Recent studies suggest that development of diabetic nephropathy (DN) is partly caused by ER dysfunction [[29]]. 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 confirmed 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 inflammatory 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 significantly 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 influence 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 hyperfiltration. 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 fluctuate 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 filtration 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 clarified. 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 influence 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 reflect any time-dependent fluctuations 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.