Routes of Albumin Overload Toxicity in Renal Tubular Epithelial Cells

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.


Introduction
Chronic kidney disease (CKD) affects more than 10% of the population worldwide and is one of the leading causes of death [1]. Consequently, there is an urgent need to apply the existent and discover new preventive and therapeutic measures. Typically, the glomerular filtration rate was used for CKD definition and classification. Still, since 2011, an albuminuria stage was added as the presence and quantity of albuminuria can predict the risk for CKD progression to end-stage renal disease and mortality [2].
Normally, a relatively small quantity of albumin passes the glomerular filtration barrier. Most of it is reabsorbed by the renal proximal tubular epithelial cells (RPTECs) and either catabolized or transcytosed. Thus, albuminuria occurs in kidney diseases that affect the proximal tubule, and, more frequently, the glomeruli [3,4].
Blockers of the renin-angiotensin system (RAS), and, more recently, sodium-glucose co-transporter-2 (SGLT-2) inhibitors are used for decreasing CKD progression. These agents reduce intraglomerular pressure, which may have a direct beneficial effect on the glomeruli, but, by doing so, they also reduce albuminuria [5][6][7]. The latter may play a significant role in the beneficial impact of RAS blockers and SGLT-2 inhibitors. Besides being a kidney, especially glomerular, disease severity marker, albuminuria is toxic to the renal tubules, contributing to CKD progression.
Regarding the mechanisms involved in albumin toxicity in RPTECs, most experimental studies suggest that albumin overload elicits an unfolded protein response (UPR) and endoplasmic reticulum stress (ERS) in RPTECs [8][9][10][11]. This is interesting since various anti-ERS agents are under development, and some of them have already been approved for clinical use, albeit in clinical entities other than CKD [12,13]. On the other hand, a recent study showed that albumin overload induces DNA damage in RPTECs, and the subsequent DNA damage response (DDR) results in cellular senescence [14].
Since albuminuria is a significant prognosticator of renal outcome in patients with CKD [2], a clarification of the exact mechanisms involved in albumin-induced toxicity in RPTECs is of particular interest as it may identify specific targets for new therapeutic agents. For this purpose, in the current study, we evaluated in RPTEC cultures whether albumin induces a UPR or a DDR, as well as the downstream pathways that may lead to the deleterious outcomes of apoptosis, senescence, or epithelial-to-mesenchymal transition (EMT) [15,16]. With the introduction of anti-ERS agents into clinical practice [12,13], it would be beneficial to elucidate the significance of this pathway, as well as the role of DDR, in the toxicity of albumin overload in RPTECs. This clarification would provide a more accurate understanding of the potential impact of anti-ERS agents on patients with albuminuria.
RPTEC apoptosis contributes to CKD progression obviously by decreasing the functional renal tissue [17]. Cellular senescence is a state of permanent cell cycle arrest. A large proportion of senescent RPTECs precludes the regeneration of apoptotic RPTECs, contributing to the progression of CKD. In addition, senescent cells acquire a senescence-associated secretory phenotype and produce various proinflammatory and profibrotic cytokines, aggravating renal damage [18]. The profibrotic cytokines may act in adjacent cells and transform them into myofibroblasts inducing tubulointerstitial fibrosis, in which all longlasting kidney diseases culminate [19]. In the case of RPTECs, EMT may take place [20]. Interestingly, even in various glomerular diseases, the degree of tubulointerstitial fibrosis is the best prognosticator of renal outcome [19,21].

Albumin Overload Triggers ROS Production and UPR
Lactate dehydrogenase (LDH) release assay showed that albumin overload does not induce cell necrosis. In addition, the anti-ERS agents tauroursodeoxycholic acid (TUDCA) and 4-Phenylbutyric acid (4-PBA) were not cytotoxic at the used concentrations ( Figure 1A).
High albumin concentration induced a reactive oxygen species (ROS) burst in RPTECs. Neither TUDCA nor 4-PBA affected albumin overload-induced ROS production ( Figure 1B). As a result of oxidative stress, protein modification occurred as the level of 4-Hydroxynonenal (4-HNE)-modified proteins increased. The anti-ERS agents partially decreased protein modification ( Figure 1C,D).
Protein modification in RPTECs exposed to a high albumin concentration triggered a UPR since the phosphorylated PKR-like ER kinase (p-PERK) level increased ( Figure 1C,E), the level of its substrate phosphorylated eukaryotic translation initiation factor-2α (p-eIF2α) was enhanced ( Figure 1C,G), and the activating transcription factor-3 (ATF3) level was upregulated ( Figure 1C,I). TUDCA and 4-PBA were ameliorated but did not eliminate all the above changes.

Albumin Overload Induces Cellular Senescence and EMT
Albumin overload increased the expression of the cell cycle arrest inducers p21 ( Figure 4A,B) and p16 ( Figure 4A,C). In addition, it decreased the level of the cell proliferation marker Ki-67 ( Figure 4A,D) and enhanced the cellular senescence marker βgalactosidase (GLB-1) ( Figure 4A,E). TUDCA and 4-PBA ameliorated but did not eliminate all the aforementioned albumin-induced changes.
The exposure of RPTECs to high albumin concentration resulted in EMT as the level of α-smooth muscle actin (α-SMA) increased ( Figure 4A,H). TUDCA and 4-PBA ameliorated the albumin-induced α-SMA upregulation.
NAC prevented UPR and DDR induced by the high albumin concentration since it normalized the albumin-induced increase in the p-PERK ( Figure 5C,D) and γ-H2AX levels ( Figure 5C,F), respectively.

Discussion
Albuminuria frequently accompanies CKD. The presence, and the greater the degree of albuminuria, the worse the renal outcome [2]. Besides being a marker of the severity of kidney, particularly glomerular, disease, albuminuria exerts a direct toxic effect on RPTECs. The latter is significant because almost all long-lasting kidney diseases culminate in tubulointerstitial fibrosis [19]. The degree of the latter is the best pathological marker for predicting CKD progression to ESRD [19,21].
In this study, we evaluated the routes of albumin overload toxicity in RPTECs. Using a cell culture system, we showed that albumin overload induces ROS production in RPTECs. It is known that albumin overload causes ROS production, but the exact mechanism is not well established [9,22]. Generally, lysosomal overload induces ROS production with an unclarified mechanism [23]. Since most studies attribute albumin toxicity in RPTECs to ERS [8][9][10][11], we used two well-established ERS inhibitors, TUDCA and 4-PBA [15]. Both compounds inhibit ERS due to their chaperoning activity [24,25], and experimental studies have confirmed that they protect RPTECs from ERS [26][27][28][29]. The fact that these substances are already in clinical use, 4-PBA to treat congenital diseases in the urea cycle [30], and TUDCA for treating primary biliary cholangitis [31], makes them particularly appealing for further investigation since we already know much about their pharmacologic properties and side effects. Currently, both medications are under investigation as anti-ERS agents for the treatment of various diseases [24,30], and recently a combination of them has been approved for the treatment of amyotrophic lateral sclerosis [13]. LDH release assay indicated that these anti-ERS substances are not toxic at the concentrations used in our experiments. Another interesting result from the LDH release assay was the absence of cell necrosis in RPTECs exposed to high albumin concentrations. On the contrary, other conditions that trigger ROS production, such as anoxia-reoxygenation, are known to induce ferroptotic cell necrosis [32]. Neither TUDCA nor 4-PBA affected ROS production. In RPTECs exposed to albumin, oxidative stress-induced protein modification was assessed by the level of 4-HNE-modified proteins. Both TUDCA and 4-PBA decreased to a great extent the level of 4-HNE-modified proteins. Protein modification may alter protein conformation resulting in UPR. Indeed, albumin overload induces a UPR assessed by the level of PERK phosphorylation, an early event in the UPR cascade. Activated p-PERK phosphorylates eIF2a, altering the translational program of the cell. The above results in the upregulation of the transcription factor ATF3 [12,15,33], which has also been detected in our experiments. TUDCA and 4-PBA ameliorated the p-PERK, p-eIF2a, and ATF3 upregulation.
Although most studies focus on albumin-induced RPTEC toxicity through the UPR pathway, at least one suggests that a DDR is elicited and may lead to cellular senescence [14]. Indeed, our experiments showed that albumin-induced ROS overproduction causes DNA damage, assessed by the level of γ-H2AX. Subsequently, DDR starts as ATM is phosphorylated. The latter activates ATM, which phosphorylates p53 [34]. Phosphorylated p53 dissociates with E3 ubiquitin-protein ligase Mouse double minute 2 homolog (Mdm2), which protects p53 from degradation by the proteasome, increasing its level [35]. Both TUDCA and 4-PBA lessen, to some extent, the above changes, possibly due to the known interplay between UPR and DDR [36]. However, an additional, unestablished pharmacological action of these compounds cannot be excluded.
Apoptosis is a possible deleterious outcome of RPTEC exposure to high albumin concentration. Our results showed that albumin overload induces apoptosis in RPTECs assessed by the level of activated cleaved caspase-3 in which all the apoptotic pathways converge [37]. Then we evaluated which of the two apoptotic pathways, the intrinsic or the extrinsic, are triggered by the high albumin concentration. Surprisingly, although albumin overload led to p53 increase-which induces the transcription of many proapoptotic genes, including Bax [35], and UPR upregulates CHOP, which does the same [38,39]-we did not find increased levels of CHOP and Bax in RPTECs exposed to albumin. In addition, the level of activated cleaved caspase-9, the signature caspase of the intrinsic apoptotic pathway, remained unaffected [37]. On the contrary, we found that albumin overload triggers the extrinsic apoptotic path since the level of activated cleaved caspase-8 was upregulated [37]. The latter is likely the result of DR5 upregulation, a known target of the p53 transcription factor [40]. As noted, albumin increased p53 through the DDR [35,41]. UPR can also upregulate p53 since ATF3 interacts with p53 preventing its degradation [41,42]. The latter may explain the observed beneficial effect of TUDCA and 4-PBA in ameliorating, to some extent, apoptosis.
Another deleterious outcome that cells can encounter under stress conditions is senescence. Senescent cells enter a permanent cell cycle arrest condition, and, in addition, acquire a senescence-associated secretory phenotype, secreting proinflammatory and profibrotic cytokines and affecting the adjustment cells. In the case of CKD, senescent cells cannot dedifferentiate and proliferate to replace apoptotic cells. In addition, they secrete proinflammatory cytokines, aggravating inflammation and accelerating kidney injury. Finally, the profibrotic cytokines accelerate tubulointerstitial fibrosis, in which most kidney diseases culminate [18,43]. In our experiments, albumin overload caused RPTEC senescence. It increased p21 and p16, critical cell cycle arrest factors, and senescence markers. The upregulation of p21 can be attributed to the p53 increase since the latter transcribes the p21 gene [35,44]. The rise of p16 is the result of ATF3 upregulation [45,46]. In addition, oxidative stress upregulates p16 through a p38 stress-activated protein kinase-dependent pathway [47]. High albumin concentration reduced RPTEC proliferation, assessed by the cell proliferation marker Ki-67 [48,49], and increased the typical cellular senescence marker GLB-1 [48,50]. In addition, RPTECs acquired a senescence-associated secretory phenotype as they overproduced IL-1β and TGF-β1. TUDCA and 4-PBA ameliorated, but did not eliminate, the above albumin-induced changes.
As noted, RPTECs exposed to high albumin concentrations overproduce TGF-β1, the archetypical profibrotic cytokine. It is likely that TGF-β1 upregulation, acting in an autocrine manner, induced EMT, assessed by the α-SMA expression in RPTECs [20]. Thus, albumin overload by inducing EMT favors renal fibrosis. Certainly, the same profibrotic cytokine acting in a paracrine manner may favor fibrosis further by promoting the transformation of regional fibroblasts to myofibroblasts or the mesenchymal transition of endothelial cells or pericytes [20,51]. Again, TUDCA and 4-PBA reduced, but did not eliminate, the albumin-induced EMT.
To confirm whether albumin-induced UPR and DDR are the major pathways that induce cellular apoptosis, senescence, and EMT in RPTECs, we repeated the experiments using the potent antioxidant NAC [52]. NAC was used in a high but not toxic concentration, as portrayed by the LDH release assay. Indeed, in RPTECs exposed to high albumin concentration, NAC normalized ROS levels and prevented UPR and DDR, assessed by p-PERK and γ-H2AX levels, respectively. Consequently, NAC protected RPTECs from albumin-induced apoptosis, evaluated by cleaved caspase-3, senescence assessed by GLB-1, and EMT evaluated by α-SMA.
A limitation of our study lies in its in vitro nature. However, the strict conditions of our experimental system allowed us to evaluate the effects of high albumin concentration in isolated RPTECs, excluding other confounding factors. For instance, hemodynamic changes, inflammation, or high glucose concentration that characterize various CKD experimental models might trigger similar pathways to those induced by high albumin concentration, making the study of the latter problematic. Certainly, the simultaneous assessment of markers associated with DDR, ERS, apoptosis, senescence, and EMT in clinical samples obtained from patients with albuminuria would be highly intriguing.
Our results are depicted in Figure 6 and support that albumin overload causes cellular apoptosis, senescence, and EMT in RPTECs by triggering both UPR and DDR. Promising anti-ERS factors, such as TUDCA and 4-PBA, may be beneficial but cannot eliminate the albumin-induced deleterious effects because DDR also occurs. Research on factors that suppress ROS overproduction due to exposure to high albumin concentration may result in finding more effective agents that are capable of halting both UPR and DDR.
concentration, as portrayed by the LDH release assay. Indeed, in RPTECs exposed to high albumin concentration, NAC normalized ROS levels and prevented UPR and DDR, assessed by p-PERK and γ-H2AX levels, respectively. Consequently, NAC protected RPT-ECs from albumin-induced apoptosis, evaluated by cleaved caspase-3, senescence assessed by GLB-1, and EMT evaluated by α-SMA.
A limitation of our study lies in its in vitro nature. However, the strict conditions of our experimental system allowed us to evaluate the effects of high albumin concentration in isolated RPTECs, excluding other confounding factors. For instance, hemodynamic changes, inflammation, or high glucose concentration that characterize various CKD experimental models might trigger similar pathways to those induced by high albumin concentration, making the study of the latter problematic. Certainly, the simultaneous assessment of markers associated with DDR, ERS, apoptosis, senescence, and EMT in clinical samples obtained from patients with albuminuria would be highly intriguing.

Detection of Cell Necrosis, ROS Production, IL-1β, and TGF-β1
Cell necrosis due to exposure to high albumin concentration and the toxicity of the used compounds was assessed colorimetrically with lactate dehydrogenase (LDH) release assay in RPTECs cultured in 96-well plates. The Cytotox Non-Radioactive Cytotoxic Assay kit (Promega Corporation, Madison, WI, USA) was used, and cytotoxicity was calculated using the equation Cytotoxicity (%) = (LDH in the supernatant: Total LDH) × 100. These experiments were repeated three times.
Reactive oxygen species (ROS) production was evaluated in RPTECs cultured in 96-well plates. Following a 24-h period of cell culture, RPTECs were treated with 5 µM of the fluorogenic probe CellROX Deep Red Reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) for 30 min at 37 • C. Subsequently, the RPTECs were rinsed with phosphatebuffered saline (Sigma-Aldrich; Merck Millipore), and the fluorescence signal intensity was measured using an EnSpire Multimode Plate Reader (PerkinElmer, Waltham, MA, USA). These experiments were conducted in triplicate.
Interleukin-1β (IL-1β) and transforming growth factor-β1 (TGF-β1) were measured in the supernatant of RPTECs cultured in 6-well plates. For IL-1β, the Hunan IL-1β PLATINUM ELISA kit (ca. no. BMS224/2, Bender MedSystems, Vienna, Austria) with a sensitivity of 0.3 pg/mL was used. The concentration of TGF-β1 was quantified using the Human TGF-beta-1 ELISA Kit (AssayPro, St. Charles, MO, USA). This kit has a detection range of 31-2000 pg/mL. ELISA measurements were conducted using the EnSpire Multimode Plate Reader (Perkin Elmer). The experiments were replicated three times.

Statistical Analysis
Statistical analysis was performed using IBM SPSS Statistics for Windows, Version 26 (IBM Corp., Armonk, NY, USA). The one-way analysis of variance (ANOVA) was used to compare means. Error bars represent the standard error of means, and p-values less than 0.05 were considered statistically significant. The GraphPad Prism, Version 9 (GraphPad Software, Boston, MA, USA) was used for the graphical presentation of the results. Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
Institutional Review Board Statement: Not applicable. The study did not involve human participants or animals.
Informed Consent Statement: Not applicable. The study did not involve humans.

Data Availability Statement:
The analyzed datasets generated during the study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.