H. William Schnaper

Cross-talk between ERK MAP kinase and Smad signaling pathways enhances TGF-beta-dependent responses in human mesangial cells.

Transforming growth factor β (TGF‐β) stimulates renal cell fibrogenesis by a poorly understood mechanism. Previously, we suggested a synergy between TGF‐β1 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‐β1 induced Smad3 activity. Total serine phosphorylation of Smad2/3, but not phosphorylation of the C‐terminal SSPXSP motif, was decreased by pretreatment with the MEK/ERK inhibitors, PD98059 (10 µM) or U0126 (25 µM). 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‐β stimulated phosphorylation of an expressed Smad3A construct, with a mutated C‐terminal SSPXSP 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 α2(I) collagen promoter activity, an effect blocked by co‐transfected Smad3EPSM, but not Smad3A. The effects of caMEK and TGF‐β1 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.

Sp1 and Smad Proteins Cooperate to Mediate Transforming Growth Factor-β1-induced α2(I) Collagen Expression in Human Glomerular Mesangial Cells

The mechanism(s) by which Smads mediate and modulate the transforming growth factor (TGF)-β signal transduction pathway in fibrogenesis are not well characterized. We previously showed that Smad3 promotes α2(I) collagen gene (COL1A2) activation in human glomerular mesangial cells, potentially contributing to glomerulosclerosis. Here, we report that Sp1 binding is necessary for TGF-β1-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-β1-induced α2(I) collagen promoter activity. TGF-β1 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-β1 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-β1-induced transcriptional activity of Gal4-Smad3, Gal4-Smad4 (266), 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-β1, but it becomes responsive to this factor when Smad3 is coexpressed. Finally, combined Sp1 and Smad3 overexpression induces marked ligand-independent and ligand-dependent promoter activity of COL1A2. Thus, Sp1 and Smad proteins form complexes and their synergy plays an important role in mediating TGF-β1-induced α2(I) collagen expression in human mesangial cells.

The Phosphatidylinositol 3-Kinase/Akt Pathway Enhances Smad3-stimulated Mesangial Cell Collagen I Expression in Response to Transforming Growth Factor-β1

Transforming growth factor (TGF)-β has been associated with renal glomerular matrix accumulation. We previously showed that Smad3 promotes COL1A2 gene activation by TGF-β1 in human glomerular mesangial cells. Here, we report that the PI3K/Akt pathway also plays a role in TGF-β1-increased collagen I expression. TGF-β1 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-β1 treatment. The PI3K inhibitor LY294002 decreases TGF-β1-stimulated α1(I) and α2(I) collagen mRNA expression. Similarly, LY294002 or an Akt dominant negative construct blocks TGF-β1 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-β1-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-β1-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-β receptor I target site. Thus, although the PI3K-PDK1-Akt pathway alone is insufficient to stimulate COL1A2 gene transcription, its activation by TGF-β1 enhances Smad3 transcriptional activity leading to increased collagen I expression in human mesangial cells. This cross-talk between the Smad and PI3K pathways likely contributes to TGF-β1 induction of glomerular scarring.

A Proposed Taxonomy for the Podocytopathies: A Reassessment of the Primary Nephrotic Diseases

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.

Estrogens and the Vascular Endothelium

Abstract: Estrogens exert important regulatory functions on vessel wall components, which may contribute to the increased prevalence and severity of certain chronic inflammatory and autoimmune diseases in females and the lower cardiovascular risk observed in premenopausal women. Endothelial cells have been recently identified as targets for estrogens, and estrogen receptors have been demonstrated in endothelial cells from various vascular beds. This review focuses on the regulatory function of estrogens in endothelial cell responses relevant to vessel inflammation, injury, and repair; estrogen effects on nitric oxide production and release; estrogen modulation of endothelial cell adhesion molecule expression; and estrogen regulation of angiogenesis. The mechanisms through which estrogen regulates endothelial cell functions are complex and involve both genomic and nongenomic mechanisms.

Tubulointerstitial injury and the progression of chronic kidney disease

In chronic kidney disease (CKD), once injury from any number of disease processes reaches a threshold, there follows an apparently irreversible course toward decline in kidney function. The tubulointerstitium may play a key role in this common progression pathway. Direct injury, high metabolic demands, or stimuli from various other forms of renal dysfunction activate tubular cells. These, in turn, interact with interstitial tissue elements and inflammatory cells, causing further pathologic changes in the renal parenchyma. The tissue response to these changes thus generates a feed-forward loop of kidney injury and progressive loss of function. This article reviews the mechanisms of this negative cycle mediating CKD.

TGF-beta signal transduction in chronic kidney disease

Transforming growth factor (TGF)-beta is a central stimulus of the events leading to chronic progressive kidney disease, having been implicated in the regulation of cell proliferation, hypertrophy, apoptosis and fibrogenesis. The fact that it mediates these varied events suggests that multiple mechanisms play a role in determining the outcome of TGF-beta signaling. Regulation begins with the availability and activation of TGF-beta and continues through receptor expression and localization, control of the TGF-beta family-specific Smad signaling proteins, and interaction of the Smads with multiple signaling pathways extending into the nucleus. Studies of these mechanisms in kidney cells and in whole-animal experimental models, reviewed here, are beginning to provide insight into the role of TGF-beta in the pathogenesis of renal dysfunction and its potential treatment.

Interdependence of HIF-1α and TGF-β/Smad3 signaling in normoxic and hypoxic renal epithelial cell collagen expression

Increasing evidence suggests that chronic kidney disease may develop following acute kidney injury and that this may be due, in part, to hypoxia-related phenomena. Hypoxia-inducible factor (HIF) is stabilized in hypoxic conditions and regulates multiple signaling pathways that could contribute to renal fibrosis. As transforming growth factor (TGF)-β is known to mediate renal fibrosis, we proposed a profibrotic role for cross talk between the TGF-β1 and HIF-1α signaling pathways in kidney cells. Hypoxic incubation increased HIF-1α protein expression in cultured human renal tubular epithelial cells and mouse embryonic fibroblasts. TGF-β1 treatment further increased HIF-1α expression in cells treated with hypoxia and also increased HIF-1α in normoxic conditions. TGF-β1 did not increase HIF-1α mRNA levels nor decrease the rate of protein degradation, suggesting that it enhances normoxic HIF-1α translation. TGF-β receptor (ALK5) kinase activity was required for increased HIF-1α expression in response to TGF-β1, but not to hypoxia. A dominant negative Smad3 decreased the TGF-β-stimulated reporter activity of a HIF-1α-sensitive hypoxia response element. Conversely, a dominant negative HIF-1α construct decreased Smad-binding element promoter activity in response to TGF-β. Finally, blocking HIF-1α transcription with a biochemical inhibitor, a dominant negative construct, or gene-specific knockdown decreased basal and TGF-β1-stimulated type I collagen expression, while HIF-1α overexpression increased both. Taken together, our data demonstrate cooperation in signaling between Smad3 and HIF-1α and suggest a new paradigm in which HIF-1α is necessary for normoxic, TGF-β1-stimulated renal cell fibrogenesis.

Advances in the Biology and Genetics of the Podocytopathies: Implications for Diagnosis and Therapy

CONTEXT Etiologic factors and pathways leading to altered podocyte phenotype are clearly numerous and involve the activity of different cellular function. OBJECTIVE To focus on recent discoveries in podocyte biology and genetics and their relevance to these human glomerular diseases, named podocytopathies. DATA SOURCES Genetic mutations in genes encoding for proteins in the nucleus, slit diaphragm, podocyte cytoplasm, and cell membrane are responsible for podocyte phenotype and functional abnormalities. Podocyte injury may also derive from secondary stimuli, such as mechanical stress, infections, or use of certain medications. Podocytes can respond to injury in a limited number of ways, which include (1) effacement, (2) apoptosis, (3) arrest of development, and (4) dedifferentiation. Each of these pathways results in a specific glomerular morphology: minimal change nephropathy, focal segmental glomerulosclerosis, diffuse mesangial sclerosis, and collapsing glomerulopathy. CONCLUSIONS Based on current knowledge of podocyte biology, we organized etiologic factors and morphologic features in a taxonomy of podocytopathies, which provides a novel approach to the classification of these diseases. Current and experimental therapeutic approaches are also discussed.

High Ambient Glucose Enhances Sensitivity to TGF-β1 via Extracellular Signal—Regulated Kinase and Protein Kinase Cδ Activities in Human Mesangial Cells

High ambient glucose activates intracellular signaling pathways to induce cytokines such as TGF-beta1 in the extracellular matrix accumulation of diabetic nephropathy. These same pathways also may directly modulate TGF-beta1 signaling. R-Smad phosphorylation, association with Smad4, and nuclear accumulation after TGF-beta1 treatment (1.0 ng/ml) were significantly higher in mesangial cells that were conditioned to 20 mM glucose for 72 h than mesangial cells in 6.5 mM glucose, suggesting that high glucose enhanced responsiveness to TGF-beta1. Neither TGF-beta1 bioactivity nor TGF-beta receptor binding was significantly different between in 6.5 and 20 mM glucose-conditioned cultures. Furthermore, adding a neutralizing anti-TGF-beta1 antibody during glucose conditioning did not affect the enhanced Smad responsiveness, indicating that enhancement likely did not result from increased TGF-beta expression. In contrast, a mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK inhibitor, PD98059, completely abrogated the effect of high glucose. Glucose stimulation of ERK was inhibited by the general protein kinase C (PKC) inhibitor calphostin C and by the PKCdelta-specific inhibitor rottlerin, whereas Gö6976, an inhibitor of conventional PKC, had no effect on ERK activity. Specificity of the PKC inhibitors was further verified by PKCbeta and delta kinase assay. High glucose increased expression of several PKC isozymes, but only PKCdelta showed proportionally increased membrane translocation and kinase activity in cells that were conditioned to 20 mM glucose. Finally, both ERK and PKCdelta inhibition during glucose conditioning abrogated enhanced alpha1(I) collagen mRNA and promoter induction by TGF-beta1. Taken together, these data strongly suggest that heightened ERK and PKCdelta activity in high ambient glucose conditions interact with the Smad pathway, leading to enhanced responsiveness to TGF-beta1 and increased extracellular matrix production in mesangial cells.