Targeting Endoplasmic Reticulum for Novel Therapeutics and Monitoring in Acute Kidney Injury

Background: Endoplasmic reticulum (ER) stress response is a conservative mechanism involving a complex network of different molecular branches to determine cell fate through specific transcription factors and downstream executors. Emerging evidence shows that ER stress is implicated in the occurrence and progression of acute kidney injury (AKI) in different animal models and human patients. However, there is still a lack of therapeutics targeting the ER in AKI. Summary: Several therapeutic chemicals, including a compound that induces activating transcription factor 6 (ATF6) and chemical chaperones, have been developed to target the ER in the treatment of AKI. Meanwhile, ER stress-inducible secreted proteins, mesencephalic astrocyte-derived neurotrophic factor (MANF), and cysteine-rich with EGF-like domains 2 (CRELD2) could serve as potential ER stress biomarkers in the early diagnosis and treatment response monitoring of human patients with AKI. Key Messages: Experimental and clinical evidence suggests the critical role of ER in the pathogenesis and progression of AKI, and ER is a novel target in AKI therapy.

© 2022 S. Karger AG, Basel

Introduction

Endoplasmic reticulum (ER) is a continuous membrane organelle that controls protein synthesis, folding, and degradation. The disruption of ER proteostasis leads to an accumulation of unfolded and misfolded proteins in the ER, which activates the unfolded protein response (UPR) or ER stress. Several common features involved in human diseases, including hypoxia, oxidative stress, and glucose deprivation, can activate ER stress. Emerging evidence has also demonstrated that ER stress or the derangement of ER proteostasis contributes to the development and progression of glomerular and tubular diseases [1].

Protein folding in the ER occurs with the help of ER-resident molecular chaperones and enzymes, such as immunoglobulin binding protein (BiP), which is also a key sensor linked to the regulation of UPR [2]. The UPR is initiated by three protein sensors, including PKR-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6) [3]. PERK phosphorylates eukaryotic initiation factor 2α (eIF2α), leading to attenuation of protein translation and activation of the transcription factor ATF4. IRE1 is a dual-activity enzyme with an endoribonuclease and a kinase domain. IRE1-mediated cleavage of x-box binding protein 1 (XBP1) mRNA leads to a spliced form of XBP1 mRNA, encoding a potent transcription activator (XBP1s). IRE1 also recruits tumor necrosis factor receptor associated factor 2 (TRAF2) and apoptosis signal-regulating kinase 1 (ASK1), thus allowing it to signal to c-Jun N-terminal kinases (JNK) and cause apoptosis. ATF6 promotes transcription of chaperones after being cleaved by site-1 and site-2 proteases in Golgi from a 90 kD full-sized protein to a 50 kD N-terminus fragment. The consequence of the UPR is to reduce the translation of misfolded proteins, to promote the transcription of the UPR target genes, to increase ER-associated degradation, and to activate ER stress-mediated apoptosis, which involves caspase-12, JNK, and C/EBP homologous protein (CHOP). Procaspase-12 localized to the ER is activated by ER calcium depletion. CHOP transcription is induced by ATF4, XBP1s, and p50 ATF6 under ER stress conditions. CHOP decreases mitochondrial antiapoptotic proteins, including Bcl-2, Bcl-xl, and Mcl-1, and increases mitochondrial pro-apoptotic proteins, such as BIM. CHOP also promotes transcription of extrinsic apoptosis initiators, death receptors 4 and 5, through interaction with JUN transcription factor, which can be phosphorylated by protein kinase JNK. In this mini-review, we summarize the experimental therapies that target the ER in the treatment of acute kidney injury (AKI) and the discovery of ER stress biomarkers in AKI.

ER Stress in AKI

ER stress activation in AKI can be driven by various factors, including renal ischemic/reperfusion (I/R) injury that can occur following renal vascular obstruction, cardiac arrest, and kidney transplantation [4]. Multiple ER stress markers have been investigated in the AKI animal models and renal biopsies of AKI patients. BiP and p-PERK levels are significantly increased in the tubular compartment of patients with acute tubular necrosis and acute interstitial nephropathy compared to those in normal controls. In addition, the intensity of CHOP staining in the tubules is inversely correlated with the renal function of these patients, as indicated by peak eGFR and peak serum creatinine (sCr) [5]. In another study, elevated levels of p-PERK and CHOP are observed in the kidney post unilateral I/R surgery in the animal model [6]. The phosphorylated eIF2α, a p-PERK downstream target, is also increased in the I/R-injured kidneys and the greatest staining of phosphorylated eIF2α is localized at the corticomedullary junction [7]. Furthermore, XBP1s is strongly and exclusively upregulated in renal tubular cells of sepsis-mediated AKI mouse models induced by lipopolysaccharide or cecal ligation and puncture [8].

ER-Targeted Experimental Treatment in AKI

Currently, there is no treatment for AKI. Thus, there is a critical need to develop mechanism-based novel therapies. 147 is a newly developed chemical compound that specifically activates ATF6. The effect of 147 was examined when the kidney was subjected to ischemic injury via unilateral renal pedicle clamping for 30 min. Administration of 147 at the time of reperfusion markedly decreased kidney infarction size and reduced sCr level at 24 h after I/R. Mechanistically, following 147 administration, expression of ATF6 target genes, BiP and catalase, a prominent antioxidant gene, was significantly increased [9]. Meanwhile, chemical chaperones were investigated in the AKI animal models. Pretreatment with tauroursodeoxycholic acid (250 mg/kg) 30 min before I/R was shown to attenuate tubular damage in ischemic AKI and to inhibit ER stress-induced activation of apoptotic molecules, including CHOP and caspase-12 [10]. Additionally, when mice were pretreated with another chemical chaperone 4-phenylbutyrate (4-PBA) (1 g/kg/day) for 7 days, followed by 3 days of co-treatment of 4-PBA and tunicamycin (TM), an ER stress inducer, 4-PBA can inhibit TM-caused AKI [11]. Moreover, 4-PBA significantly mitigated TM-induced CHOP upregulation and protected the ultrastructure of proximal tubules against TM-induced tubular damage [11].

ER Stress-Induced Biomarkers in AKI

It is imperative to develop noninvasive biomarkers for detecting tubular ER stress at the early stage of AKI, which is critical for early diagnosis and therapeutic intervention, as well as treatment response monitoring. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a novel, ER stress-regulated secreted protein. We find that MANF is induced and secreted in response to ER stress in AKI. In addition, for the first time, we have discovered MANF as a urinary ER stress biomarker in the AKI mouse models [12].

We have further identified cysteine-rich with EGF-like domains 2 (CRELD2) as a urinary ER stress biomarker in both AKI mouse models and human patients. In TM- and I/R-induced AKI mouse models, we show that compared to controls, CRELD2 level is significantly elevated in the kidneys under ER stress and in the urine early in the disease course, preceding clinical or histologic manifestation of AKI [13]. We further conduct a pilot case-control study of 23 pediatric patients with normal baseline renal function, who has received cardiopulmonary bypass surgery for their congenital cardiac disease to evaluate whether early postoperative elevation of urine CRELD2 is associated with a higher risk of severe AKI postoperatively. In this cohort, preoperative urinary CRELD2 excretion is absent in the 21 participants. Postoperatively, in patients with severe AKI, as defined by either receipt of acute dialysis or postoperative doubling of sCr during hospital stay, urine CRELD2 concentration peaks at the first collection, which is the postoperative 0–6 h time point, and declines over the first 3 postoperative days (Fig. 1a). In addition, ELISA shows that the first postoperative urine CRELD2/Cr ratio is significantly higher in the 8 patients with severe AKI than in the 15 non-AKI patients (Fig. 1b). No difference in sCr concentrations on day 1 was observed between the two groups (Fig. 1c). Finally, 66% of patients with detectable urine CRELD2 levels versus 14% of patients with undetectable levels within the first postoperative 6 h develop severe AKI, indicating that an early postoperative increase of urinary CRELD2 is strongly correlated with severe AKI after pediatric cardiac surgery [13]. Urine CRELD2/Cr values at the postoperative 0–6 h are also moderately associated with longer hospital stay and duration of mechanical ventilation (Fig. 1d).

Fig. 1.

Early post-op measure of urine CRELD2 can stratify pediatric patients for developing severe AKI and other adverse outcomes after CPB surgery. AKI in a–c denotes severe AKI. a Representative pre-op and post-op WB analysis of CRELD2 (arrow) in crude urine specimens obtained from non-AKI and severe AKI patients at the indicated time points. Time points 0–6 h, 6–12 h, 12–18 h, day 2, and day 3 are the sample collection times after the CPB surgery. The urinary excretion of CRELD2 was normalized to 2 µg of urine Cr excretion. b ELISA assay of urine CRELD2 concentrations normalized to urine Cr excretion in unconcentrated urine specimen before surgery and at the first post-op collection (0–6 h) in severe AKI (n = 8) and non-AKI (n = 15) patients. **p < 0.01 by Wilcoxon rank-sum test. c Pre- and post-op sCr measurements in severe AKI (n = 8) and non-AKI (n = 15) patients. NS, not significant by Wilcoxon rank-sum test. d Length of stay in hospital and length of ventilation in patients with undetectable and detectable urine levels of CRELD2 within post-op 6 h, respectively. Figure 1 was originally published in JCI Insight [13] used with permission. AKI, acute kidney injury; CRELD2, cysteine-rich with EGF-like domains 2; CPB, cardiopulmonary bypass; pre-op, preoperative; post-op, postoperative.

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To detect low abundant protein biomarkers, we have developed an ultrasensitive, plasmon-enhanced fluorescence-linked immunosorbent assay (p-FLISA), which is based on a newly invented ultrabright fluorescent nanoconstruct, named plasmonic fluor [14]. By employing p-FLISA, we have identified secreted BiP as a urinary ER stress biomarker in tubular ER stress-induced autosomal dominant tubulointerstitial kidney disease [15]. This novel technology will enable us to identify more biomarkers in AKI patients.

Conclusion

As ER stress has emerged as a signaling platform underlying the pathogenesis of AKI, there is an urgent need to develop ER-targeted therapeutics and discover more ER stress biomarkers at the early stage of AKI in human patients.

Acknowledgment

Y.M.C. is a member of the Washington University Diabetes Research Center (supported by NIH P30 DK020579), the Washington University Musculoskeletal Research Center (supported by NIH P30AR057235), and the Washington University Institute of Clinical and Translational Sciences (UL1 TR000448).

Conflict of Interest Statement

A patent (US 10,156,564) entitled “methods of detecting biomarkers of ER stress-associated kidney diseases” with Chen Y. and Kim Y. being inventors was issued by the US Patent and Trademark Office on December 18, 2018.

Funding Sources

Y.M.C. is supported by NIH Grants R01 DK105056, R03DK106451, and K08DK089015, the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program under Award W81XWH-19-1-0320, George M. O’Brien Kidney Research Core Center (NU GoKidney, NIH P30 DK114857; UAB/UCSD, NIH P30 DK079337), Office of the Vice Chancellor for Research (OVCR) Seed Grant, Washington University in St. Louis, Mallinckrodt Challenge Grant, Washington University Center for Drug Discovery, Investigator Matching Micro Grant and Faculty Scholar Award (MD-FR-2013-336) from the Children’s Discovery Institute of Washington University and St. Louis Children’s Hospital.

Author Contributions

Chuang Li and Ying Maggie Chen wrote the manuscript, and Siva Krothapalli and Ying Maggie Chen edited the manuscript. All the authors have read and agreed to the published version of the manuscript.

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