromal vascular smooth muscle cell precursors and developing blood vessels suggests a role for the TGF-γ type II receptors in the formation of the renal vascular smooth muscle compartment. The continued intense expression in juxtaglomerular cells argues for a role in renin synthesis and/or release. The absence of ALK-5, ALK-1, and ALK-2 in developing vascular smooth muscle and mature juxtaglomerular cells indicates that the canonical view of TGF-γ signaling may not hold in these locations

Chronic in vitro shear stress stimulates endothelial cell retention on prosthetic vascular grafts and reduces subsequent in vivo neointimal thickness

AbstractObjective: The absence of endothelial cells at the luminal surface of a prosthetic vascular graft potentiates thrombosis and neointimal hyperplasia, which are common causes of graft failure in humans. This study tested the hypothesis that pretreatment with chronic in vitro shear stress enhances subsequent endothelial cell retention on vascular grafts implanted in vivo. Methods: Cultured endothelial cells derived from Fischer 344 rat aorta were seeded onto the luminal surface of 1.5-mm internal diameter polyurethane vascular grafts. The seeded grafts were treated for 3 days with 1 dyne/cm2 shear stress and then for an additional 3 days with 1 or 25 dyne/cm2 shear stress in vitro. The grafts then were implanted as aortic interposition grafts into syngeneic rats in vivo. Grafts that were similarly seeded with endothelial cells but not treated with shear stress and grafts that were not seeded with endothelial cells served as controls. The surgical hemostasis time was monitored. Endothelial cell identity, density, and graft patency rate were evaluated 24 hours after implantation. Endothelial cell identity in vivo was confirmed with cells transduced in vitro with β-galactosidase complementary DNA in a replication-deficient adenoviral vector. Histologic, scanning electron microscopic, and immunohistochemical analyses were performed 1 week and 3 months after implantation to establish cell identity and to measure neointimal thickness. Results: The pretreatment with 25 dyne/cm2—but not with 0 or 1 dyne/cm2—shear stress resulted in the retention of fully confluent endothelial cell monolayers on the grafts 24 hours after implantation in vivo. Retention of seeded endothelial cells was confirmed by the observation that β-galactosidase transduced cells were retained as a monolayer 24 hours after implantation in vivo. In the grafts with adherent endothelial cells that were pretreated with shear stress, immediate graft thrombosis was inhibited and surgical hemostasis time was significantly prolonged. Confluent intimal endothelial cell monolayers also were present 1 week and 3 months after implantation. However, 1 week after implantation, macrophage infiltration was observed beneath the luminal cell monolayer. Three months after the implantation in vivo, subendothelial neointimal cells that contained α–smooth muscle actin were present. The thickness of this neointima averaged 41 ± 12 μm and 60 ± 23 μm in endothelial cell–seeded grafts that were pretreated with 25 dyne/cm2 shear stress and 1 dyne/cm2 shear stress, respectively, and 158 ± 46 μm in grafts that were not seeded with endothelial cells. Conclusion: The effect of chronic shear stress on the enhancement of endothelial cell retention in vitro can be exploited to fully endothelialize synthetic vascular grafts, which reduces immediate in vivo graft thrombosis and subsequent neointimal thickness. (J Vasc Surg 1999;29:157-67.

Rat mesangial cell hypertrophy in response to transforming growth factor-β1

Rat mesangial cell hypertrophy in response to transforming growth factor-β1. Central features of progressive glomerular sclerosis are initial glomerular hypertrophy and subsequent accumulation of extracellular matrix proteins. Since TGF-β1 may play a key role in this glomerular response to injury, the present study sought to explore further TGF-β1 actions and regulated expression of its receptor in rat mesangial cells. The rat TGF-β type II receptor (TGF-βRII) homolog was cloned by screening a rat kidney cDNA library with a human TGF-βRII cDNA probe, and sequenced. Expression of this receptor subtype in rat mesangial cells was then demonstrated by RNase protection assay, and by Northern blot analysis of poly (A)+ RNA, TGF-βRII expression was down-regulated in cells treated with exogenous TGF-β1. Affinity cross linking studies demonstrated presence of this receptor on cell surface. Rat mesangial cells also expressed TGF-β1 and autoinduction by TGF-β1 was observed in the same cells, suggesting that this polypeptide may act in an autocrine fashion on mesangial cells, and that it may stimulate a positive autoamplification loop. TGF-β1 inhibited mesangial cell proliferation and stimulated significant overall protein and collagen production. Furthermore, mesangial cell size increased in response to chronic TGF-β1 treatment. These findings demonstrate that rat mesangial cells express key components of the TGF-β system and raise the intriguing possibility that in the glomerular mesangium, TGF-β1 may not only induce extracellular matrix synthesis, but may also participate in the process of glomerular hypertrophy in response to injury

A human glomerular SAGE transcriptome database

Background: To facilitate in the identification of gene products important in regulating renal glomerular structure and function, we have produced an annotated transcriptome database for normal human glomeruli using the SAGE approach. Description: The database contains 22,907 unique SAGE tag sequences, with a total tag count of 48,905. For each SAGE tag, the ratio of its frequency in glomeruli relative to that in 115 non-glomerular tissues or cells, a measure of transcript enrichment in glomeruli, was calculated. A total of 133 SAGE tags representing well-characterized transcripts were enriched 10-fold or more in glomeruli compared to other tissues. Comparison of data from this study with a previous human glomerular Sau3A-anchored SAGE library reveals that 47 of the highly enriched transcripts are common to both libraries. Among these are the SAGE tags representing many podocyte-predominant transcripts like WT-1, podocin and synaptopodin. Enrichment of podocyte transcript tags SAGE library indicates that other SAGE tags observed at much higher frequencies in this glomerular compared to non-glomerular SAGE libraries are likely to be glomerulus-predominant. A higher level of mRNA expression for 19 transcripts represented by glomerulus-enriched SAGE tags was verified by RT-PCR comparing glomeruli to lung, liver and spleen. Conclusions: The database can be retrieved from, or interrogated online at http://cgap.nci.nih.gov/SAGE. The annotated database is also provided as an additional file with gene identification for 9,022, and matches to the human genome or transcript homologs in other species for 1,433 tags. It should be a useful tool for in silico mining of glomerular gene expression

Expression of rat fibroblast growth factor receptor 1 as three splicing variants during kidney development

Fibroblast growth factors (FGF) are known to participate in the processes of embryogenesis and angiogenesis. This study was undertaken to examine the transcriptional and posttranscriptional regulation of the FGF receptor 1 (FGFR-1) subclass in the embryonic rat kidney. Two full-length FGF receptor cDNAs were cloned using low-stringency screening of a neonatal rat kidney library with a chicken FGFR-1 cDNA probe. Sequencing revealed these cloned cDNAs to be rat homologues of the FGFR-1 subtype, with the two clones representing splicing variants ?? and ?? of the FGFR-1. Evidence for renal expression of a third splicing variant (??) was obtained by use of the polymerase chain reaction. Splicing variants ?? and ?? of FGFR-1 are predicted to produce cell-surface FGF receptors with three and two immunoglobulin-like domains, respectively, whereas the ??-isoform may represent an intracellular form of the receptor. Although all three splicing variants were expressed in the developing kidney at days 14, 17, and 20 of gestation, at neonatal days 1 and 7 and in mature rats the ??-isoform was present in vastly larger abundance than ??- and ??-isoforms at all stages studied. Northern blot analysis revealed enhanced expression of FGFR-1 in the neonatal compared with the mature kidney. It is concluded that FGFR-1 is expressed in the kidney predominantly as the ??-isoform splicing variant and that expression of this receptor is enhanced during kidney development.open171