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. CONCLUSION: 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.

Effects of high glucose on cytokine-induced nerve growth factor (NGF) expression in rat renal mesangial cells.

Nerve growth factor (NGF) accumulates at sites of inflammation and modulates local immune reactions. To characterize the mechanisms of cytokine-induced NGF expression under physiological and pathophysiological conditions, we have used cultured glomerular mesangial cells, which play a key role in glomerular inflammatory diseases such as diabetic nephropathy. To study the effects of high glucose on cytokine-induced NGF expression, rat mesangial cells were treated with the cytokines interleukin-1beta and tumor necrosis factor alpha under normal (1.0 g/L) and high (4.5 g/L) glucose concentrations. In the presence of high glucose concentrations, the cytokines drastically potentiated NGF protein but not mRNA expression when compared to physiological glucose levels. The specific protein kinase C inhibitors Ro31-8220 and CGP41251 suppressed cytokine-induced NGF expression. Moreover, blocking the oxidative activation of the protein kinase C pathway by N-acetylcysteine inhibited glucose effects on NGF synthesis. Neutralizing antibodies against transforming growth factor-beta inhibited cytokine-induced NGF expression under normal glucose concentrations but not under high glucose conditions. Enhanced expression of NGF under high glucose conditions may contribute to kidney diseases such as diabetic nephropathy.

Differential regulation of osteopontin expression in the clipped and nonclipped kidney of two-kidney, one-clip hypertensive rats.

BACKGROUND: Osteopontin (Opn) is highly upregulated in many different animal models of renal disease, where it is suspected to participate in progression of the disease. In some models, angiotensin II (Ang II) seems to induce the elevated Opn production. Therefore, we examined the regulation of Opn in two-kidney, one-clip (2K1C) hypertensive rats, in which Ang II mediates the elevated blood pressure. METHODS: At days 7, 14, and 28, the clipped and nonclipped kidneys of hypertensive or sham-operated rats were analyzed for osteopontin protein, mRNA expression and mononuclear cell infiltration by imumunohistochemistry, in situ hybridization, and Northern blot analysis. Rats were treated with the Ang II type 1 receptor antagonist Valsartan starting 14 days after clipping. RESULTS: In sham-operated rats, Opn was mainly localized to cells of the thin ascending limbs of the outer medulla. No significant Opn staining was observed in cortical tubules. Focally defined tubular cortical Opn staining was observed in clipped and contralateral kidneys of hypertensive animals at days 14 and 28. Osteopontin protein expression correlated with the mRNA expression detected by in situ hybridization and Northern blot. Treatment with Valsartan reduced osteopontin staining by 51%, mRNA by 47%, and mononuclear cell number by 97% in nonclipped kidneys compared to untreated two-kidney, one-clip animals. In clipped kidneys, however, Opn protein and mRNA expression was not reduced, but a 240% increase in interstitial mononuclear cell number was observed. CONCLUSIONS: Osteopontin is involved in the induction of nephrosclerosis in renovascular hypertension, probably by a mechanism augmenting monocyte infiltration. Angiotensin II appears to be an important inducer of Opn in the nonclipped kidney. Ischemic conditions may regulate Opn expression in the clipped kidney.

CC-chemokine RANTES is increased in serum and urine in the early post-transplantation period of human renal allograft recipients.

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.

A SAGE-based comparison between glomerular and aortic endothelial cells.

Endothelial cells have many characteristics in common, but significant morphological and functional differences exist between endothelial cells from different anatomic sites. The specific glomerular endothelial (GEn) cell transcript repertoire is unknown. We sought to determine whether endothelial cells derived from bovine glomeruli display a distinct transcriptional profile compared with bovine aortic endothelium (BAE) under identical conditions. Serial analysis of gene expression (SAGE), which includes known and unknown transcripts, was used to make the comparison. The GEn and BAE SAGE libraries contain 36,844 and 26,452 total tag sequences, respectively. Among 6,524 unique tag sequences represented at least 2 times in the 2 libraries, 2,094 (32%) were matched to well-characterized bovine cDNA sequences (358 tags) or expressed sequence tags (EST). Identification of the human homolog was achieved for 1,035 of these tags. Forty-two tags were differentially expressed in GEn. For 25 of these, the bovine cDNA or EST, and for 17 the human homolog was identified. Among all transcripts with a known bovine and human tag, seven were expressed at levels more than 10-fold higher in cultured GEn cells compared with all other SAGE libraries. The transcript "DKFZp564B076" was localized by in situ hybridization to glomerular endothelium in vivo and was shown by real-time RT-PCR to be highly abundant in glomeruli compared with aortic intima. This work supports the concept that differences in the transcriptional profile of endothelial cells from distinct origins are observed under otherwise equivalent conditions. Furthermore, we have identified the first known transcript predominant in glomerular endothelium in vivo.

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.

Glomerular endothelial fenestrae in vivo are not formed from caveolae.

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.

TIMAP, a novel CAAX box protein regulated by TGF-beta1 and expressed in endothelial cells.

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.