Mitochondria Are the Major Targets in Albumin-Induced Apoptosis in Proximal Tubule Cells

Abstract

Nephrotic-range proteinuria is considered a poor prognostic factor. A correlation between tubulointerstitial injury and the degree of proteinuria is well established. In an attempt to explain the tubular atrophy that is observed in advanced glomerulonephritides, this study investigated apoptotic mechanisms in cultured human proximal tubule cells (HKC-8) that were exposed to endotoxin-free albumin (5, 10, and 20 mg/ml). Apoptosis was detected by Hoechst 33342; annexin staining; and assays for caspases 3, 8, and 9. The apoptotic effect of albumin was maximal at 10 mg/ml albumin, and necrosis prevailed in cells that were incubated with 20 mg/ml. Increase in caspase-9 and -3 activity was observed starting at 6 and maximally at 16 to 24 h. The proapoptotic Bcl-2 protein Bax was upregulated at 6 h, associated with translocation of cytochrome-c from mitochondria to cytosol and alteration in the mitochondrial membrane potential. Production of reactive oxygen species (ROS) was significant at 6 h but declined at 16 and 24 h. Treatment with ROS scavenger dimethylthiourea or antioxidant N-acetylcysteine did not alleviate caspase-3 production. Pan protein kinase C inhibitor bisindolylmaleimide-1 protected the cells from apoptosis. It is concluded that albumin induces apoptosis in human proximal tubule cells by stimulating mitochondrial apoptotic pathway independent of ROS production.

PKC-δ Promotes Renal Tubular Cell Apoptosis Associated with Proteinuria

“…As shown in Supplemental Figure 2, transient transfection of Bcl-2 into RPTC suppressed albumin-induced cytochrome c release and apoptosis, whereas vector transfection was ineffective. Together, the results support the recent work by Erkan et al 7 that the mitochondrial pathway of apoptosis plays an important role in albumin-induced apoptosis in renal tubular cells.…”

“…Erkan and colleagues 7 have recently demonstrated a role for the mitochondrial/intrinsic pathway of apoptosis in albumininduced apoptosis in HKC-8 renal tubular cells. Consistently, we showed cytochrome c release from mitochondria during albumin incubation of RPTC, which was suppressed by Bcl-2 expression (Figure 2).…”

“…6 However, in HKC-8 human proximal tubular cells, albumin-induced apoptosis was shown to be mainly mediated by the intrinsic pathway of apoptosis, characterized by Bax translocation to mitochondria and cytochrome c release from the organelles. 7 In addition, Ohse et al 8 demonstrated endoplasmic reticulum (ER) stress in albumin-treated rat kidney proximal tubular cells and proteinuric kidneys of puromycin aminonucleoside-treated rats, suggesting the involvement of ER stress in tubular apoptosis. Recent work by Lindenmeyer et al 9 further demonstrated ER stress in proteinuric kidney biopsy of established diabetic nephropathy.…”

“…In this regard, Erkan et al 7 showed that albumininduced apoptosis in HKC-8 cells could be suppressed by bisindolylmaleimide-1, a general pharmacologic inhibitor of protein kinase C (PKC). Nonetheless, it remains unclear which PKC member(s) is activated during albumin treatment and plays a key role in the induction or regulation of tubular cell apoptosis.…”

Increased Tubular Proliferation as an Adaptive Response to Glomerular Albuminuria

“…23 Thus, it was possible that the tubular proliferation occurred in response to albuminuria-induced apoptosis and reduced tubular cell numbers. However, terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling staining at 3, 5, and 7 days after podocyte ablation with 25 ng DT revealed that there was no increase in tubular cell apoptotic rates on days 3 and 5 (i.e., preceding the proliferation observed on days 6-7) and only a modest increase in apoptotic rates on day 7 (Figure 4, D and E).…”