Diabetic nephropathy is a common microvascular complication in diabetic patients, which leads to high morbidity and mortality throughout the world [1
]. Diabetic nephropathy is characterized by structural as well as functional abnormalities. Urinary albumin excretion along with extracellular matrix accumulation, basement membrane thickening, mesangial hypertrophy, and glomerular epithelial cell (podocyte) loss within the glomeruli are characteristic pathological features of diabetic nephropathy [3
Especially, changes in glomeruli such as fibrosis and apoptosis of mesangial cells play important roles in the progression of diabetic nephropathy. The hyperglycemic condition induces expression of genes associated with fibrosis, such as transforming growth factor-β (TGF-β), fibronectin, collagen type IV, α2 (Col4a2), and plasminogen activator inhibitor-1 (PAI-1) [5
]. Moreover, activation of apoptotic programs such as nuclear condensation, caspase activation, and release of cytochrome c from mitochondria are observed in high glucose-treated mesangial cells [7
Intensive blood glucose control and anti-hypertensive agents such as angiotensin converting enzyme inhibitors and angiotensin receptor-1 antagonists are currently the most effective treatments for progressive diabetic nephropathy [9
]. However, there are several limitations to these drugs, such as patient resistance and heart or kidney failure [10
]. Therefore, the development of new drugs or adjuvants that act on various components of diabetic nephropathy is urgently required.
L. seed (PCS), commonly known as “Boh-Gol-Zhee” in Korea, has been used in herbal and traditional medicine for various diseases including diabetes, cancer, inflammatory disease, neurodegenerative disease, and kidney disease [12
]. Six compounds, bakuchiol, psoralen, isopsoralen, corylifolin, corylin, and psoralidin, are the major components of PCS extract [13
]. Among these, bakuchiol, which is a meroterpene, and psoralen and isopsoralen, which are coumarins, have been widely studied, and their health benefits regarding anti-oxidant, anti-tumor, and estrogenic activity have been identified [14
Previously, we found that PCS extract shows protective effects on oxidative stress-induced pancreatic beta cell apoptosis [18
] and hepatic damage [19
]. Furthermore, PCS extract shows promise as an anti-obesity agent in a high fat diet-induced obesity model [20
], suggesting that PCS extracts might also have ameliorative effects on diabetic nephropathy. Therefore, we investigated whether PCS extracts have beneficial effects on diabetic nephropathy in a streptozotocin (STZ)-induced type 1 diabetic mouse model and investigated the mechanisms involved in high glucose-treated glomerular cells.
2. Materials and Methods
Dulbecco modified eagle medium (DMEM), Ham’s F-12 (F-12) medium, and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). Bakuchiol was purchased from Enzo Life Sciences Inc. (Farmingdale, NY, USA). Antibodies against poly (ADP-ribose) polymerase (PARP), B-cell lymphoma (Bcl)-2, Bcl-2-associated death promoter (Bad), and phospho-Bad were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against beta-actin and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Streptozotocin (STZ), losartan potassium, psoralen, and isopsoralen were obtained from Sigma-Aldrich (St. Louis, MO, USA).
2.2. Preparation of PCS Extract
PCS was purchased from an oriental drug store (Kwang Myung Dang Co., Ulsan, Korea), and the extract was prepared by the standard procedure as described previously [18
]. In summary, 300 grams of dry seed were ground into small pieces and then extracted twice with distilled water under reflux. The combined water extracts were evaporated in vacuo and finally yielded 61.92 g of a dark brown residue.
Six-week-old male C57BL/6 mice were supplied by the Orient Bio Inc (Seongnam, Gyeonggi-do, Korea). Animals were maintained at animal facilities at the Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Science, under a 12-h light, 12-h dark photoperiod. All animal experiments were carried out under a protocol approved by the Institutional Animal Care and Use Committee (LCDI-2012-0029) at Lee Gil Ya Cancer and Diabetes Institute, Gachon University. After a week of adaptation, mice were injected intraperitoneally with 50 mg/kg/day STZ for five consecutive days. Age-matched control mice received an equal volume of vehicle. After one week after the fifth STZ injection, blood glucose levels were checked, and mice with blood glucose levels over 300 mg/dL were used for experiments. STZ-induced diabetic mice were treated orally with PCS extract (500 mg/kg/day) or vehicle (daily for 8 weeks), as described previously [18
]. Losartan potassium (Sigma-Aldrich) was orally administered (10 mg/kg/day) for 8 weeks as a positive control.
2.4. Periodic Acid-Schiff (PAS) Staining
After 10% formalin fixation, 4 μm sections cut from paraffin-embedded kidney samples were stained with PAS. Thirty glomeruli were randomly selected from each mouse for the PAS analysis using Image J software (version 1.48q, National Institutes of Health, Bethesda, MD, USA). The mesangial matrix index is the ratio of mesangial matrix area divided by the tuft area.
2.5. Biochemical Parameters in Blood and Urine
Urinary and serum parameters, such as creatinine, urea nitrogen, total protein, and microalbumin, were measured by an immunoturbidimetric method using an AU680 automated chemistry analyzer (Beckman Coulter, Inc., Brea, CA, USA). Creatinine clearance was calculated using following calculation formula. Creatinine clearance = urine volume (mL/min) × urine creatinine/serum creatinine
2.6. Murine Mesangial Cell Culture
SV40-transformed murine glomerular mesangial cells (MES-13) were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in a 3:1 mixture of DMEM and F-12 medium containing FBS (5%), penicillin (100 U/mL), streptomycin (100 μg/mL), HEPES (14 mM), and glucose (5.5 mM) at 37 °C in an atmosphere containing 5% CO2–95% air. To test the response of high glucose concentration, the cells were maintained in 25 mM glucose-containing medium for 24 h. Cells were cultured in the presence of 5.5 mM glucose and 19.4 mM mannitol as an osmotic control. The number of viable cells was determined using Cell Counting Kit-8 (CCK-8) assay kit (Dojindo Laboratories, Kumamoto, Japan), which measures dehydrogenase activities in cells.
2.7. Western Blot Analysis
Cells were lysed with mammalian protein extraction buffer (GE Healthcare, Milwaukee, WI, USA) containing a protease inhibitor and phosphatase inhibitor cocktail (Sigma-Aldrich). A standard amount of protein was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto membranes, and blocked [20
]. The membranes were incubated with specific primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Chemiluminescence was detected on LAS-4000 (Fuji Film, Tokyo, Japan) using Immobilon Western Chemiluminescent HRP Substrate (Millipore, St. Charles, MO, USA). The protein bands obtained by western blotting were analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA) software for Windows.
2.8. Quantitative Real-Time RT-PCR (qRT-PCR) Analysis
Total RNA was extracted using TRIZOL reagent (Invitrogen Corp., Carlsbad, CA, USA), and cDNA was synthesized using a PrimeScript 1st strand cDNA synthesis kit (Takara Bio Inc., Kyoto, Japan). qRT-PCR was performed as previously described [20
]. The relative copy number was calculated using the threshold crossing point (Ct) as calculated by ΔΔ Ct. Primer sequences were as follows: 5′-TGGAGAGCACCAAGACAGACA-3′ and 5′-TGCCGGAGTCGACAATGAT-3′ for mouse cyclophlin; 5′-TGACGATGGGAAGACCTACCA-3′ and 5′-GGAACAAATGGCTCCGAGATAT -3′ for mouse fibronectin; 5′-TCAATGACTGGGTGGAAAGG-3′ and 5′-AGGCGTGTCAGCTCGTCTAC-3′ for mouse PAI-1; 5′-GCAAAAGGTCAGGATCGAGGTA-3′ and 5′-GTGCCGAACCACAAAGAGAAAG-3′ for mouse Col4a2 and 5′-GCAGTGGCTGAACCAAGGA-3′ and 5′-AGCAGTGAGCGCTGAATCG-3′ for mouse TGF-β1.
2.9. Statistical Analyses
All data are expressed as mean ± standard error of at least three independent experiments. Data were analyzed using Analysis of Variance followed by post-hoc analysis using the Tukey range test (SPSS 10.0 statistical software). The p-values less than 0.05 were considered to be statistically significant.
Diabetic nephropathy is the most serious complication in diabetes mellitus and causes glomerular fibrosis and impairment of renal function. Currently available drugs, which control blood glucose or blood pressure, have a number of limitations, such as patient resistance and high rates of secondary failure [10
]. These lead to the search for alternative therapies from natural products that have low or no side effects and multi-target actions.
PCS extract has generated much interest due to its various biological activities, including anti-inflammation, anti-apoptosis, and anti-hyperglycemic effects [20
], suggesting further study on its effect on diabetic complications such as nephropathy. Therefore, we investigated anti-diabetic nephropathy effects of PCS extract in STZ-induced diabetic mice, which develop renal injury with similarities to human diabetic nephropathy [24
The increase in urinary albumin excretion, one of the marked renal pathological features in diabetic nephropathy, is caused by mesangial expansion due to accumulation of extracellular matrix components [25
]. Our STZ-induced diabetic mice showed an increase in urinary albumin concentration and a corresponding increase in the mesangial matrix index relative to non-diabetic control mice. In addition, the level of creatinine clearance, the most widely used clinical marker of kidney function [26
], was higher in diabetic mice than control mice, indicating the presence of diabetic nephropathy with renal hyperfiltration in our STZ-induced diabetic mice. Eight-weeks of PCS extract treatment attenuated the increased albuminuria, creatinine clearance, and mesangial expansion in the glomeruli of STZ-induced diabetic mice. These results demonstrated that PCS extract has potent effects by counteracting mesangial expansion and hyperfiltration in diabetic mice, possibly leading to the amelioration or delay in the development of advanced diabetic renal injury.
We observed that hyperfiltration induced by STZ injection led to a decrease in serum urea nitrogen levels, but administration of PCS extract did not recover this to normal levels. These results show lack of correlation with creatinine clearance and serum urea nitrogen, and this is consistent with another study in STZ-induced diabetic rats fed a high fat diet [27
Previously, we reported that PCS extract treatment showed anti-hyperglycemic effects via reduced lipid accumulation and reduced inflammation in the liver of mice fed a high fat diet [20
]. In the present study, treatment of diabetic mice with PCS extract ameliorated creatinine clearance and mesangial matrix accumulation, but had no impact on glucose homeostasis (Figure S1
). These conflicting results might be the result of the different mechanisms used to induce different diabetic mouse models [28
]: such as type 1 diabetes (present study) versus type 2 diabetes (previous study). Moreover, effect of PCS extract regarding improvement of renal injury was similar to the losartan-treated group, suggesting that PCS extract might directly improve kidney function by glucose-independent mechanisms.
Several studies have demonstrated that increased TGF-β expression in mesangial cells promotes extracellular matrix accumulation and hypertrophy during progression of diabetic nephropathy [29
]. Moreover, as increased TGF-β is known to be a potent inducer of Col4a2, fibronectin, and PAI-1 expression [31
], we investigated whether PCS extract reduced the expression of these molecules. Treatment with PCS extract inhibited the expression level of fibrosis markers both in the kidney tissue of diabetic mice and in high glucose-treated MES-13 mesangial cells, and also increased cell viability. These results show that the anti-fibrotic effects of PCS extract provided effective protection from high glucose-mediated kidney damage.
Apoptosis plays a pathological role leading to the death of mesangial cells, which is associated with progressive glomerulosclerosis [32
]. Khera et al. reported that high glucose-mediated TGF-β activation decreases nuclear factor-kB activation and in turn, alters the expression ratio of Bcl-2:Bcl-2-associated X protein favoring caspase-3 activation and increased apoptosis [8
]. We also found that expression level of apoptotic makers was increased under diabetic nephropathy conditions, and PCS extract treatment attenuated cleaved PARP and increased Bcl-2 expression and phosphorylation of Bad (Ser-112). Although we did not evaluate the correlation between fibrosis and apoptosis pathways in glucose-treated mesangial cells, these results suggest that inhibition of apoptosis under conditions of high glucose toxicity is an important mechanism of PCS extract in reducing glomerulosclerosis in diabetic nephropathy.
We found that treatment with major compounds of PCS extract (bakuchiol, psoralen, and isopsoralen) inhibited mesangial cell death, but the anti-apoptotic and anti-fibrotic responses varied. Two coumarins, psoralen and isopsoralen, increased cell viability and decreased apoptotic protein expression, and bakuchiol showed anti-apoptotic effects at much lower concentration compared with psoralen or isopsoralen. Similarly, previous studies found that psoralen or isopsoralen inhibits apoptotic cell death in H2
-treated INS-1 cells [18
] or palmitate-treated PC12 cells [34
]. Psoralen or isopsoralen decreased mRNA expression level of fibronectin and PAI-1, suggesting that the effects of PCS extract in diabetic nephropathy was mediated primarily by these compounds. However, total PCS extract treatment significantly increased the expression of PAI-1. PCS extract contains other chemical compounds such as coryfolin, corylin, and 3-hydroxybakuchiol [13
], which might have resulted in the increase of PAI-1 expression in apoptotic mesangial cells seen after PCS treatment.
It is known that the treatment of high glucose in mesangial cells increases the level of reactive oxygen species (ROS) [35
]. Previously, we reported that PCS extract has an anti-oxidative effect in hepatocytes [19
] and pancreatic beta cells [18
], and this effect is mediated by improvement of mitochondrial function. As oxidative stress plays a role in the pathogenesis of diabetic nephropathy [37
], possible involvement of the anti-oxidative effect of PCS extract in the inhibition of diabetic nephropathy will be investigated.