Angiostatin, a proteolytic fragment of plasminogen, is a potent antagonist of angiogenesis and an inhibitor of endothelial cell migration and proliferation. To determine whether the mechanism by which angiostatin inhibits endothelial cell migration and͞or proliferation involves binding to cell surface plasminogen receptors, we isolated the binding proteins

Transforming growth factor beta (TGF-beta) stimulates renal cell fibrogenesis by a poorly understood mechanism. Previously, we suggested a synergy between TGF-beta1 activated extracellular signal-regulated kinase (ERK) and Smad signaling in collagen production by human glomerular mesangial cells. In a heterologous DNA binding transcription assay, biochemical or dominant-negative ERK blockade reduced TGF-beta1 induced Smad3 activity. Total serine phosphorylation of Smad2/3, but not phosphorylation of the C-terminal SS(P)XS(P) motif, was decreased by pretreatment with the MEK/ERK inhibitors, PD98059 (10 microM) or U0126 (25 microM). This effect was not seen in the mouse mammary epithelial NMuMG cell line, indicating that ERK-dependent activation of Smad2/3 occurs only in certain cell types. TGF-beta stimulated phosphorylation of an expressed Smad3A construct, with a mutated C-terminal SS(P)XS(P) motif, was reduced by a MEK/ERK inhibitor. In contrast, MEK/ERK inhibition did not affect phosphorylation of a Smad3 construct mutated at consensus phosphorylation sites in the linker region (Smad3EPSM). Constitutively active MEK (caMEK) induced alpha2(I) collagen promoter activity, an effect blocked by co-transfected Smad3EPSM, but not Smad3A. The effects of caMEK and TGF-beta1 on collagen promoter activity were additive. These results indicate that ERK-dependent R-Smad linker region phosphorylation enhances collagen I synthesis and imply positive cross talk between the ERK and Smad pathways in human mesangial cells.

These studies demonstrate that, in vitro and in vivo, estradiol enhances endothelial cell activities important in neovascularization and suggest a promoting influence of estrogens on angiogenesis.

Transforming growth factor-β (TGF-β) is closely associated with progressive renal fibrosis. Significant progress has been accomplished in determining the cellular signaling pathways that are activated by TGF-β. This knowledge is being applied to glomerular mesangial cell models of extracellular matrix (ECM) accumulation. A central component of TGF-β-stimulated mesangial cell fibrogenesis is the TGF-β family-specific Smad signal transduction pathway. However, while Smads play an important role in collagen accumulation, recent findings indicate that cross talk among a variety of pathways is necessary for maximal stimulation of collagen expression. Further investigation of these multiple interactions will provide insight into possible ways to interrupt cellular mechanisms of glomerular fibrogenesis.

Transforming growth factor (TGF)-␤ has been associated with renal glomerular matrix accumulation. We previously showed that Smad3 promotes COL1A2 gene activation by TGF-␤1 in human glomerular mesangial cells. Here, we report that the PI3K/Akt pathway also plays a role in TGF-␤1-increased collagen I expression. TGF-␤1 stimulates the activity of phosphoinositide-dependent kinase (PDK)-1, a downstream target of PI3K, starting at 1 min. Akt, a kinase downstream of PDK-1, is phosphorylated and concentrates in the membrane fraction within 5 min of TGF-␤1 treatment. The PI3K inhibitor LY294002 decreases TGF-␤1-stimulated ␣1(I) and ␣2(I) collagen mRNA expression. Similarly, LY294002 or an Akt dominant negative construct blocks TGF-␤1 induction of COL1A2 promoter activity. However, PI3K stimulation alone is not sufficient to increase collagen I expression, since neither a constitutively active p110 PI3K construct nor PDGF, which induces Akt phosphorylation, is able to stimulate COL1A2 promoter activity or mRNA expression, respectively. LY294002 inhibits stimulation of COL1A2 promoter activity by Smad3. In a Gal4-LUC assay system, blockade of the PI3K pathway significantly decreases TGF-␤1-induced transcriptional activity of Gal4-Smad3. Activity of SBE-LUC, a Smad3/4-responsive construct, is stimulated by over-expression of Smad3 or Smad3D, in which the three C-terminal serine phospho-acceptor residues are mutated. This induction is blocked by LY294002, suggesting that inhibition of the PI3K pathway decreases Smad3 transcriptional activity independently of C-terminal serine phosphorylation. However, TGF-␤1-induced total serine phosphorylation of Smad3 is decreased by LY294002, suggesting that Smad3 is phosphorylated by the PI3K pathway at serine residues other than the direct TGF-␤ receptor I target site. Thus, although the PI3K-PDK1-Akt pathway alone is insufficient to stimulate COL1A2 gene transcription, its activation by TGF-␤1 enhances Smad3 transcriptional activity leading to increased collagen I expression in human mesangial cells. This crosstalk between the Smad and PI3K pathways likely contributes to TGF-␤1 induction of glomerular scarring.

A spectrum of proteinuric glomerular diseases results from podocyte abnormalities. The understanding of these podocytopathies has greatly expanded in recent years, particularly with the discovery of more than a dozen genetic mutations that are associated with loss of podocyte functional integrity. It is apparent that classification of the podocytopathies on the basis of morphology alone is inadequate to capture fully the complexity of these disorders. Herein is proposed a taxonomy for the podocytopathies that classifies along two dimensions: Histopathology, including podocyte phenotype and glomerular morphology (minimal-change nephropathy, focal segmental glomerulosclerosis, diffuse mesangial sclerosis, and collapsing glomerulopathy), and etiology (idiopathic, genetic, and reactive forms). A more complete understanding of the similarities and differences among podocyte diseases will help the renal pathologist and the nephrologist communicate more effectively about the diagnosis; this in turn will help the nephrologist provide more accurate prognostic information and select the optimal therapy for these often problematic diseases. It is proposed that final diagnosis of the podocytopathies should result from close collaboration between renal pathologists and nephrologists and should whenever possible include three elements: Morphologic entity, etiologic form, and specific pathogenic mechanism or association.Clin J Am Soc Nephrol 2: 529-542, 2007. doi: 10.2215/CJN.04121206 T he spectrum of primary nephrotic syndrome includes a variety of causes, presentations, histopathologic findings, and outcomes. These disorders may appear at any age, may be exquisitely sensitive or highly resistant to therapy, and have varied implications for long-term renal function. Past efforts to develop an organized approach to these diseases have largely centered on histopathology. Although these approaches were helpful in defining lesions, for each morphologic entity there is a wide range in response to treatment and outcome. This observation suggests that traditional pathologic description alone is insufficient to classify these disorders.Here, we propose a new taxonomy for podocyte diseases. The word "taxonomy" was coined by Carl Linnaeus, the 18th century Swedish scientist who proposed the system for naming and classifying organisms that remains in use today. A taxonomy is organized into multiple levels, each of which represents a taxon with one or more elements. The ideal taxonomy separates the elements of each taxon (the taxa) into mutually exclusive, unambiguous, and all-encompassing categories. In practice, a good taxonomy should be simple, easy to remember, and easy to use. Taxonomies provide classification and frequently more: A conceptual framework for analysis, discussion, and hypothesis generation. Our proposed podocyte taxonomy has two dimensions-histopathology and etiology-and includes additional modifiers related to biomarkers that have been identified through recent progress in the genetics, cell biology, and pathophysiol...

These data indicate that MAP kinase pathways can be activated by TGF-beta1 in mesangial cells and that the ERK MAP kinase plays a role in TGF-beta-stimulated collagen I expression. Because we have shown previously that SMADs mediate TGF-beta1-stimulated collagen I expression, our findings raise the possibility of interactions between the MAP kinase and the SMAD pathways.

The mechanism(s) by which Smads mediate and modulate the transforming growth factor (TGF)-␤ signal transduction pathway in fibrogenesis are not well characterized. We previously showed that Smad3 promotes ␣2(I) collagen gene (COL1A2) activation in human glomerular mesangial cells, potentially contributing to glomerulosclerosis. Here, we report that Sp1 binding is necessary for TGF-␤1-induced type I collagen mRNA expression. Deletion of three Sp1 sites (GC box) between ؊376 and ؊268 or mutation of a CAGA box at ؊268/؊260 inhibited TGF-␤1-induced ␣2(I) collagen promoter activity. TGF-␤1 inducibility was also blocked by a Smad3 dominant negative mutant. Chemical inhibition of Sp1 binding with mithramycin A, or deletion of the GC boxes, inhibited COL1A2 activation by Smad3, suggesting cooperation between Smad3 and Sp1 in the TGF-␤1 response. Electrophoretic mobility shift assay showed that Sp1 and Smads form complexes with ؊283/؊250 promoter sequences. Coimmunoprecipitation experiments demonstrate that endogenous Sp1, Smad3, and Smad4 form complexes in mesangial cells. In a Gal4-LUC reporter assay system, Sp1 stimulated the TGF-␤1-induced transcriptional activity of Gal4-Smad3, Gal4-Smad4 (266 -552), or both. Using the transactivation domain B of Sp1 fused to the Gal4 DNA binding domain, we show that, in our system, the transcriptional activity of this Sp1 domain is not regulated by TGF-␤1, but it becomes responsive to this factor when Smad3 is coexpressed. Finally, combined Sp1 and Smad3 overexpression induces marked ligand-independent and liganddependent promoter activity of COL1A2. Thus, Sp1 and Smad proteins form complexes and their synergy plays an important role in mediating TGF-␤1-induced ␣2(I) collagen expression in human mesangial cells. Glomerulosclerosis is a scarring process involving extracellular matrix (ECM)1 accumulation and obliteration of glomerular capillaries. It is considered to be the final pathway leading to the progressive loss of renal function in several kidney diseases. Mechanical factors such as hyperfiltration and intraglomerular hypertension, as well as a variety of mediators including cytokines, growth factors, and eicosanoids derived from circulating or glomerular cells, have been implicated in initiating or maintaining sclerosis (1). However, little information is available regarding the cellular mechanisms by which these factors affect matrix turnover. Our laboratory has been studying the mechanisms by which transforming growth factor (TGF)-␤ stimulates ECM accumulation. Previously, we determined that the Smad pathway plays a role in activating type I collagen gene expression in human glomerular mesangial cells.