Routes of Albumin Overload Toxicity in Renal Tubular Epithelial Cells

Abstract

Besides being a marker of kidney disease severity, albuminuria exerts a toxic effect on renal proximal tubular epithelial cells (RPTECs). We evaluated whether an unfolded protein response (UPR) or DNA damage response (DDR) is elicited in RPTECs exposed to high albumin concentration. The deleterious outcomes of the above pathways, apoptosis, senescence, or epithelial-to-mesenchymal transition (EMT) were evaluated. Albumin caused reactive oxygen species (ROS) overproduction and protein modification, and a UPR assessed the level of crucial molecules involved in this pathway. ROS also induced a DDR evaluated by critical molecules involved in this pathway. Apoptosis ensued through the extrinsic pathway. Senescence also occurred, and the RPTECs acquired a senescence-associated secretory phenotype since they overproduced IL-1β and TGF-β1. The latter may contribute to the observed EMT. Agents against endoplasmic reticulum stress (ERS) only partially alleviated the above changes, while the inhibition of ROS upregulation prevented both UPR and DDR and all the subsequent harmful effects. Briefly, albumin overload causes cellular apoptosis, senescence, and EMT in RPTECs by triggering UPR and DDR. Promising anti-ERS factors are beneficial but cannot eliminate the albumin-induced deleterious effects because DDR also occurs. Factors that suppress ROS overproduction may be more effective since they could halt UPR and DDR.

Keywords: DNA damage; albumin; apoptosis; endoplasmic reticulum stress; epithelial-to-mesenchymal transition; renal tubular epithelial cells; senescence.

Figure 1

Albumin overload does not induce cell necrosis but triggers ROS production and UPR. LDH release assay showed that albumin overload does not cause cell necrosis. TUDCA and 4-PBA were not cytotoxic (A). High albumin concentration induced a ROS burst in RPTECs. Neither TUDCA nor 4-PBA affected ROS production (B). Panel (C) depicts the results of one out of three performed experiments. BSA treatment increased the level of 4-HNE-modified proteins. TUDCA and 4-PBA partially ameliorated protein modification (D). In RPTECs, high albumin concentration triggered a UPR since the p-PERK level increased (E), whereas the total PERK remained unaffected (F). p-eIF2α level also enhanced (G), whereas the total eIF2α remained stable (H). ATF3 level upregulated (I). TUDCA and 4-PBA ameliorated but did not eliminate all the above changes. * p < 0.05 vs. control; # p < 0.05 vs. RPTECs treated with 4-PBA; ^ p < 0.05 vs. RPTECs treated with TUDCA; + p < 0.05 vs. RPTECs exposed to BSA; & p < 0.05 vs. RPTECs exposed to BSA and 4-PBA; ! p < 0.05 vs. RPTECs exposed to BSA and TUDCA. 4-PBA, 4-Phenylbutyric acid; 4-HNE, 4-Hydroxynonenal; ATF-3, activating transcription factor-3; BSA, bovine serum albumin; eIF2α, eukaryotic translation initiation factor-2α; PERK, PKR-like ER kinase; ROS, reactive oxygen species; TUDCA, tauroursodeoxycholic acid.

Figure 2

Albumin overload induces a DDR. Panel (A) depicts the results of one out of three performed experiments. Albumin overload caused DNA damage detected by the enhanced γ-H2AX level. TUDCA and 4-PBA decreased to some extent DNA damage (B). DNA damage in RPTECs exposed to albumin elicited a DDR since the p-ATM level increased (C) on a stable total ATM background (D). As a result of ATM activation, p-p53 increased (E), resulting in total p53 upregulation (F). TUDCA and 4-PBA reduced to some extent all the above albumin overload-induced changes. * p < 0.05 vs. control; # p < 0.05 vs. RPTECs treated with 4-PBA; ^ p < 0.05 vs. RPTECs treated with TUDCA; + p < 0.05 vs. RPTECs exposed to BSA; & p < 0.05 vs. RPTECs exposed to BSA and 4-PBA; ! p < 0.05 vs. RPTECs exposed to BSA and TUDCA. 4-PBA, 4-Phenylbutyric acid; γ-H2AX, phosphorylated at Ser139 histone H2AX; ATM, ataxia telangiectasia mutated kinase; BSA, bovine serum albumin; p53, tumor suppressor p53; TUDCA, tauroursodeoxycholic acid.

Figure 3

Albumin overload triggers the extrinsic but not the intrinsic apoptotic pathway. Panel (A) depicts the results of one out of three performed experiments. Albumin overload left the intrinsic apoptotic pathway unaffected since the CHOP (B), Bax (C), and CC-9 (D) levels did not change significantly. TUDCA and 4-PBA did not change the levels of the above proteins. Exposure of the RPTECs to high albumin concentration triggered the extrinsic apoptotic pathway as it upregulated DR5 (E) and CC-8 (F). Eventually, apoptosis ensued since the level of CC-3 increased (G). TUDCA and 4-PBA ameliorated but did not eliminate the above changes. * p < 0.05 vs. control; # p < 0.05 vs. RPTECs treated with 4-PBA; ^ p < 0.05 vs. RPTECs treated with TUDCA; + p < 0.05 vs. RPTECs exposed to BSA; & p < 0.05 vs. RPTECs exposed to BSA and 4-PBA; ! p < 0.05 vs. RPTECs exposed to BSA and TUDCA. 4-PBA, 4-Phenylbutyric acid; BSA, bovine serum albumin; Bax, Bcl-2-associated X protein; CC-3, cleaved caspase-3; CC-8, cleaved caspase-8; CC-9, cleaved caspase-9; CHOP, C/EBP homologous protein; DR5, death receptor-5; TUDCA, tauroursodeoxycholic acid.

Figure 4

Albumin overload induces cellular senescence and EMT. Panel (A) depicts the results of one out of three performed experiments. Albumin overload increased the expression of the cell cycle arrest inducers p21 (B) and p16 (C). In addition, it decreased the level of the cell proliferation marker Ki-67 (D) and enhanced the cellular senescence marker GLB-1 (E). RPTECs exposed to high albumin concentration overproduced IL-1β (F) and TGF-β1 (G). Exposure of RPTECs to high albumin concentration resulted in EMT as the level of α-SMA increased (H). TUDCA and 4-PBA ameliorated but did not eliminate all the changes above. * p < 0.05 vs. control; # p < 0.05 vs. RPTECs treated with 4-PBA; ^ p < 0.05 vs. RPTECs treated with TUDCA; + p < 0.05 vs. RPTECs exposed to BSA; & p < 0.05 vs. RPTECs exposed to BSA and 4-PBA; ! p < 0.05 vs. RPTECs exposed to BSA and TUDCA. 4-PBA, 4-Phenylbutyric acid; α-smooth muscle actin; BSA, bovine serum albumin; GLB-1, β-galactosidase; IL-1β, interleukin-1β; Ki-67, marker of proliferation Ki-67; p16, p16 INK4A; p21, p21 Waf1/Cip1; TGF-β1, transforming growth factor-β1; TUDCA, tauroursodeoxycholic acid.

Figure 5

NAC at a non-cytotoxic concentration prevents ROS upregulation, initiation of UPR and DDR, and eventually apoptosis, senescence, and EMT. LDH release assay revealed that NAC was not cytotoxic for the RPTECs at the used concentration (A). NAC completely abolished the rise of ROS levels in RPTECs exposed to high albumin concentration (B). Panel (C) depicts the results of one out of three performed experiments. NAC prevented UPR since it normalized the albumin overload-induced increase in the p-PERK level (D) without affecting total PERK (E). NAC prevented DDR as it normalized the albumin overload-induced increase in the γ-H2AX level (F). NAC prevented the harmful effects of apoptosis, senescence, and EMT. NAC normalized the albumin overload-induced upregulation of CC-3 (G), GLB-1 (H), and α-SMA (I). * p < 0.05 vs. control; # p < 0.05 vs. RPTECs treated with NAC; ^ p < 0.05 vs. RPTECs exposed to BSA; + p < 0.05 vs. RPTECs exposed to BSA and NAC. α-SMA, α-smooth muscle actin; γ-H2AX, phosphorylated at Ser139 histone H2AX; BSA, bovine serum albumin; CC-3, cleaved caspase-3; GLB-1, β-galactosidase; NAC, N-acetylcysteine; PERK, PKR-like ER kinase; ROS, reactive oxygen species.

Figure 6

In RPTECs, albumin overload triggers a UPR and DDR, resulting in apoptosis, senescence, and EMT. Albumin overload causes ROS overproduction. ROS induces protein modification triggering a UPR. In addition, ROS causes DNA damage eliciting a DDR. Both DDR and UPR result in p53 upregulation, which induces apoptosis through the extrinsic apoptotic pathway. Both DDR and UPR, through p53, upregulate p21, while p16 increases due to the UPR-dependent ATF-3 or ROS. Upregulation of the above cell cycle arrest inducers causes cellular senescence and a SASP. TGF-β1 overproduction because of the SASP may contribute to the albumin overload-induced EMT of the RPTECs. 4-HNE, 4-Hydroxynonenal; α-SMA, α-smooth muscle actin; γ-H2AX, phosphorylated at Ser139 histone H2AX; ATF-3, activating transcription factor-3; ATM, ataxia telangiectasia mutated kinase; CC-3, cleaved caspase-3; CC-8, cleaved caspase-8;, CC-9, cleaved caspase-9; DR5, death recetror-5; eIF2α, eukaryotic translation initiation factor-2α; GLB-1, β-galactosidase; IL-1β, interleukin-1β; Ki-67, marker of proliferation Ki-67; p16, p16 INK4A; p21, p21 Waf1/Cip1; p53, tumor suppressor p53; PERK, PKR-like ER kinase; ROS, reactive oxygen species; RPTEC, renal proximal tubular epithelial cell; SASP, senescence-associated secretory phenotype; TGF-β1, transforming growth factor-β1.