Introduction: Diabetic nephropathy (DN) is a long-term loss of renal function occurring in the diabetic patients, leading to 5 million deaths in 2015, and this number is dramatically growing annually. Due to unsatisfied outcome of current treatment, there is urgent need to develop more effective therapeutic drugs for DN. Methods: Approximately 150 kinds of natural small molecule drugs that have been used on the market or in the clinical trials in the presence of high glucose were tested individually on the same batch of human renal glomerular endothelial cells (GECs) and human kidney 2 (HK-2) cells with triplicated wells by using a robotic pipetting workstation to screen for the potential drug candidate. Cell viability and oxidative stress were examined in the GECs and HK-2 cells. DN mouse model was established and treated with 25 mg/kg xanthohumol. Results: By measuring cell viability, xanthohumol was selected as our predicted drug candidate for DN because it could mostly protect renal cells from high glucose with about 90% survived GECs and HK-2 cells, about 2.12- and 2.37-fold increase compared to glucose group which was with 42.78% and 37.69% survived GECs and HK-2 cells, respectively. Then, xanthohumol inhibited high glucose-induced oxidative stress through nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in vitro. Moreover, xanthohumol (25 mg/kg) significantly decreased the levels of serum creatinine, blood urea nitrogen, urea protein, and kidney weight/body weight ratio in DN mice. In addition, the increase of reactive oxygen species production and the decrease of superoxide dismutase and catalase activities in DN mice were partially reversed by xanthohumol. mRNA levels of Nrf2, Hmox1, and Nqol genes were all decreased by xanthohumol DN mice. Conclusion: Xanthohumol could ameliorate DN-related impairments via Nrf2 signaling pathway, which might serve as a promising drug candidate for treatment of DN.
Diabetic nephropathy (DN), also known as diabetic kidney disease, is a major microvascular complication of type 1 diabetes mellitus and type 2 diabetes mellitus (T2DM), which is characterized by long-term loss of renal function, and also one of the leading causes of kidney failure, end-stage renal disease, as well as chronic kidney disease . In 2015, approximately 415 million people worldwide suffered from diabetes and caused 5 million deaths. By 2040, the prevalence of diabetes is estimated to increase to 642 million . About 30% of type 1 diabetes mellitus patients and about 40% of T2DM patients will develop DN . Moreover, the number of deaths caused by DN increased by 94% from 1990 to 2012 , which becomes one of the diseases with the highest increase among all reported chronic diseases.
The metabolic changes caused by diabetes can result in glomerular hypertrophy, glomerular sclerosis, tubular interstitial inflammation, and fibrosis; however, due to the lack of effectively therapeutic drugs, the occurrence and progression of DN are still a huge threat to diabetic patients. Therefore, it is required to develop more effective drugs to improve the health of patients with DN . Screening of natural small molecule drugs that have been applied on the market or in the stage of clinical trials have high safety; therefore, new use of old drugs or multiple uses of one drug are highly effective strategy for rapid application to treat DN patients.
In our study, human renal tubular epithelial and glomerular endothelial cells (GECs) were stimulated with high glucose and then treated with approximately 150 kinds of natural small molecule drugs, respectively, that have been utilized on the market or in the clinical trials, to screen the candidate one that had protective effects on tubules and glomerular cells. Next, in vitro experiment and streptozotocin (STZ)-induced DN mouse model were combined together to confirm its role on kidney injury and also to explore the molecular mechanism of candidate drug to improve DN. Our study will provide a new therapeutic strategy for treating DN patients.
Methods and Materials
DN Mouse Model
Eight-week-old male C57BL/6 mice (Beijing Vital River Laboratory Animal Technology Co., China) were intraperitoneally injected with 60 mg/kg STZ (V900890; Sigma-Aldrich, St. Louis, MO, USA) for 5 consecutive days to induce diabetes, and blood glucose levels were measured from the tail vein using a glucometer (Roche, Mannheim, Germany) 16–25 days after the last dose of STZ injection. Mice with blood glucose over 16.7 mmol/L were selected as DN mice and used for the following studies. Animal study was approved by the Ethical Committee of Daqing Longnan Hospital.
STZ-induced diabetic mice were randomly divided into four groups by equal average body weight: control (saline + 0.5% DMSO), XN only (25 mg/kg Xanthohumol [XN] dissolved in saline + 0.5% DMSO), DN (STZ + saline + 0.5% DMSO), and DN + XN (STZ + 25 mg/kg XN in saline + 0.5% DMSO). Xanthohumol was dissolved in saline plus 0.5% DMSO and intraperitoneally injected into mice daily after the last dose of STZ until the end of experiment on week 20 (day 140).
On week 20, mice were placed in the metabolic cages individually with free access to water and food for 24 h to acquire urine samples. Next, after 12 h fasting, mice were weighted, and blood glucose was measured from the tail vein using a glucometer (Roche). Blood samples were collected from the tail vein and centrifuged at 3,000 g for 10 min to get serum for analyzing the level of serum creatinine and blood urea nitrogen. Serum and urinary parameters, including serum creatinine, blood urea nitrogen, and urine protein, were evaluated by an immunoturbidimetric method using an AU680 automated chemistry analyzer (Beckman Coulter, Inc., Brea, CA, USA). Both sides of kidney tissues were also collected, weighted, homogenized, and centrifuged at 3,000 g for 10 min to get supernatant for the further studies .
The library (HY-L021P, MedChem Express) contains about 150 kinds of natural small molecule drugs that have been applied on the market or in the stage of clinical trial. Xanthohumol was purchased from MedChem Express (MCE, HY-N1067), and ML385 was purchased from Selleck (S8790).
In vitro Experiments
Human renal GECs (ScienCell, Carlsbad, CA, USA) were cultured in endothelial cell medium containing 5.6 mm glucose, 5% endothelial cell growth supplement, and 5% fetal bovine serum . Human kidney 2 (HK-2) cells (CRL-2190, ATCC) were cultured according to the protocol from ATCC. When reaching a density of 50%–60% (about 5 × 104 cells/well) in a 96-well plate with 100 μL culture medium, GECs/HK-2 cells were treated with 25 mm glucose to establish a diabetic model in vitro and exposed to every natural compound (50 μm) at the same time for 72 h. Cell viabilities were evaluated using CCK-8 kit following the manufacturer’s protocol (C0038, Beyptime). Compounds were tested individually on the same batch of GECs and HK-2 cells with triplicated wells for each compound in a 96-well plate by using a robotic pipetting workstation to limit errors for pipetting. Triplicates were done in the same 96-well plate. For oxidative stress analysis in vitro, GECs/HK-2 cells were exposed to 25 mm high glucose and 50 μm xanthohumol for 48 h, then cells were collected for measuring oxidative levels.
Oxidative Stress Analysis
Treated cells were collected, lysed, and centrifuged at 3,000 g for 10 min to get supernatant. Supernatant of homogenized kidney tissues and cells was used to measure superoxide dismutase (SOD) and catalase (CAT) activity using the Superoxide Dismutase Activity Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) and Catalase Activity (CAT) Assay (Elabscience), respectively.
Total protein was extracted from treated cells or kidney tissues, and then Western blotting was performed as previously described . All the primary antibodies used were purchased from Abcam, including Nrf2 (1:1,500) and GAPDH (1:2,000).
Total mRNA was extracted from treated cells or kidney tissues using TRIzol reagent according to the manufacturer’s instruction, and then transcripted into cDNA using HiScript RT SuperMix (Vazyme, China). RT-PCR was performed as previously described . The sequences of primers were as below:
Nrf2 (mouse): F, TCTTGGAGTAAGTCGAGAAGTGT; R, GTTGAAACTGAGCGAAAAAGGC.
Hmox1 (mouse): F, AAGCCGAGAATGCTGAGTTCA; R, GCCGTGTAGATATGGTACAAGGA.
Nqo1 (mouse): F, AGGATGGGAGGTACTCGAATC; R, AGGCGTCCTTCCTTATATGCTA.
Gapdh (mouse): F, AGGTCGGTGTGAACGGATTTG; R, TGTAGACCATGTAGTTGAGGTCA.
GAPDH (human): F, GGAGCGAGATCCCTCCAAAAT; R, GGCTGTTGTCATACTTCTCATGG.
HMOX1 (human): F, AAGACTGCGTTCCTGCTCAAC; R, AAAGCCCTACAGCAACTGTCG.
NQO1 (human): F, GAAGAGCACTGATCGTACTGGC; R, GGATACTGAAAGTTCGCAGGG.
Data were analyzed by one-way ANOVA with Tukey’s multiple-comparisons test in GraphPad Prism 7 and showed as means ± SD. It was regarded as statistically significant when p < 0.05.
Xanthohumol Protected Human GECs and HK-2 Cells from the Stimulation of High Glucose
First, we screened approximately 150 kinds of natural small molecule drugs in the library (online suppl. Table S1; for all online suppl. material, see www.karger.com/doi/10.1159/000528650) and added these drugs into GECs, respectively, in the presence of 25 mm high glucose for 72 h and then selected the top 10 compounds that protected GECs against high glucose by evaluating GECs’ viabilities (Fig. 1a, b). Moreover, we also selected the top 10 compounds that protected HK-2 cells from the stimulation of high glucose (Fig. 1c, d). In the two figures, xanthohumol had the best protective effect against the stimulation of glucose on GECs and HK-2 cells, with about 90% survived GECs and HK-2 cells and about 2.12- and 2.37-fold increase compared to glucose group which was with 42.78% and 37.69% survived GECs and HK-2 cells, respectively (Fig. 1a–d. Therefore, we selected xanthohumol (Fig. 1e) as our predicted candidate drug for treating DN.
Xanthohumol Inhibited High Glucose-Induced Oxidative Stress in GECs and HK-2 Cells
Numerous studies revealed that oxidative stress was associated with the critical pathways that participated in the development and progression of macro- and microvascular complications of diabetes, including DN; therefore, a large body of oxidants were implicated to improve oxidative stress-related diabetic complications . Previous publications indicated that xanthohumol alleviated oxidative stress as well as oxidative stress-associated cardiovascular complications in the liver and kidney of T2DM mice . Hence, here we explored whether it could inhibit oxidative stress in GECs and HK-2 cells. Xanthohumol did not change the basal levels of reactive oxygen species (ROS), SOD activity, or CAT activity. But after high glucose stimulation, oxidative stress was significantly enhanced, as judged of increased ROS levels and decreased SOD and CAT activities, which were all reversed by xanthohumol treatment both in GECs (Fig. 2a–c) and HK-2 cells (Fig. 2d–f. All these data confirmed that xanthohumol effectively reduced high glucose-induced oxidative stress in GECs and HK-2 cells.
Xanthohumol Inhibited Oxidative Stress via NRF2 Signaling Pathway in vitro
Nuclear factor erythroid 2-related factor 2 (Nrf2) was reported to regulate the expressions of anti-oxidant proteins to protect against oxidative stress-caused damage, and several studies indicated that Nrf2 signaling pathway might be involved in the progression of xanthohumol-reduced oxidative stress. The basal protein levels of Nrf2 were not altered in the supplement of xanthohumol in GECs and HK-2 cells. But high glucose significantly decreased the protein expressions of Nrf2, which could be remarkably restored by xanthohumol treatment in GECs (Fig. 3a–b) and HK-2 cells (Fig. 3e, f). Next, we measured the expressions of Nrf2-downstream genes, including HMOX1 (heme oxygenase 1, HO-1) and NQO1 (NADPH dehydrogenase [Quinone 1]), and their mRNA levels were all changed in the same pattern with Nrf2 protein levels. Xanthohumol significantly increased their expressions that were inhibited by high glucose in GECs (Fig. 3c, d) and in HK-2 cells (Fig. 3g, h). Therefore, xanthohumol inhibited high glucose-induced oxidative stress by regulating Nrf2 signaling pathway.
Inhibition of NRF2 Abolished the Anti-Oxidative Effect of Xanthohumol in vitro
To further confirm whether xanthohumol suppressed high glucose-induced oxidative stress through Nrf2 signaling pathway, we used Nrf2 inhibitor – ML385 to block Nrf2 signaling pathway. Like xanthohumol, ML385 alone did not affect oxidative levels with no change of ROS levels, SOD, and CAT activities. But in the presence of high glucose, ML385 could further increase oxidative stress in GECs and HK-2 cells, as judged of upregulated ROS levels (Fig. 4a–d) and downregulated SOD activities (Fig. 4b, e) and CAT activities (Fig. 4c, f). Notably, oxidative stress inhibited by xanthohumol, in the presence of high glucose was greatly reserved after ML385 treatment in GECs and HK-2 cells, which suggested that ML385 effectively abolished the anti-oxidative effect of xanthohumol. All these data further demonstrated that the anti-oxidative effect of xanthohumol was mediated by Nrf2 signaling pathway.
Xanthohumol Ameliorated STZ-Induced Kidney Injury in Mice
As followed, we evaluated the protective effect of xanthohumol in STZ-induced DN mice (Fig. 5a). Compared to control and xanthohumol-treated mice, STZ-induced DN mice displayed an increased level of blood glucose (Fig. 5b) and severe kidney injury with elevated kidney injury markers, including serum creatinine (Fig. 5c), blood urea nitrogen (Fig. 5d), urine protein (Fig. 5e), and kidney weight/body weight (Fig. 5f). Xanthohumol significantly ameliorated kidney injury, as judged by all the decreased expressions of kidney injury markers, with no alternation of blood glucose levels. Thus, these data suggested that although xanthohumol could not improve diabetes, it effectively ameliorated DN in mice.
Xanthohumol Alleviated Oxidative Stress in DN Mice by Nrf2 Signaling Pathway
Next, we assessed the anti-oxidative effect of xanthohumol in DN mice. Compared to control and xanthohumol-treated mice, STZ injection significantly induced oxidative stress in mice with increased ROS levels and decreased anti-oxidant enzyme activity, such as SOD and CAT, which could be remarkably alleviated by xanthohumol injection (Fig. 6a–c). Hence, xanthohumol promoted the anti-oxidative activity in the kidney of DN mice. The mRNA levels of Nrf2, Hmox1, and Nqo1, which were greatly reduced in STZ-induced DN mice, were significantly reversed in xanthohumol-treated DN mice (Fig. 7a–c). Therefore, xanthohumol increased anti-oxidative activity by activating Nrf2 signaling pathway, leading to the improvement of oxidative stress-associated DN.
In our current study, human renal GECs and tubular epithelial cells were cultured under the stimulation of high glucose to establish diabetic model in vitro and then treated with approximately 150 kinds of natural small molecule drugs, respectively, that have been applied on the market now or in the stage of clinical trials to screen a potential candidate drug that could treat DN. By evaluating cell viability, we found that xanthohumol had the best protective effect on both human renal tubules and glomerular cells; therefore, we selected xanthohumol as our predicted candidate drug to treat STZ-induced DN mice, and eventually it ameliorated kidney injury by alleviating oxidative stress via Nrf2 signaling pathway. Owing to the dramatic effect of xanthohumol in treating DN in mice, our study might provide a novel therapeutic strategy.
This commercial library contains approximately 150 kinds of the natural small molecule drugs that have been in use on the market or in the stage of clinical trials, which indicated that all these drugs we used in our project have been studied well and applied to treat human diseases. Instead of developing a new drug, exploring new uses of old drugs or investigating one drug with multiple uses are significantly effective strategies with high safety and efficacy . If the candidate drug we predicted worked well for treating DN patients, it would save much time for the predicted candidate drug to utilize in the clinical trials or on the market, which will be the quickest way to solve the urgent need for the growing DN patients. However, it should be noted that only one concentration (50 μm) of the drugs was used from the library. Some potential compounds may be missed due to improper concentration or other work condition.
To find the potential drug that could protect against DN, we used human renal GECs and tubular epithelial cells to screen all the drugs in the library. We used two different kidney cells to screen out the potential drugs that have the greatest protective effect on the kidney. By measuring the cell viability after each drug treated, the top 13 drugs that protect both GECs and HK-2 cells were selected, namely xanthohumol, curcumin, limonene, aldosterone, quercetin, isoquercitrin, lycopene, ALCAR (AMPK activator), ellagic acid, fraxin, papain, fisetin, and pinocembrin. Notably, 8 of 13 have been reported to have therapeutic effects on DN, including curcumin, Aldosterone, quercetin, lycopene, ALCAR, ellagic acid, fisetin, and pinocembrin, which also indicated that our screening system was effective [13, 16]. The other five compounds, including xanthohumol, limonene, isoquercitrin, fraxin, and papain, have no related studies indicating that they play a role in the occurrence and development of DN . Among all these five compounds that have not been reported to be related to DN, we selected xanthohumol as our predicted potential candidate drug because it was the top one that protect both GECs and HK-2 cells from the stimulation of high glucose.
Xanthohumol is a prenylated chalconoid derived from female inflorescences of Humulus lupulus, also known as hops, and is synthesized in the glandular trichromes of hop cones . Plenty of studies reported that xanthohumol possessed anti-oxidant and anti-inflammatory properties . Published literature indicated that xanthohumol ameliorated type 2 diabetes-related oxidative stress. Additionally, xanthohumol improved skin wound healing by regulating oxidative stress, inflammation, and angiogenesis in type 1 diabetic rat. All these studies revealed its relationship with diabetes; however, there is no direct evidence proved that xanthohumol had a protective role on DN. Our current study not only demonstrated that xanthohumol could effectively ameliorated DN in STZ-induced mice and high glucose-stimulated GECs and HK-2 cells but also revealed xanthohumol protected the kidney by reducing oxidative stress through Nrf2 signaling pathway. According to only a few reports, xanthohumol may be the inhibitor of diacylglycerol acyltransferase . However, due to the lack of enough studies on xanthohumol’s target and the relationship of diacylglycerol acyltransferase and Nrf2, the detailed mechanisms and the target of xanthohumol need to be further explored, especially, whether it functions via diacylglycerol acyltransferase to regulate Nrf2 signaling.
During the progression of DN, hyperglycemia-mediated excess ROS production is well linked between the disrupted renal hemodynamics and metabolic pathways . ROS activates a variety of cellular responses, which play a key role on the pathogenesis of hyperglycemia-induced kidney injury. Under hyperglycemia conditions, oxidative stress caused by excessive ROS stimulates the production of growth factors, cytokines, and transcriptional factors in the kidney, which in turn leads to chronic inflammation, tubular interstitial fibrosis, and renal hypertrophy [11, 21, 22]. Because oxidative stress causes and exacerbates inflammation in the kidney, though xanthohumol has both anti-inflammatory and anti-oxidant activities, in our study we explored its anti-oxidant effect instead of anti-inflammatory role.
Our study for the first time discovered that xanthohumol could effectively ameliorate kidney injury in STZ-induced DN mice, considering that xanthohumol is already on the market or in the stage of clinical trials , which proved that it has been studied well. If more studies further confirmed its protective effect on DN, plenty of time will be saved to apply xanthohumol in the clinical trials for DN patients. Xanthohumol might serve as a promising candidate therapeutic drug for treating DN patients in the near future.
Our study screened about 150 kinds of natural small molecule drugs that have been used on the market or in the stage of clinical trials and selected xanthohumol due to its protective effect on human renal cells and in STZ-induced DN mice by activating Nrf2 signaling pathway. Therefore, xanthohumol might serve as a promising therapeutic drug candidate for treating DN patients in the future.
Statement of Ethics
Animal study was approved by the Ethical Committee of Daqing Longnan Hospital; the approval number is cdf/23. This study was performed in strict accordance with the NIH guidelines for the care and use of laboratory animals (8th edition, NIH). Only commercial cell lines were used. Consent to participant is not applicable in this case.
The study has no external funding sources.
Conflict of Interest Statement
The authors have no conflicts of interest to declare.
Fenglin Li, Jinling Zhang, and Le Luo: data curation, data analysis, drafting of the article, and final approval of the version to be published. Jing Hu: study supervision, coordination, funding support, design of this study, drafting of the article, and final approval of the version to be published.
Data Availability Statement
All data generated or analyzed during this study are included in this article and its online supplementary material files. Further inquiries can be directed to the corresponding author.
Fenglin Li and Jinling Zhang contributed equally to this work.