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

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

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

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

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

Proximal tubular cell exosomes contain OPG.

<p><b>A)</b> Exosomes from HK-2 conditioned serum-free cell culture media. Representative Western blots of TRAIL, TWEAK and exosome markers. Each lane contains 5 µg exosomal protein. <b>B)</b> OPG is observed in HK-2-derived exosomes when reducing, denaturizing conditions are applied. Each lane contains 10 µg exosomal protein. <b>C)</b> OPG expression in HK-2-derived exosomes detected by ELISA. Results expressed as pg/µg of total protein. Mean+SEM of 3 independent experiments. <b>D)</b> OPG analysis by selected reaction monitoring (SRM) in a LC-(QQQ)-MS/MS showing two different transitions corresponding to the same precursor peptide which coelute in time. The mass and charge of the precursor and its fragments are shown. A single peptide (<u>YLHYDEETSHQLL</u>) and a single precursor were measured under two different charge state (1025.9624+2 and 684.3107+3), each of them with its own fragment (1230.5783+ and 1138.4687+, respectively), thus yielding two different peaks or transitions.</p

OPG immunohistochemistry in human kidneys.

<p>Control, CKD and autosomal dominant polycystic kidney disease kidneys samples were studied. For ADPKD a cortical cyst wall is shown. <b>A)</b> OPG mainly stained the basolateral aspect of proximal tubules in control kidneys (arrowheads). <b>B)</b> In CKD whole tubular cells are intensely stained in the cortex (arrows). <b>C)</b> A similar pattern of intense whole cell staining is observed in cyst epithelial lining (arrows). Original magnification ×200.</p

Exosomes from human urine contain OPG.

<p><b>A)</b> Transmission electron microscopy representative images showing microvesicles purified from human urine. i) Scale bar = 200 nm; ii) Scale bar = 50 nm; iii) Scale bar = 200 nm; iv)Scale bar = 50 nm. ii&iv) show amplified areas from i&iii). <b>B)</b> Western blot. Representative images. Urine: whole urine collected from healthy donors. Supernat.: Urine supernatant from the last exosome isolation step. 5 µg protein were loaded per well.</p