Fig 1.
hpa-TG mice are protected from Adriamycin-induced albuminuria and nephrotic syndrome.
(A) Male wild type BALB/c mice (wt) and hpa-TG mice were injected with Adriamycin (ADR) or kept as control (sham). Prior to injection (week 0) or one and two weeks post injection, urine was collected for 24h and used to determine albumin/creatinine ratio concentrations as described (n = 7 per each experimental group). **, P<0.001 vs. hpa-TG sham; ***, P<0.0001 vs. wt sham. (B) Blood samples were drawn from cardiac puncture at the day of sacrifice. Sera were analyzed for albumin and cholesterol (n = 13, 9, 12, and 11 for wt sham, wt ADR, hpa-TG sham, and hpa-TG ADR mice, respectively). **, P<0.001 vs. wt sham.
Fig 2.
Expression of key slit diaphragm proteins is reduced in wild type but not in hpa-TG Adriamycin-injected mice.
Podocyte markers expression is reduced in wild type but not in hpa-TG Adriamycin-injected mice. wt or hpa-TG mice were injected with Adriamycin (ADR) or served as control (sham). Two weeks post injection, the animals were sacrificed. (A, D) Representative immunofluorescence staining performed on cryostat sections from kidneys of the indicated experimental groups, using anti-podocin or anti-nephrin primary antibodies, and Dylight 488-conjugated anti-rabbit IgG. Nuclei were stained with DAPI (Confocal microscope, scale bar = 25 μm). The extent of podocin (B) and nephrin (E) staining and its intensity (C, F) in glomerular tuft were quantified. For podocin we analyzed n = 30, 23, 15 and 24 glomeruli, for wt sham, wt ADR, hpa-TG sham, and hpa-TG ADR mice, respectively), whereas for nephrin n = 22, 23, 17 and 27 glomeruli, for wt sham, wt ADR, hpa-TG sham, and hpa-TG ADR mice, respectively. Glomeruli were evaluated at least from 3–4 mice per each experimental group. *, P<0.01 vs. wt sham. (G) Protein was extracted from kidney cortex or isolated glomeruli and analyzed by Western blotting using the indicated antibodies. P97 was used as loading control. A representative immunoblot is depicted in panel G (n = 7, 11, 7 and 11 for wt sham, wt ADR, hpa-TG sham, and hpa-TG ADR mice, respectively from three independent experiments) (H, I) Densitometry of immunoblotting of isolated glomeruli lysates (n = 8, 7, 3 and 6 for wt sham, wt ADR, hpa-TG sham, and hpa-TG ADR mice, respectively). *, P = 0.03 vs. wt sham **, P = 0.02 vs. wt sham.
Fig 3.
Adriamycin causes renal damage in wild type mice but not in hpa-TG mice.
wt or hpa-TG mice were injected with Adriamycin (ADR) or served as control (sham). Two weeks post injection, the animals were sacrificed. Representative images of H&E (A) and Masson’s trichrome (B) staining of paraffin-embedded kidney sections from various experimental groups (scale bar = 100 μm). n = 6 mice per each experimental group, from three independent experiments.
Fig 4.
Heparanase protects podocytes from Adriamycin induced injury.
wt or hpa-TG mice were injected with Adriamycin (ADR) or served as control (sham). Two weeks post injection, the animals were sacrificed. (A) Representative images of transmission electron microscopy of ultrathin sections of kidney tissue. Magnification X15 000; scale bar = 1μm. (B) Quantification of foot process width (n = 12 glomeruli per each wt group, 13 for hpa-TG, and 11 for hpa-TG ADR group, obtained from 6, 3, 4, and 5 animals, respectively). *, P<0.01 vs. all other groups.