Representational difference analysis of the glomerular endothelial cell response to transforming growth factor-beta1 (TGF-beta1) revealed a novel gene, TIMAP (TGF-beta-inhibited membrane-associated protein), which contains 10 exons and maps to human chromosome 20.q11.22. By Northern blot, TIMAP mRNA is highly expressed in all cultured endothelial and hematopoietic cells. The frequency of the TIMAP SAGE tag is much greater in endothelial cell SAGE databases than in nonendothelial cells. Immunofluorescence studies of rat tissues show that anti-TIMAP antibodies localize to vascular endothelium. TGF-beta1 represses TIMAP through a protein synthesis- and histone deacetylase-dependent process. The TIMAP protein contains five ankyrin repeats, a protein phosphatase-1 (PP1)-interacting domain, a COOH-terminal CAAX box, a domain arrangement similar to that of MYPT3, and a PP1 inhibitor. A green fluorescent protein-TIMAP fusion protein localized to the plasma membrane in a CAAX box-dependent fashion. Hence, TIMAP is a novel gene highly expressed in endothelial and hematopoietic cells and regulated by TGF-beta1. On the basis of its domain structure, TIMAP may serve a signaling function, potentially through interaction with PP1.

Abstract. Previous reports indicate that endothelial fenestrae in vitro can form by fusion of caveolae or caveolae-like vesicles. The principal aim of this study was to determine whether formation of glomerular endothelial cell fenestrae in vivo similarly involves caveolae and caveolin-1. Whereas caveolin-1 immunofluorescence was found around the circumference of human and mouse glomerular capillary loops, it co-localized only partially with the endothelium-specific lectin Ulex Europaeus I in human glomeruli, leaving portions of the endothelium devoid of caveolin-1. Immunogold electron microscopy, used to definitively localize caveolin-1 in glomeruli, showed that caveolin-1 was completely excluded from the fenestrated portion of the endothelium. Moreover, in caveolin-1-deficient mice, which cannot form caveolae, the ultrastructure of glomerular endothelial fenestrae appeared entirely normal. Interestingly, strong caveolin-1 immunogold labeling was observed in podocytes, where some caveolin-1 localized to filtration slits. Caveolin-1 co-immunoprecipitated with the podocyte slit diaphragm proteins nephrin and CD2AP, and dual immunofluorescence confirmed co-localization of caveolin-1 and nephrin. Nevertheless, in caveolin-1-deficient mice, podocyte ultrastructure appeared normal, and the podocyte proteins synaptopodin, nephrin, and podocin were expressed normally. In addition, blood urea nitrogen concentrations and urinary protein excretion in these mice were similar to those in wild-type mice. Thus, unlike caveolae formation, glomerular endothelial cell fenestrae formation in vivo does not require caveolin-1, ruling out the previous hypothesis that endothelial fenestrae represent fused caveolae, at least for glomerular endothelial cells. Localization of caveolin-1 to podocytes and their filtration slits is consistent with the view that the filtration slit plasma membrane represents a type of lipid raft microdomain.

Abstract. Transforming growth factor-␤ (TGF-␤) stimulates endothelial cell apoptosis in vitro, and inhibition of TGF-␤1 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-␤1-dependent endothelial cell apoptosis. Neutralizing anti-TGF-␤1 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-␤1 antibody or control IgY, the frequency of endocapillary apoptotic cells was 0.03 and 0.09/loop, respectively (P Ͻ 0.001, 2 ). Dual labeling with TUNEL and antivon 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-␤1 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-␤1. Taken together with the previous finding, that TGF-␤1 blockade blunts glomerular capillary lumen formation in vivo, it is proposed that TGF-␤1-dependent apoptosis serves to open capillary lumens in this vascular bed during glomerular development.

. A SAGEbased comparison between glomerular and aortic endothelial cells.

BackgroundTo 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.DescriptionThe 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.ConclusionThe 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.

Volume-sensitive and chemosensitive cardiopulmonary reflexes modulate volume homeostasis via renal sympathetic nerve activity (RSNA). Blunting of volume-sensitive cardiopulmonary reflexes is associated with volume retention, e.g., in hypertension, whereas the role of chemosensitive cardiopulmonary reflexes is largely unknown. To elucidate the possible role of chemosensitive cardiopulmonary reflexes in control of volume homeostasis, we investigated whether subthreshold stimulation of 5-HT3 receptors modulates the control of RSNA by volume-sensitive cardiopulmonary reflexes or the arterial baroreceptor reflex in rats. Phenyl biguanide (PBG) was infused intravenously to stimulate 5-HT3 receptors. Higher doses of PBG lowered RSNA, but a dose of 6 micrograms/min, given as a background infusion throughout the experiment, did not change arterial pressure, heart rate (HR), or RSNA. Ten minutes after beginning the 6 micrograms/min PBG infusion, a 15-min volume expansion (0.9% saline, 5 or 10% body weight) was started to stimulate volume-sensitive cardiopulmonary reflexes. In separate experiments, 5-min ramp infusions of methoxamine and nitroglycerin to stimulate the arterial baroreceptor reflex (evaluated by a 4-parameter logistic regression) were performed 15 min after beginning the PBG background infusion (6 micrograms/min). During PBG infusion, the RSNA responses to volume expansions were significantly impaired (5% body weight: PBG -6 +/- 6%, n = 7 vs. control -39 +/- 9%, n = 6, P < 0.001; 10% body weight: PBG -33 +/- 6%, n = 8 vs. control -52 +/- 5%, n = 7, P < 0.05). The 5-HT3 receptor antagonist odansetron (GR-38032F) abolished these effects of PBG. The maximum HR gain of the arterial baroreceptor reflex was impaired but the arterial baroreceptor control of RSNA was unaffected by PBG background infusion. We conclude that 5-HT3-serotonergic cardiopulmonary chemoreceptors blunt the RSNA decrease to volume loading. This mechanism may facilitate volume retention when cardiac serotonin is increased.

Background: The chemokine RANTES is a potent chemoattractant for T cells and monocytes that has been shown to enhance inflammation. The aim of our study was to investigate whether RANTES is upregulated within the early post-transplantation period that may influence short-time allograft function rate. Methods: Serum and urine samples from transplanted renal allograft recipients (n = 17) were obtained from specimens taken for diagnostic reasons. Four patients developed biopsy-proven rejection episodes within the first month. Time course of RANTES was studied within the first 12 days after renal transplantation using ELISA technique. Data were tested for significances between patients with rejection and without rejection, compared to healthy volunteers as controls, and correlated with clinical data. Results: In the control group RANTES concentration was 37.2 ± 2.7 ng/ml (serum) and 8.1 ± 1.3 pg/ml (urine), respectively. In transplanted recipients serum RANTES was significantly upregulated up to 132 ± 28 ng/ml on day 1 after transplantation and remained elevated within the first 12 days (n = 17). Time course of urine RANTES demonstrated elevated concentrations with 754 ± 115 pg/ml on day 1 followed by an continuous decrease to 22.3 ± 7 pg/ml on day 12 (n = 17). No significant differences could be detected between patients with rejection and without rejection episodes. Conclusions: In contrast to data of other urinary marker molecules (like IL-6), there are no significant differences between the rejection and non-rejection group. RANTES is therefore not suitable for early detection of rejection. Nevertheless, serum and urine RANTES concentrations were highly elevated in freshly transplanted renal allograft recipients reflecting an activated immune system.

The efficacy of an early and late treatment with the angiotensin converting enzyme inhibitor lisinopril or the angiotensin II receptor blocker ICI D8731 was investigated in uninephrectomized spontaneously hypertensive rats (SHR). Rats that underwent uninephrectomy (UNX) at six weeks of age were randomly assigned to receive no treatment, lisinopril shortly after UNX, lisinopril starting 16 weeks after UNX, ICI D8731 shortly after UNX, and ICI D8731 starting 16 weeks after UNX. Blood pressure was normalized with both treatments. After six months inulin clearance was not significant different, while proteinuria and prevalence of interstitial fibrosis were significantly reduced in all treatment groups. Immunohistochemical studies revealed an interstitial, periglomerular and perivascular increase of extracellular matrix proteins in all rats, but a markedly reduced expression of collagen I, IV and fibronectin after early and late treatment compared to untreated controls. We found a significant reduction of infiltrating macrophages and T-lymphocytes in all treated animals compared to untreated controls after 2, 4 and 6 months. Especially early treatment was associated with lower numbers of infiltrating cells. Both treatments reduced proliferation of tubular and interstitial cells. There were no striking differences with regard to nephroprotection between the ACE inhibitor and angiotensin II receptor blocker. These findings show that both treatments have beneficial effects on kidney structure and function. They suggest that both ACE inhibition and angiotensin II blockade decrease renal cell proliferation and suppress the infiltration of mononuclear cells that may trigger expression of extracellular matrix proteins and progressive nephrosclerosis.