Endoplasmic reticulum (ER) stress plays a significant role in the progression of diabetic kidney disease (DKD), and dipeptidyl peptidase-4 (DPP4) inhibitors are widely used antihyperglycemic agents, exerting renal beneficial effects in DKD. Here, we investigated the role of DPP4 inhibitor Sitagliptin (Sita) in ER homeostasis in the kidneys of diabetic DBA2/J (D2) mice and in albumin-stimulated HK-2 cells.
ER stress was observed both
The results suggest that a novel mechanism links the DPP4 enzyme to ER stress during tubular injury in DKD and highlight that SIRT1 may be a potential target for managing DKD.
- diabetic kidney disease
- ER stress
- tubular injury
Diabetic kidney disease (DKD) has become the leading cause of chronic kidney disease (CKD) . Among the changes in the progression of DKD, renal tubular injury plays a critical role, which is more closely related to renal outcomes during end-stage renal disease (ESRD), presenting tubulointerstitial compartments with intrarenal vasculature thickening and hyalinization [2, 3]. Although the pathogenetic mechanism of diabetic tubular injury remains unclear, several studies have validated that cellular apoptosis largely contributes to its development [4, 5].
Accumulating evidence indicates that endoplasmic reticulum (ER) stress could be induced by several factors, such as hypoxia, hyperglycemia, reactive oxygen species (ROS), and proteinuria, which accelerate the progression of DKD. . In DKD, a variety of stimulations break ER homeostasis, subsequently inducing unfolded protein response (UPR), and eventually activating the cell death pathway [6, 7].
Glucose-regulated protein of 78 kDa (GRP78) serves as a central regulator of three main UPR sensors, namely, activating transcription factor (ATF6), inositol-requiring enzyme (IRE)-1α, and protein kinase RNA-like ER kinase (PERK), which initiate the UPR signaling pathway under ER stress . Several past studies have shown that ER stress contributes to the development of CKD and DKD, while inhibition of the ER stress could restore the progression of various renal diseases [9, 10]. In our past studies, we also found that inhibition of ER stress by tauroursodeoxycholic acid (TUDCA) significantly alleviated renal tubular injury and ER stress-associated cellular apoptosis in diabetic db/db mice . It is likely that elevated ER stress processes contribute to the renal functional deterioration and tubular injury that occur in the context of DKD, although the precise mechanism underlying the disruption of these processes remains unclear.
Dipeptidyl peptidase-4 (DPP4) inhibitors are widely used anti-hyperglycemic agents that prolong the effects of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic poly-peptide (GIP), which further stimulate the secretion of insulin [11,12,13]. Notably, DPP4 inhibitors exert positive renal effects in different renal diseases: Sitagliptin (Sita) alleviates kidney oxidative stress in STZ-induced diabetic rats,  and linagliptin shows obvious effects in preventing CKD progression in rats with 5/6 nephrectomy  and in GLP-1R knockout mice with CKD,  indicating that hypoglycemic effects may play a minor role underlying the beneficial renal effects of DPP4 inhibition. In our previous study, we found that Sita delays the progression of DKD in STZ-induced D2 mice, ameliorating mitochondrial abnormalities, preventing renal interstitial fibrosis, and attenuating tubular cell apoptosis . However, whether DPP4 inhibitors regulated ER stress in diabetic STZ-induced mice remains unclear.
Sirtuin 1 (Sirt1), an NAD+-dependent protein deacetylase, plays multiple roles in cells involving hypoxic stress, DNA repair, cell senescence, inflammation, and mitochondrial regulation ; meanwhile, its degradation has essential effects on the development of DKD. In our past experiments, we found that SIRT1 was notably decreased in the kidneys of diabetic db/db mice . Recent studies, however, show that SIRT1 participates in the ER stress response related to hyperglycemia and hypoxia, [19,20,21] but whether SIRT1 is involved in the protection of DPP4 inhibitors is still unknown.
Therefore, our current study aimed to determine whether the DPP4 inhibitor Sita could ameliorate renal tubular injury in STZ-induced diabetic mice through inhibiting ER stress, and whether this protective effect is mediated by SIRT1. Our studies may provide additional mechanistic insights into the pathogenesis of DKD.
Eight-week-old DBA2/J (D2) male mice were purchased from Beijing HFK Bioscience and housed under a constant 12-h light-dark cycle; they were free to eat and drink in the SPF room. D2 mice were randomly divided into three experimental groups (
The measurements of blood glucose and the collection of urine of these mice were carried out monthly. Blood glucose was measured by the Roche meter. Serum creatinine levels were measured through the Creatinine Assay Kit (BioAssay Systems, DICT-500) according to the manufacturer's instructions. Mice urine was collected over 24 h and urine creatinine and albumin levels were detected using ELISA kits (Abcam, ab204537 and ab108792). Urinary albumin/creatinine ratio (ACR) (in milligrams per gram) was calculated as follows: urine albumin (milligrams per decilitre)/urine creatinine (grams per decilitre).
For the morphometric studies, 3-μm-thick sections of paraffin-embedded tissues were stained with HE, PAS, and Masson solutions. To assess and calculate the renal histological damage, digital images of the glomeruli and tubules were obtained using light microscopy (original magnification 400×). A total of 20 images were randomly selected by two blinded researchers for quantitative and histochemical quantitation. The glomerular injuries were graded as follows: 0 represents no lesion; 1+ represents sclerosis of <25% of the glomerulus; and 2+, 3+, and 4+ represent sclerosis of >25–50, >50–75, and >75% of the glomerulus . Tubular injury was defined and calculated as follows: score 0: no tubular injury; score 1: <10% of tubules injured; score 2: 10–25% of tubules injured; score 3: 25–50% of tubules injured; score 4: 50–74% of tubules injured; and score 5: >75% of tubules injured, as previously described [24, 25].
Conditionally immortalized human renal proximal tubular epithelial cell line (HK-2) was cultured in DMEM/F-12 containing 10% fetal bovine serum at 37°C. We then treated HK-2 cells with human serum albumin (HSA) at 20 mg/ml to mimic the injury caused by hyperglycemia or diabetic state.
To pharmacologically inhibit the DPP4 enzyme activity, cells were pretreated with Sita alone (5 μm, Biovision) for 4 h, and then the cells were exposed to the culture medium with albumin and Sita for 72 h. For gene disruption, Lipofectamine 3000 and P3000 reagents (Invitrogen, Grand Island, NY) were used to transfect cells according to the manufacturer's instructions at 60–70% confluent conditions. The cells were treated with SIRT1 siRNA (riboBio, Guangzhou, China), and the special sequences of Sirt1-siRNA were as follows: sense: 5′-CCAGUAGCACUAAUUCCAATT-3″ and antisense: 5′-UUGGAAUUAGUGCCACUGGTT-3′. For overexpression of SIRT1, pCMV3.0 cDNA plasmids (Sino Biological, Beijing, China) were used according to the manufacturer's instructions.
Total protein was extracted from cells and renal tissues with radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime Tech, Shanghai, China) with mixed inhibitors of. phosphatase and protease at 4°C. The protein suspension was centrifuged at 14,000×
The results were expressed as means ± SEM. Comparison of the results between the two groups was evaluated by one-way analysis of variance (ANOVA). If any statistically significant difference was detected, post hoc comparisons were performed using the least significant difference test. Statistical analysis was performed with the SPSS (version 25.0) or GraphPad Prism (version 6.0) software, while
Sita, the inhibitor of DPP4 enzyme, was gavage fed at 20 mg/kg daily to inhibit the activity of the DPP4 enzyme. Eight-week-old male D2 mice were randomly divided into three groups: control group, STZ-induced diabetes group, and Sita-treated diabetic group. Consistent with our previous studies,  compared with the control mice, the STZ-treated diabetic D2 mice had higher blood glucose levels, more obvious urinary ACRs, and more elevated serum creatinine levels, but reduced body weight
To further determine whether the renal beneficial effects of Sita in STZ-induced diabetic mice were through ER stress, we examined the expression of ER stress-associated markers in the kidneys by WB analysis. Compared with the kidneys from control mice, the kidneys of STZ mice presented more increased phosphorylation of PERK at the Thr980 site (p-PERK), and higher expressions of GRP78 and CHOP
It has been reported that excessive elevation of the DPP4 enzyme may lead to the progression of DKD, in which SIRT1 may be involved [26,27,28]. Thus, we further determined whether the protective roles of Sita in the kidneys of diabetic mice were likely related to the SIRT1. First, we found that the protein expression of DPP4 in the kidneys was obviously increased in the STZ-induced diabetic mice compared with the control mice, and this increase was notably suppressed after Sita treatment
Given that Sita exerts nephroprotective effects on diabetic STZ mice, the apoptosis-related protein was also determined. Compared with the control mice, diabetic mice presented more elevated c-CASP3 in the renal lysates, while this disorder was effectively attenuated by Sita
Since our animal experiments suggest that Sita had a significant protective effect on the kidneys of diabetic mice, we identified the potential mechanism of this effect
Then, we further examine the potential reason underlying its protective effects. Consistent with our
To validate the role of SIRT1 in albumin-overloaded ER stress, we further confirmed whether regulation of SIRT1 influences imbalanced ER stress in HSA-treated HK-2 cells through using SIRT1 cDNA plasmids (oxp-SIRT1) and SIRT1 siRNA. Compared with the control cells, si-SIRT1 in HK-2 cells yielded significantly decreased SIRT1 expression, higher p-PERK, and elevated expressions of GRP78 and CHOP
Renal tubulointerstitial fibrosis is the most common lesion of tubular injuries, some studies have reported that tubulointerstitial fibrosis appears earlier than glomerular fibrosis and is more closely related to renal outcomes during DKD progression . Thus, it is important to understand the exact mechanism of tubular injury to develop specific therapies for diabetic tubular changes. However, in animal studies, it is difficult to successfully create significant tubular damages in diabetic models. Recent studies show that D2 mice may have a diabetes-susceptible background , which was confirmed in our past studies. In our previous work, tubular injuries and tubulointerstitial fibrosis were observed in STZ-induced D2 mice with unilateral nephrectomy , which was significantly reversed by Sita administration. Thus, in our present study, we used STZ-injected D2 mice to study the potential mechanism of the DPP4 inhibitor Sita on tubular cell injury and abnormal ER stress of DKD.
ER stress exerts significant effects in the progression of DKD, while inhibition of ER stress through TUDCA presented obvious renoprotection in diabetic db/db mice [5, 7]. Proteinuria is a critical player in DKD and is positively correlated with the progression to ESRD [30, 31]. It has been reported that the exposure of proximal tubular epithelial cells to excess albumin causes renal tubular fibrosis in the progression of DKD , thus we use HSA-treated HK-2 cells to mimic the diabetic renal states. In our current work, we observed highly pronounced ER stress and its associated cellular apoptosis in the kidneys of STZ-diabetic D2 mice and HSA-treated HK-2 cells, showing the elevated expressions of GRP78, CHOP, p-PERK, and c-caspase3, which were consistent with our past studies in db/db mice .
Recently, several new drugs have been approved for the treatment of T2DM, including DPP4 inhibitors. Recent studies also found that DPP4 inhibitors play beneficial effects on nondiabetic renal diseases, [32, 33] but the protective mechanism is still not well defined so far. In our study, we confirmed that Sita, a DPP4 inhibitor, improved renal functions and alleviated renal histological changes in the kidneys of STZ-treated diabetic mice without exerting hypoglycemic effects. The finding that Sita failed to exert a hypoglycemic role in STZ-diabetic mice is not surprising, as its effects are largely induced by activating GLP-1R, GIP, or other possible pathways that promote insulin secretion [27, 34]. However, the STZ-injected diabetic mice that we used are insulin-deficient mouse models , as STZ destroys the function of islets.
In this study, we found that ER stress-associated markers were ameliorated after Sita treatment in both diabetic mouse kidneys and HSA-treated HK-2 cells, as reflected by the decreased CHOP, GRP78, and p-PERK. Besides, ER stress-related apoptosis was also ameliorated by Sita, showing attenuated levels of cleaved-caspase3. The results provide evidence that the DPP4-mediated signaling pathway may partly be associated with ER stress-induced injury in diabetic tubules, but the potential mechanism underlying it is still unclear.
Recent studies have focused on SIRT1, a deacetylase that involves various signaling pathways, including cell repair and mitochondrial function [35,36,37]. Recent studies also confirm its beneficial effects in managing DKD;  for example, upregulation of SIRT1 protects cells from apoptosis by inhibiting ER stress in hepatocytes and pancreatic β cells;  SIRT1 also improved palmitate-induced insulin resistance via regulation of ER stress in HepG2 cells . In our work, we demonstrated that SIRT1 expression was significantly decreased, whereas Sita treatment can effectively restore its expression. We further investigated the possibility that SIRT1 regulates ER stress in HSA-treated HK-2 cells via using the overexpressed SIRT1 plasmids and SIRT1 siRNA. The increased SIRT1 alleviated ER stress-associated markers and cellular apoptosis induced by albumin overloading in HK-2 cells, indicating that SIRT1 inhibited ER stress in the HSA state. Furthermore, si-SIRT1 confirmed the link between SIRT1, increased expression of c-CASP3, and activation of ER stress. These results are in agreement with previous studies, [41,42,43,44] which described the renal beneficial effects and anti-ER stress of SIRT1.
However, how DPP4-mediated the changes in SIRT1 and regulated its downstream ER stress and its exact role in the tubular cell injury has not been fully studied. In our study, we confirmed the destruction of ER homeostasis of HK-2 cells induced by overloaded albumin, and these effects were significantly attenuated by Sita treatment. Sita preserved the expression of SIRT1, and the downregulation of SIRT1 in HK-2 cells enhanced the HSA-induced ER stress in HK-2 cells, suggesting that SIRT1 may be regulated by DPP4.
There are several limitations in our work. This current work studied the beneficial effects induced by Sita via inhibiting ER stress, but we cannot exclude the possibility that these effects may also be attributed to other substrates of the DPP4 enzyme, given that the DPP4 enzyme cleaves a large number of chemokines, neuropeptides, and peptide hormones, like SDF-1α, substance P, peptide YY, and neuropeptide Y . We have studied the role of SDF-1α, a substrate of the DPP4 enzyme, in maintaining mitochondrial dynamic homeostasis in DKD . The effects of these substrates on DKD progression are still unclear, and further investigations are needed to address these pathways individually and in detail. Another limitation of our study is that the study design is not usually comparable to the clinical conditions.
Collectively, in our present study, we found that the DPP4 inhibitor Sita prevented ER stress in STZ-induced diabetic D2 mice and HSA-treated HK-2 cells, whose protective effects may be through upregulating SIRT1. Thus, our results demonstrate a novel mechanism linking the DPP4 enzyme to ER stress during tubular injury in DKD.