Angiotensin II Contributes to Renal Fibrosis Independently of Notch Pathway Activation

<div><p>Recent studies have described that the Notch signaling pathway is activated in a wide range of renal diseases. Angiotensin II (AngII) plays a key role in the progression of kidney diseases. AngII contributes to renal fibrosis by upregulation of profibrotic factors, induction of epithelial mesenchymal transition and accumulation of extracellular matrix proteins. In cultured human tubular epithelial cells the Notch activation by transforming growth factor-β1 (TGF-β1) has been involved in epithelial mesenchymal transition. AngII mimics many profibrotic actions of TGF-β1. For these reasons, our aim was to investigate whether AngII could regulate the Notch/Jagged system in the kidney, and its potential role in AngII-induced responses. In cultured human tubular epithelial cells, TGF-β1, but not AngII, increased the Notch pathway-related gene expression, Jagged-1 synthesis, and caused nuclear translocation of the activated Notch. In podocytes and renal fibroblasts, AngII did not modulate the Notch pathway. In tubular epithelial cells, pharmacological Notch inhibition did not modify AngII-induced changes in epithelial mesenchymal markers, profibrotic factors and extracellular matrix proteins. Systemic infusion of AngII into rats for 2 weeks caused tubulointerstitial fibrosis, but did not upregulate renal expression of activated Notch-1 or Jagged-1, as observed in spontaneously hypertensive rats. Moreover, the Notch/Jagged system was not modulated by AngII type I receptor blockade in the model of unilateral ureteral obstruction in mice. These data clearly indicate that AngII does not regulate the Notch/Jagged signaling system in the kidney, <em>in vivo</em> and <em>in vitro</em>. Our findings showing that the Notch pathway is not involved in AngII-induced fibrosis could provide important information to understand the complex role of Notch system in the regulation of renal regeneration vs damage progression.</p> </div

Role of Epidermal Growth Factor Receptor (EGFR) and Its Ligands in Kidney Inflammation and Damage

Chronic kidney disease (CKD) is characterized by persistent inflammation and progressive fibrosis, ultimately leading to end-stage renal disease. Although many studies have investigated the factors involved in the progressive deterioration of renal function, current therapeutic strategies only delay disease progression, leaving an unmet need for effective therapeutic interventions that target the cause behind the inflammatory process and could slow down or reverse the development and progression of CKD. Epidermal growth factor receptor (EGFR) (ERBB1), a membrane tyrosine kinase receptor expressed in the kidney, is activated after renal damage, and preclinical studies have evidenced its potential as a therapeutic target in CKD therapy. To date, seven official EGFR ligands have been described, including epidermal growth factor (EGF) (canonical ligand), transforming growth factor-α, heparin-binding epidermal growth factor, amphiregulin, betacellulin, epiregulin, and epigen. Recently, the connective tissue growth factor (CTGF/CCN2) has been described as a novel EGFR ligand. The direct activation of EGFR by its ligands can exert different cellular responses, depending on the specific ligand, tissue, and pathological condition. Among all EGFR ligands, CTGF/CCN2 is of special relevance in CKD. This growth factor, by binding to EGFR and downstream signaling pathway activation, regulates renal inflammation, cell growth, and fibrosis. EGFR can also be “transactivated” by extracellular stimuli, including several key factors involved in renal disease, such as angiotensin II, transforming growth factor beta (TGFB), and other cytokines, including members of the tumor necrosis factor superfamily, showing another important mechanism involved in renal pathology. The aim of this review is to summarize the contribution of EGFR pathway activation in experimental kidney damage, with special attention to the regulation of the inflammatory response and the role of some EGFR ligands in this process. Better insights in EGFR signaling in renal disease could improve our current knowledge of renal pathology contributing to therapeutic strategies for CKD development and progression

Data of the experimental models of spontaneously hypertensive rats (SHR) and diabetic nephropathy induced by streptozotocin injection (STZ).

<p>Data of the experimental models of spontaneously hypertensive rats (SHR) and diabetic nephropathy induced by streptozotocin injection (STZ).</p

TGF-β1, but not AngII, increased Jagged-1 synthesis in cultured human tubular epithelial cells.

<p>Cultured human tubular epithelial cells (HK-2) were treated with 10⁻⁷ mol/L AngII or 10 ng/mL TGF-β1 for increasing times. <b>A.</b> Results of total protein expression were obtained from densitometric analysis and expressed as ratio protein/GAPDH as n-fold over control as mean ± SEM of 3 independent experiments. *p<0.05 vs control. Figure <b>B</b> shows a representative Western blot experiment. <b>C. </b><b>Dose-response of AngII.</b> HK-2 cells were stimulated with AngII (10⁻⁶ to 10⁻¹¹ mol/L) for 48 hours and Jagged-1 protein levels were determined by Western blot. Figure shows a representative experiment of 3 done. <b>D. TGF-β1, but not AngII, upregulated Notch-related genes in tubular epithelial cells.</b> Gene expression levels of jagged-1, delta-1 and notch1/3 were determined by Real Time PCR. Data are expressed as mean ± SEM of 5 experiments. *p<0.05 vs control. <b>E.</b> Nuclear localization of activated Notch-1 (NICD) is only observed in TGF-β1 treated cells for 48 hours (green staining), while in control and AngII-treated cells there is no positive NICD staining. Nuclei are in blue (DAPI staining). Figure shows a representative experiment of 2 done by confocal microscopy. Magnification 200x.</p

Blockade of the Notch pathway inhibited TGF-β1-, but not AngII- induced EMT changes in cultured tubular epithelial cells.

<p>HK-2 cells were pretreated with the gamma-secretase inhibitor, DAPT (3×10⁻⁸ mol/L) for 1 hour and then stimulated with 10⁻⁷ mol/L AngII or 10 ng/mL of TGF-β1 for 24 or 48 hours (gene and protein studies, respectively). Figure <b>A</b> shows a representative Western blot experiment <b>B.</b> Results of total protein expression were expressed as mean ± SEM of 3 independent experiments. Figure <b>C</b> shows gene expression levels, determined by Real Time PCR, were shown as mean ± SEM of 5 experiments. *p<0.05 vs control, #p<i><</i>0.05 vs TGF-β1.</p

AT1 antagonism increased renal Jagged-1 protein levels in the model of unilateral ureteral obstructed kidneys in mice.

<p>The figure <b>A</b> shows a representative experiment of Jagged-1 protein levels evaluated by western blot and in <b>B</b> data as mean ± SEM of 6 animals per group. *p<0.05 vs contralateral kidneys.</p

Jagged-1 induced EMT changes in cultured tubular epithelial cells

<p>. <b>A.</b> HK-2 cells were treated with 50 ng/mL Jagged-1 for 48 hours. Left panel: total protein levels as mean ± SEM of 3 independent experiments. *p<0.05 vs control; #p<i><</i>0.05 vs Jagged-1. Right panel: representative Western blot experiment. <b>Blockade of the Notch pathway inhibited TGF-β1-induced upregulation of Notch components.</b> Cells were pretreated for 1 hour with 3×10⁻⁸ mol/L DAPT and then incubated with 10 ng/mL TGF-β1 or 10⁻⁷ mol/L AngII for 24 or 48 hours (gene and protein studies, respectively). <b>B.</b> Gene expression levels are expressed as mean ± SEM of 5 experiments. Figure <b>C</b> shows a representative western blot of Jagged-1 and data as of mean ± SEM of 3 independent experiments. *p<0.05 vs control; #p<i><</i>0.05 vs TGF- β1.</p

AngII increased TGF-β1 production in renal cells.

<p>The different cell types, human tubular epithelial cells (HK-2), murine renal fibroblasts (TFBs) and human podocytes, were treated with 10⁻⁷ mol/L AngII for 48 hours. Then, supernatants were collected, and active TGF-β1 was determined by ELISA. Figure <b>A</b> shows TGF-β1 protein levels as mean ± SEM of 3 independent experiments analyzed by duplicate. *p<0.05 vs control. <b>B. Low doses of TGF-β1 did not increase Jagged-1 protein production in tubular epithelial cells.</b> HK-2 cells were stimulated with TGF-β1 (10 to 0.5 ng/mL) for 48 hours and Jagged-1 protein levels were determined by Western blot. Figure shows a representative experiment and data as mean ± SEM of 3 experiments. *p<0.05 vs control.</p

TGF-β1, but not AngII, increased Jagged-1 expression in human podocytes and murine renal fibroblasts.

<p>Cells were treated with 10⁻⁷ mol/L AngII or 10 ng/mL TGF-β1 for 24 or 48 hours (gene and protein studies, respectively). In some points cells were pretreated with the gamma-secretase inhibitor, 3×10⁻⁸ mol/L DAPT, for 1 hour. <b>A.</b> In human podocytes, gene expression levels of Notch components are expressed as mean ± SEM of 5 experiments. *p<0.05 vs control, #p<i><</i>0.05 vs TGF- β1. <b>B.</b> Representative Western blot of Jagged-1 levels in podocytes and fibroblasts of 3 independent experiments.</p