Fig 1.
Levels of blood urea nitrogen and serum creatinine.
The levels of blood urea nitrogen (A) and serum creatinine (B) were decreased in rats treated with Nec-1, zVAD or both Nec-1 and zVAD compared with the control group.(n = 6 rats per group, **p<0.01 versus the SNx+vehicle group). SNx: subtotal nephrectomy.
Fig 2.
Morphological changes in renal tubules.
(A) Representative images of H&E-stained kidney tissue sections exhibiting tubular dilatation, atrophy, desquamation of epithelial cells and hyaline deposition in the tubular lumen from the SNx rats, Scale bar, 50μm.(B) Tubular damage scores were decreased in rats treated with Nec-1,zVAD, or both Nec-1 and zVAD. (C)The proportion of necrotic dying cells and apoptotic cells. (n = 6 rats per group, **p<0.01 versus the SNx+vehicle group). SNx: subtotal nephrectomy.
Fig 3.
Necroptotic renal tubular epithelial cells were evaluated in vivo and in vitro using TEM.
(A) TEM photomicrographs of renal tubular epithelial cells from sham-operated rats and SNx rats treated with vehicle, Nec-1, zVAD, or Nec-1 and zVAD. Scale bar, 2μm. (B) TEM photomicrographs of cultured renal tubular epithelial cells pretreated for 30 min with DMSO (1%), Nec-1(30 mmol/L), zVAD (25 mmol/L), or Nec-1(30 mmol/L) and zVAD(25 mmol/L) and subsequently treated with TNF-a (100 ng/mL) for 24 h. (Scale bar, 2μm). (C) Quantification of the necroptotic incidence and apoptotic incidence in tubular epithelial cells in vivo. (*p<0.05, **p<0.01versus the tubular epithelial cells treated with only TNF-a, ##p < 0.01 necroptotic incidence versus apoptotic incidence in tubular epithelial cells treated with only TNF-a). SNx: subtotal nephrectomy.
Fig 4.
Detection of dead renal tubular epithelial cells using TUNEL staining in vivo and in vitro.
(A) TUNEL (green) and DAPI (blue) staining in renal tubular epithelial cells in sham-operated rats and SNx rats treated with vehicle, Nec-1, zVAD, or Nec-1 and zVAD. Scale bar, 50μm. (B) Immunofluorescence staining of cleaved caspase-3 (red),TUNEL staining (green) and DAPI (blue) staining in renal tubular epithelial cells pretreated for 30 min with DMSO (1%), Nec-1(30 mmol/L), zVAD(25 mmol/L), or Nec-1(30 mmol/L) and zVAD(25 mmol/L) and subsequently treated with TNF-a (100 ng/mL) for 24 h.(Scale bar, 50μm). (C) Quantification of TUNEL-positive cells in sham-operated rats and SNx rats treated with vehicle, Nec-1, zVAD, or Nec-1 and zVAD (n = 6 rats per group, **p<0.01 versus the SNx+vehicle group). SNx: subtotal nephrectomy. (D) Quantification of TUNEL-positive, cleaved caspase-3-positive cells and TUNEL-positive, cleaved caspase-3-negative cells in vivo (*p<0.05, **p<0.01versus the tubular epithelial cells treated with TNF-a alone, ##p <0.01 necroptotic incidence versus apoptotic incidence in tubular epithelial cells treated with TNF-a alone). SNx: subtotal nephrectomy.
Fig 5.
Western blot analysis of RIPK3 and caspase-3 protein levels in the remaining kidney tissue of SNx rats.
RIPK3 (A) and caspase-3(B) protein expression in the remaining kidney tissue of sham-operated rats and SNx rats treated with vehicle, Nec-1, or zVAD. (n = 6 rats per group, **p<0.01 versus the SNx+vehicle group). SNx: subtotal nephrectomy.
Fig 6.
Immunohistochemistry analysis of RIPK3 and cleaved caspase-3 protein expression in the remaining kidney tissues of SNx rats.
RIPK3 (A) and cleavedcaspase-3(B) in sham-operated rats and SNx rats treated with vehicle, Nec-1, or zVAD. Scale bar, 50μm. Semi-quantitative analysis of RIPK3 (C)and cleaved caspase-3(D) expression in renal tubular epithelial cells derived from SNx rats treated with vehicle, Nec-1or zVAD. (n = 6 rats per group, **p<0.01 versus the SNx+vehicle group). SNx: subtotal nephrectomy.