Expression of Serum GRP78 and CHOP in Endoplasmic Reticulum Stress Pathways of Chinese Type 2 Diabetic Kidney Disease Patients

Background: The kidney has a rich endoplasmic reticulum system. A close relationship exists between endoplasmic reticulum stress (ERS) and diabetic kidney disease (DKD). The current study aimed to investigate serum glucose-regulated protein 78 (GRP78) as well as CCAAT/enhancer binding protein homologous protein (CHOP) concentrations in type 2 diabetes mellitus (T2DM) Chinese patients, especially those with microalbuminuria. Methods: We evaluated the relationships between serum GRP78 or CHOP levels and DKD. We recruited 67 patients with T2DM and 63 control subjects. We determined serum GRP78 and CHOP concentrations by ELISA, collected anthropometric data, and measured biochemical parameters in a clinical laboratory. Results: Compared with control groups, Chinese T2DM patients showed decreased serum levels of GRP78 [0.21 (0.16–0.24) vs. 0.16 (0.16–0.19) ng/mL, p < 0.01] and CHOP [3.8 (3.0–5.5) vs. 5.5 (3.7–7.9) ng/mL, p < 0.01]. Reduction in GRP78 and CHOP serum levels was more pronounced in patients with more severe categories of microalbuminuria. Amounts of serum GRP78 correlated directly with serum fasting c-peptide, cystatin-c (cys-c), creatinine (Cr), blood urea nitrogen (BUN), and uric acid, and inversely with glomerular �ltration rates. Serum CHOP level was positively correlated with age, Cr, BUN, cys-c, urinary microalbumin/creatinine (UmALB/Cr), and eGFR. Serum GRP78 was predicted independently by Cr, BUN, serum uric acid, eGFR, and cys-c, while CHOP depended on age, Cr, BUN, serum uric acid, eGFR, UmALB/Cr, and cys-c. After controlling for confounding factors, GRP78 and CHOP expression was signi�cantly associated with DKD (binary logistic


Background
Diabetic kidney disease (DKD) represents an important health problem worldwide with millions of people affected.As a microvascular complication of diabetes mellitus (DM), it is responsible for substantial proportion of end-stage kidney disease cases.It is estimated that there are approximately 120 million people with chronic kidney disease in China [ [1]].DKD accounts for 30-47% of cases of end-stage renal disorders (ESRD) and is a major cause of death in DM patients [ [2]].However, the underlying pathophysiological mechanisms of DKD remain incompletely understood, hampering the development of new therapeutic approaches.Over the years, numerous basic and clinical studies have con rmed that advanced glycation end products (AGEs), oxidative stress, in ammation, as well as activation of protein kinases C, renin-angiotensin aldosterone system, and others have made valuable contributions to the pathogenesis and development of DKD.Among these, it is believed that increased production of reactive oxygen species (ROS) and subsequent oxidative stress contributes signi cantly to DKD development [ [3]].
In attempt to counteract numerous environmental stressors and preserve normal cell function, kidney cells in DM patients developed delicate signaling systems, such as a homeostatic pathway for regulating membrane structure and secretory activity of endoplasmic reticulum (ER) (unfolded protein response, UPR).It was reported that activation of UPR in kidneys of DM patients contributes to ER's functional restoration and preserved cell viability [ [4]].Growing evidence now suggests that endoplasmic reticulum stress (ERS) plays critical roles in the pathophysiological mechanisms of DKD.Studies have shown that changes in ER regulation of protein folding pathways cause ROS imbalance and increase their production, indirectly interfering with ER and redox balance [ [5]].
ER's main function in normal conditions is related to folding, modi cation and degradation of secretory binding proteins of the plasma membrane [[6, 7]].We know that disturbed homeostasis in DM due to various factors, such as ROS, high serum glucose, free fatty acids, etc. lead to ERS re ected in ER accumulation of unfolded proteins [[8]].Earlier studies showed that ERS plays a key role in diabetes [ [9][10][11][12][13]]. By identifying disease-causing mutations in epithelial-restricted genes, recent studies have indicated the importance of severe or prolonged ERS for multiple organs' degenerative diseases and brosis, including the pro-brosis role of UPR signaling in different cell types [ [14]].Many stressors encountered upon kidney injury may trigger ERS.In the kidney, despite ERS involvement in both acute and chronic histological damage, it may have nephroprotective effects and promote cellular adaptation [[15]].Yet, pathological ERS activation may lead to in ammatory response, cell apoptosis, and alteration in protective processes, such as autophagy and mammalian target of rapamycin complex (mTORC) activation [ [4]].However, the way ERS participates in the generation and development of DKD is still not fully understood.
Glucose-regulated protein 78 (GRP78) belongs to the family of heat-shock proteins (HSP70 family).It is also known as the immunoglobulin heavy chain binding protein (BiP).It is an ER lumen protein whose expression is induced during ERS and plays a novel protective role in preventing ERS-induced cell death [[16]].As an ER chaperone, GRP78 modulates the UPR signaling network.In conditions of ERS, it dissociates from protein kinase R-like ER kinase (PERK) and binds to unfolded or misfolded proteins [ [17]].
CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) is another essential player in ERSinduced apoptotic cell death [[18]].CHOP contains a C-terminal alkaline zinc nger (bZIP) domain and an N-terminal transcriptional activation threshold.The expression of CHOP can signi cantly affect cell survival.It is also known as growth arrest and DNA damage inducible gene 153 (GADD153) [[19]].Each of the three ERS pathways can induce C/EBP source protein CHOP -is a translocation factor unique to ERS [[19]].CHOP mainly exists in the cytoplasm, and its expression level is very low.When ERS is induced, the expression of CHOP increases substantially and is activated and translocated into the nucleus [ [20]].Overexpressed CHOP promotes cell cycle stagnation or apoptosis [ [21]], but CHOP can also protect cells from apoptosis [ [22]].
Acute kidney injury leads to perturbations of kidney cell pathways, which causes ER accumulation of unfolded/misfolded proteins and subsequent UPR or ERS.The cell fate in ERS depends on the balance between the UPR adaptive and apoptotic pathway [ [23]].The involvement of serum GRP78 and/or CHOP in the development of DKD through ERS pathway has not yet been elucidated.Although some observations have been made in animal studies, few have yet explored ERS in humans with DKD.Therefore, here we investigated the relationships of serum concentrations of GRP78 and CHOP in T2DM patients from China, particularly in patients with different severity categories of microalbuminuria, to test the hypothesis that ERS potently affects the pathophysiological mechanisms of DKD.

Subjects
We enrolled 67 T2DM patients hospitalized at Lianyungang No. 1 People's Hospital and 63 healthy patients from a medical examination center that were included as controls.T2DM was diagnosed as per American Diabetes Association diagnostic criteria from 2014 [ [24]]: fasting glucose of 7.0 mmol/L or higher, glycosylated hemoglobin of 6.5% or higher, or oral glucose tolerance test (OGTT) showing plasma glucose of 11.1 mmol/L or higher at 2 hours after glucose load.Based on cystatin-c levels, two groups were de ned, including Group A (Cys-c ≤ 1.03) and Group B (Cys-c > 1.03).Standard OGTT was also conducted in control group to con rm normal glucose tolerance.Histories of disease, smoking, and alcohol consumption were collected via a detailed questionnaire.The following were considered as exclusion: type 1 DM, secondary diabetes, pregnancy, thyroid diseases, endogenous or exogenous corticosteroid excess, acute or chronic virus hepatitis, malignant tumor, failure of major organs (such as heart, liver, kidneys), infection or in ammation.The study was performed in accordance with the Helsinki Declaration, and the Ethics Committee of our hospital approved the study.Each subject provided a written informed consent after understanding the study details.We followed the methods of Xing-bo Cheng et al 2017 [25].

Anthropometric data collection
Based on hospital case les, the data on body height, body weight, waist circumference (WC), and hip circumference (HC) were obtained.We calculated waist-to-hip ratio (WHR) by dividing WC by HC.After resting in a sitting position for 10 minutes, before blood pressure was measured using Omron electronic sphygmomanometer.The average of three measurements of blood pressure was calculated.

Indices of insulin secretion and insulin sensitivity/resistance
Insulin resistance status was assessed based on homeostasis model assessment of insulin resistance index (HOMA-IR) which was calculated as a product of fasting glucose (mmol/L) and fasting insulin (mIU/L), divided by 22.

Measurements of serum GRP78 and CHOP
After centrifuging of blood samples, serum samples were preserved at − 80 °C for further analyses.
Commercial ELISA kits (Cloud-Clone Corp., Wuhan, China) were used to determine serum concentrations of GRP78 and CHOP proteins, strictly complying with the instruction manual.The detection ranges of the GRP78 and CHOP assays were 0.312-20 ng/mL and 0.156-10 ng/mL, respectively, while minimum detectable doses were typically lower than 0.129 ng/mL (GRP78) and lower than 0.065 ng/mL (CHOP).
The interassay and intraassay coe cients of variation were < 12% and 10% for both proteins.

Serum GRP78 and CHOP concentrations
As shown in Fig. 1a, according to the cys-c, serum GRP78 level was signi cantly higher in DKD (p = 0.008).As shown in Fig. 1b, serum CHOP concentrations also showed signi cant differences (p = 0.011).
The biochemical and clinical parameters and of patients with DKD are shown in Table 2.

Correlations and regression analysis between serum GRP78 and CHOP concentrations and clinical parameters
Serum GRP78 level was negatively correlated with eGFR and positively correlated with fasting c-peptide, Cr, BUN, cys-c, and serum uric acid.Serum CHOP level was positively correlated with age, Cr, BUN, cys-c, UmALB/Cr, and eGFR (Tables 3-6).].High glucose may induce ERS in podocytes.ERS upregulates GRP78 expression, activates the CHOP pathway and caspase-12 pathway, and causes apoptosis of mouse podocytes, which may be related to the development process of DKD [ [30]].There is already evidence for the involvement of ERS-mediated apoptosis in development of diabetic complications in eyes and kidneys, but also in pathogenesis of non-diabetic neurodegenerative changes.For instance, a study in hippocampal neurons of diabetic mice induced by streptozocin (STZ) showed a reduced expression of GRP78 along with higher expression of the UPR-associated pro-apoptotic regulator CHOP [ [31]].Wu et al. have shown that GRP78 levels in renal tissue are higher than CHOP, JUK, and the caspase-12 pathway.The parallel relationship between expression and transcription of death signals suggests that excessive ERS promotes progressive damage of DKD by increasing apoptosis [[18]].Expression of nuclear transcription factor rBp65, CHOP, and GRP78 were increased in DN rats with myocardial infarction compared with control rats with myocardial infarction.In addition, the degree of podocyte damage caused by high-glucose-mediated ERS was more severe, which deformed the structure and function of the glomerulus [ [32]].Cao et al.. induced a DN model by unilateral nephrectomy combined with single STZ (65 mg/kg) injection intraperitoneally in rats.GRP78 was found by histochemical staining in diabetic rats compared with controls, and the expression levels of renal glomerular and tubular epithelial cells were upregulated[ [33]].Lindenmeye and colleagues con rmed that, compared with mild diabetes, mRNA expression of GRP78, oxyregulatory protein 150, and transcription molecule X-box binding protein-1(Xbp-1) of diabetic patients increased in the kidneys, indicating that ERS was stimulated in human DN [ [34]].These studies suggest that ERS is a central link in the development of a variety of systemic chronic metabolic diseases including T2DM, and it is also coupled with in ammatory responses, oxidative stress, autophagy, apoptosis, and other signaling pathways [ [35]].
In this study, the classic proteins of ERS, GRP78, and CHOP, were measured and compared with cys-c, urinary microalbumin, eGFR, and other indicators for prediction of DKD.We found higher serum concentrations of GRP78 and CHOP in T2DM group than in controls (p < 0.01).GRP78 and CHOP concentrations were signi cantly increased during DKD (GRP78: p = 0.008; CHOP: p = 0.011).Urinary micro albumin creatinine ratio (UACR) or eGFR are usually chosen as a standard, but in this study cystatin-c (Cys-c) was used as a grouping indicator.Increased UACR and decreased eGFR are closely related to higher risk of adverse cardiovascular events and death.UmALB/Cr is usually used as the evaluation index of DKD, but UACR measures the in uence of various factors, e.g., hypertension, heart failure, infection, hyperglycemia.Microalbuminuria as a marker of DKD progression has been challenged [[36, 37]].Early DKD is often associated with eGFR, a phenomenon known as high glomerular hyper ltration.A cross-sectional survey showed that some diabetic patients did not have abnormal urinary albumin excretion but had decreased eGFR [[38, 39]].Calculation of eGFR requires information on patient age and sex, as well as serum Cr level.When a patient's eGFR < 60 mL, a decrease in eGFR can be diagnosed.However, the eGFR value may uctuate and should be reviewed when a decrease occurs to determine the DKD stage.eGFR decline is closely associated with a higher cardiovascular risk and risk of death.Recent studies from China have shown that even mild eGFR decline can increase cardiovascular risk [ [40]].Cys-c, a low molecular weight protein that can be produced by all nucleated cells in the body, was not glycosylated, and its production rate is constant and is not affected by the patient's age, gender, etc.Therefore, serum Cys-c level mainly depends on the ltration rate of the glomerulus, which, together with urine α-microglobulin, IgG or IgM, and IV collagen, are sensitive indicators for early diagnosis of DKD [ [41]].In this study, we therefore used Cys-c as a grouping indicator.
Hitherto, the mechanisms behind lower levels in DKD have not been clari ed.Notably, GRP78 and CHOP are closely related to DKD as animal studies have shown; herein, we demonstrated that GRP78 and CHOP in human serum correlated with DKD.Hence, we assumed a possible variation in levels of GRP78 between the subgroups divided by cys-c.Together with previous studies, the results reported herein suggest that GRP78 and CHOP levels may have potential to be used as biomarkers of the DKD risk.
Nevertheless, this study had a few limitations.Based on cross-sectional design of the study the potential in uence of increased GRP78 and CHOP levels on the development of T2DM could not be evaluated, and further studies are warranted to further clarify this issue.The strength of our conclusions and wide extrapolation to the general population is limited by a relatively small sample size and single center study design.In addition, the study encompassed single measurements of fasting serum GRP78 and CHOP levels.That approach was based on limited funds and does not re ect any time-dependent uctuations in GRP78 and CHOP levels, which is of particular interest after macronutrient consumption.Therefore, further studies are still necessary.In summary, GRP78 and CHOP serum levels are increased in T2DM patients from China.Furthermore, DKD patients had greater reductions in GRP78 and CHOP levels.

Conclusions
Here we showed evidence for the importance of ERS as well as associations of GRP78 and CHOP with DKD, which may lead to new therapeutic directions for renal complications of diabetes.With consideration of the roles of GRP78 and CHOP and involvement of ERS in other diabetic microvascular complications, it will be needed to further analyze the exact roles of ERS/UPR in DM-related complications, as well as evaluate interactions of ERS and biochemical parameters and their relationship with DKD.Our data highlight the possibility of using serum indicators of ERS as biomarkers of DKD.Therefore, with further study to elucidate the underlying mechanisms behind these effects, there may be a chance to improve treatment of DKD through improved regulation of ERS.

Figure 2 (
Figure 2 power analysis showed that the effect size for GRP78 concentrations was 0.686 [95% CI 0.558-0.813]and for CHOP concentrations was 0.670 [95% CI 0.524-0.816]. of factors independently associated with GRP78 and CHOP and control for covariates, we performed multiple stepwise regression.Data not tting to normal distribution underwent log-transformation (log-GRP78, CHOP) before correlation and regression analyses.A receiver operating characteristic (ROC) curve analysis was applied to determine the area under curve and cutoff value for potential of serum GRP78 and CHOP levels as biomarkers for DKD.A two-tailed p value below 0.05 was considered as signi cant.In addition, the

Table 1
shows the clinical parameters of the 67 T2DM and 63 health control patients.The groups did not differ in age, sex, and BMI.Serum GRP78 and CHOP concentrations were signi cantly lower (p < 0.01) in T2DM than in control group.WC waist circumference; WHR waist-hip ratio; SBP systolic blood pressure; DBP diastolic blood pressure; QUICKI: Quantitative Insulin Check Index.The enumeration data were compared with χ 2 test.a:Data normally distributed are shown as mean ± SD.Independent sample T test was performed.

Table 2
General clinical and laboratory parameters in patients with DKD BMI, body mass index; WC, waist circumference; WHR, waist-hip ratio; SBP, systolic blood pressure; DBP, diastolic blood pressure; QUICKI: Quantitative Insulin Check Index.The enumeration data were compared with χ 2 test.a: Data normally distributed are shown as mean ± SD.Independent sample T test was performed.b:Data with skewed distribution are shown as median (IQR, 25th-75th).Mann-Whitney U test was performed.

Table 3
Bivariate correlation between GRP78 levels and other variables.