Fig 2: Leukocytes and flow synergistically induce PIEZO1 activation to stimulate endothelial downstream signaling. (A) HUVECs were preactivated with TNFα, loaded with Fluo-4, and exposed to PMNs alone, low flow (1.2 dynes/cm2) alone, or both. [Ca2+]i was determined as fluorescence intensity (RFU, relative fluorescence units). (B) HUVECs transfected with control (siCtrl) or PIEZO1-specific siRNA (siPIEZO1) were preactivated with TNFα, loaded with Fluo-4, and exposed to PMNs and low flow (1.2 dynes/cm2) given together. [Ca2+]i was determined as fluorescence intensity. Five traces representative of the traces of 1 experiment are shown in panels A and B, and the time point of addition of PMNs is indicated by an arrow. The bar diagrams in panels A and B show the area under the curve (AUC) of the [Ca2+]i traces from 6 independent experiments (20-40 cells were analyzed per experiment). (C-D) Currents from MLECs of wild-type (control) or EC-Piezo1-KO mice were recorded in the whole cell patch clamp configuration. The holding potential was −80 mV, and the MLECs were exposed to PMNs, low flow (1.2 dynes/cm2), or both (n = 8-9 independent measurements per condition). (E-H) Immunoblot analysis of total and phosphorylated PYK2, SRC, and MLC in lysates of TNFα-activated HUVECs transfected with control siRNA (siCtrl) or siRNA directed against PIEZO1 and incubated without or with human PMNs in the absence or presence of low flow (1.2 dynes/cm2) (E) or without or with 5 μM Yoda1 (G). Immunoblot analysis of PIEZO1 and GAPDH served as controls. Bar diagrams (F,H) show the densitometric analysis of 3 independent experiments. (I) Transmigration of human PMNs across TNFα-activated HUVECs preincubated for 30 minutes with the PYK2 and SRC inhibitors PF431396 (10 μM) and PP2 (10 μM), respectively (n = 5 independent experiments). (J) HUVECs transfected with control (siCtrl) or PIEZO1-specific siRNA (siPIEZO1) were preactivated with TNFα and exposed to PMNs alone, low flow (1.2 dynes/cm2) alone, or both. After 15 minutes, VE-cadherin internalization was determined as described in Methods (n = 4 independent experiments). Shown are mean values ± SEM. *P ≤ .05; **P ≤ .01; ***P ≤ .001 (1-way ANOVA [A,D]; unpaired t test [B,F,H-J]).

Fig 3: Autophagy regulated OS and senescence in PTCs of Aldo-induced rats. (A) Senescence-associated-galactosidase (SA-β-Gal) Staining in rat renal cortex (n = 6). SA-β-Gal is labeled by bright-blue in the PTCs. (B) Bar graph indicating the percentage of SA-β-Gal positive cells per field in tubular. (C) DHE staining of rat kidney sections (n = 6). (D) Bar graph indicating the mean DHE intensity per field in rat tubular cells. (E) Immunohistochemical staining for NOX4 in rat kidney tissues from various groups, as indicated (n = 6). (F) Bar graph indicating NOX4 immunoreactivity per field in rat tubular cells. (G) Western blot analysis revealed the expression of NOX4, p21, and GAPDH proteins after various treatments in rats (n = 3). (H) Graphical presentation shows the relative abundance levels of NOX4 and p21 after normalization with GAPDH (n = 3). #p < 0.05 vs. normal control, *p < 0.05 vs. Aldo alone.

Fig 4: Rap improved Aldo-induced OS and senescence in HK-2 cells. (A) Immunofluorescence staining for LC3 in HK-2 cells, which were incubated in media containing buffer (Control) or Aldo with or without Rap for 24 h as indicated (n = 3). (B) Western blot analysis revealed the expression of LC3-II/LC3-I, p62, and GAPDH proteins in HK-2 cells after various treatments as indicated. (C) Graphical presentation indicated the relative abundance levels of LC3-II/LC3-I and p62 after normalization with GAPDH (n = 3). (D) SA-β-gal activity, which appears as bright-blue granular staining in the cytoplasm of HK-2 cells in various groups as indicated (n = 3). (E) Quantification of 2’, 7’-dichlo-rofluorescein (DCF) fluorescence in various groups as indicated (n = 3). (F) Western blot analysis showed the expression of p21, NOX4, and GAPDH proteins in HK-2 cells after various treatments as indicated (n = 3). (G) Graphical presentation shows the relative abundance levels of p21 and NOX4 after normalization with GAPDH (n = 3). #p < 0.05 vs. normal control, *p < 0.05 vs. Aldo alone.

Fig 5: MNRR1 and cellular growth. a S473 pAKT levels in MNRR1-knockdown (R1-KD) and MNRR1-OE (R1-OE) cells compared to WT controls. Total AKT levels do not change. GAPDH is used as a loading control. b Total Rictor protein levels in WT cells expressing either an empty vector (EV), Flag-tagged WT-MNRR1 (WT), or flag tagged transcriptionally active MNRR1 (TA). Tubulin is used as a loading control. Flag levels indicate transfection efficiency between WT and TA MNRR1. c PGC-1α and Rictor protein levels in MCF7 cells with a knockout of MNRR1. GAPDH is used as a loading control. d Cell growth between MCF10A cells transfected with either an empty vector (EV) or MNRR1 (R1) expression plasmid. The Cell Titre96 assay was measured spectrophotometrically. p = 0.03