Fig. 8. Tat-beclin 1 peptide upregulates autophagy flux and protects against phosphotoxicity in renal proximal tubular cells.
GFP-LC3 reporter (LC3) mice at 10 weeks-old were intraperitoneally injected TB-11 or TB-Sc (2 mg/kg daily for 4 weeks). Four hours prior to sacrifice, mice were intraperitoneally injected chloroquine (A-C). Each treatment consisted of 5 mice. (A) Plasma Pi (left panel) and fractional excretion of phosphate (FEPi) (right panel) of LC3 mice. The data is presented at mean ± S.D. with scatter plots of individual data points. (B) Immunoblots for αKlotho, LC3, p62, and β-actin protein in the kidneys. Left panel shows representative immunoblots. Right panel is quantitation of all blots from each treatment. (C) Autophagic flux in the kidneys of LC3 mice. Each treatment has 5 mice. Upper panel is representative images of GFP-LC3 immunofluorescence in the renal tubules. Scale bars = 50 μm. Bottom panel summarizes quantitation of GFP-LC3 punctas in renal tubules. The data are presented as mean ± S.D. with scatter plots of individual data points. *P<0.05; **P<0.01 between 2 groups by unpaired t-test for A-C. TB-11 or TB-Sc (10 μM for 24 hours) were added to OKP cells with normal (0.96 mM) or high (3.0 mM) Pi (D and E). (D) Immunoblots of total cell lysates for LC3 and p62 protein in the OKP cells. Upper panel is representative immunoblots from each treatment. Bottom panel is summary of data from all of blots. (E) LDH in culture media. Data are presented as mean ± S.D. with scatter plots of individual data points from 5 independent experiments. *P<0.05; **P<0.01 between 2 groups by two-way ANOVA for D and E.