Uric acid, proteinuria, and renal function

Blood samples were collected from the rats and centrifuged at 4000 g for 10 min to obtain serum. Next the levels of serum UA, BUN, creatinine, and reactive oxygen species (ROS) were determined using commercial assay kits. Urine samples were collected from the rats and used for proteinuria analysis. All assays were performed according to instructions provided with the assay kit.

Cell culture and transfection

HK-1 cells were purchased from ATCC (Manassas, VA, USA) and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum (FBS) and 1% streptomycin-penicillin (Gibco, Waltham, MA, USA) in a 37°C chamber containing 5% CO₂. Cells in their log-growth phase were used for further experiments. HK-1 cells were cultured in vitro and divided into four groups: blank group; hyperuricemia group; hyperuricemia + GTP group (siRNA NC); hyperuricemia + GTP + siRNA group (siRNA-jagged1). The cells were transfected for 72 hours and then collected for use in subsequent assays. The nucleotide fragments used for transfection were: siRNA-jagged1 sense: 5’-ACACUUUGAAGUAUGUGUCAC-3’; si-NC sense: 5’-ACACUUUGAAGUAUGUGUCAC-3’ (Ruibo Bio, Beijing, China).

Renal histology

Specimens of rat renal tissue were fixed in 4% paraformaldehyde for 24 h and then embedded in paraffin. Hematoxylin-eosin (H&E), periodic acid Schiff (PAS), and Masson’s trichrome staining were performed on 4 μm tissue sections. All analyses of tissue samples were performed in blinded manner.

Glomerulosclerosis (GS) index scores and tubulointerstitial (TI) index scores were based on semi-quantitative scoring systems. The score assessments were made by viewing periodic-acid Schiff (PAS)-stained tissue sections at a magnification of ×200 [18]. The GS index was assessed in 20 glomeruli per animal and graded on a scale of 0 to 4 according to the percentage of glomeruli displaying obliteration or hyalinosis: grade 0 = no sclerosis; grade 1 = 0-25%; grade 2 = 25-50%; grade 3 = 50-75%; grade 4 = 75-100%. The TI index was assessed in 20 fields per kidney section, and the lesions were graded on a scale of 0 to 5 according to the amount of tissue area that displayed tubule interstitial changes (tubular atrophy, casts, interstitial inflammation, and fibrosis): grade 0 = normal; grade 1 = 0-10%; grade 2 = 10-25%; grade 3 = 25-50%; grade 4 = 50-75%; grade 5 = 75-100%. The final index score for each rat was determined by calculating the mean value of all the obtained scores.

Images of arcuate (×200), interlobular (×200), and afferent (×400) arteries were analyzed as previously described [4], and the results were used to evaluate the wall thickening of preglomerular arteries. Briefly, an outline of the vessel and its internal lumen (excluding the endothelium) were studied with a Motic Med 6.0 digital medical image analysis system (Motic Group Co., Xiamen, Fujian, China). The total medial area was calculated by subtracting the lumen area from the outline area. Next, the M/L ratio was calculated in 10 arteries of each type to obtain the mean value for each rat.

Immunohistochemistry (IHC) staining

Paraffin embedded sections (4 μm) were deparaffinized and blocked for 10 min with 0.3% hydrogen peroxide at room temperature, followed by antigen retrieval. After being rinsed with PBS, the sections were incubated overnight at 4°C with rabbit polyclonal antibodies against Jagged1 (1:100, Abcam, Cambridge, MA, USA), Notch1-ICD (1:100, Anbobio, San Francisco, CA, USA), MnSOD2 (1:500, Abcam), XO (1:100, Abcam, Cambridge, MA, USA), rabbit monoclonal antibodies against Hes5 (1:200, Abcam, Cambridge, MA, USA), and mouse monoclonal antibodies against p-STAT3 (1:400, Cell Signaling Technology, Beverly, MA, USA). Negative control tissue sections were incubated without the primary antibody. Next, the sections were stained using a two-step EnVision System (Dako Cytomation, Santa Clara, CA, USA) according to manufacturer’s instructions. Each tissue section was viewed under a microscope, and ten arteries of each type (arcuate, interlobular and afferent) were analyzed using the Motic Med 6.0 digital medical image analysis system. The amount of signal-positive area relative to the vascular area was calculated, and the mean values were used to evaluate gene expression.

In situ hybridization (ISH)

Paraffin embedded sections (4 μm) were deparaffinized and treated with pepsin (Boster, Beijing, China) for 15 min at 37°C. After post-fixation in 4% paraformaldehyde (PFA), the sections were hybridized with digoxigenin (DIG)-labeled nucleic acid probes (40 ng/μL) at 42°C overnight. The oligonucleotide sequence used for Jagged1 was 5’-CGGATGTCTTCGGCAGAAATGGC-3’, and was synthesized by Sangon Bio (Shanghai, China). Negative control hybridizations were performed with the omission of probes. The slides were sequentially washed in 2×SSC, 0.5×SSC, and 0.2×SSC at 37°C and then sequentially incubated with the blocking buffer for 30 min at 37°C, with anti-DIG/biotin conjugate antibodies (Boster, Beijing, China) for 60 min at 37°C, and with streptavidin-biotin complex (SABC) for 60 min at 37°C. Finally, the slides were stained with 0.05% DAB (Boster, Beijing, China). Each section was examined under a microscope, and ten arteries of each type (arcuate, interlobular, and afferent) were analyzed using the Motic Med 6.0 digital medical image analysis system. The amount of Jagged1 positive area relative to the vascular area was calculated, and the mean values were used to evaluate Jagged1 expression.

qPCR

Tissue samples were pulverized in liquid nitrogen and the dispersed cells were collected by centrifugation at 10,000 g for 1 min. The cells were then treated with Trizol reagent (100 mg tissue: 1 mL Trizol, cells: 600 μL Trizol) (Invitrogen, Carlsbad, CA, USA). The cell solution was transferred to an Ep tube and 200 μL of chloroform was added. After vibration for 15 sec, 500 μL of isopropanol was added to the upper aqueous phase and the 2-phase solution was let sit for 10 min. The solution was then centrifuged at 12000 g for 10 min, and 1 mL of 75% ethanol was added to the precipitate. The precipitate was then pelleted by centrifugation at 7500 g for 5 min at 4°C; after which, the supernatant was removed and the tube was dried for 10 min. Next, the RNA-containing pellet was dissolved in DEPC water, and its RNA content and purity were determined by ultraviolet spectroscopy. A reaction solution containing the following ingredients was prepared according to instructions provided with the reaction kit: 2 μg total RNA, 1 μL oligo primer (50 μM), 1 μL dNTP mix (10 μM), and ddH₂O. The solution was pre-incubated for 5 min at 65°C. Next, a cDNA first chain synthesis reaction system containing the following ingredients was prepared: 2 μL 10×RT buffer, 4 μL MgCl₂ (25 μM), 2 μL DTT (0.1 M), l μL RNAase OUT (40 U/μL), 1 μL SuperScrip III RT (200 U/μL), and ddH₂O (Promega, USA). The reaction conditions were as follows: 50°C for 50 min followed by 85°C for 5 min. Real-time quantitative PCR was then performed using a SYBR Premix Ex Taq GC kit (Takara, Japan), a LightCycler 480 PCR machine (Roche Diagnostics, Indianapolis, IN, USA), and the following conditions: denaturation at 94°C for 30 sec, followed by 40 cycles of denaturation at 94°C for 5 sec and annealing at 60°C for 30 sec. The primer sequences used and amplification lengths are shown in Table 1. GAPDH was used as an internal control. The relative levels of gene expression were semiquantitatively analyzed using the 2^-ΔΔCt method; where 2^-ΔΔCt = gene copy number in the test group/gene copy number in the control. All the experiments were conducted in triplicate.

Western blotting assay

Total cellular proteins were extracted using reagents in a radio immunoprecipitation assay (RIPA) kit, and the protein extracts were centrifuged at 10000 g for 30 min. The bicinchoninic acid (BCA) assay was used to quantify the amount of total protein in each extract. Next, a 50 μg sample of protein from each extract was separated by electrophoresis on an 8% sodium dodecyl sulfate polyacrylamide gel for 3 h, and the separated protein bands were transferred onto a polyvinylidene fluoride (PVDF) membrane by electrophoresis at 200 mA for 100 min. After being blocked with 5% skimmed milk at room temperature for 60 min, the membrane was incubated with the following primary antibodies (anti-notch1, anti-jagged1, anti-hes5, anti-p-STAT3, anti-MnSOD, anti-XO, and anti-GAPDH; Abcam, USA) at 4°C overnight. Following incubation, the membrane was washed with phosphate buffered saline Tween 20 (PBST) solution and then incubated with a HRP-labeled secondary antibody (Abcam, USA) at room temperature for 60 min. The membrane was then treated with electrochemiluminescence (ECL) reagent and developed.

Hoechst staining

Cells were treated with berberine (50 μM) for 48 h and then harvested by trypsinization. After being washed twice with phosphate buffered saline (PBS) solution, the cells were resuspended in binding buffer and cultured at 37°C for 24 h; after which, they were stained with 0.1 μg/mL Hoechst 33342 (Sigma-Aldrich, Temecula, CA, USA). A fluorescence microscope (Olympus IX71; Olympus Corporation, Tokyo, Japan) equipped with a filter for Hoechst 33342 (365 nm) was used to detect any changes in nuclear morphology.

Reactive oxygen species (ROS)

Cells were digested and incubated for 20 min at 37°C with a 0.1% 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) probe (Beyotime, Beijing, China) that had been diluted with serum-free medium to a final concentration of 10 mM, as recommended in the manufacturer’s instructions The cells were then washed three times with PBS and tested with a Beckman Coulter CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA).

Effects of GTPs on preglomerular arteriopathy

Because GTPs can function as antioxidants, we investigated the effect of GTPs on preglomerular arteriopathy. The results showed that when compared with PA symptoms in the RK + OA group, the pathological symptoms of PA were remarkably alleviated following the treatment with GTPs (Figure 2A). Furthermore, the mean weight of rats in RK + OA + GTP group was significantly higher than that in the RK + OA group, and the levels of proteinuria, serum UA, BUN, and creatinine in the RK + OA + GTP group all decreased in a GTP-dose dependent manner (Table 2). Data concerning the M/L ratio of the preglomerular artery indicated that the values for the arcuate, interlobular, and afferent arteries of rats in the RK + OA + GTP group were significantly decreased when compared to those values in the RK + OA group (Table 3), and this was further confirmed by H&E and PAS staining data (Figures S1 and S2). This implied that GTP treatment could effectively prevent the damage caused by preglomerular vascular disease in the hyperuricemic CKD model.

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Figure 2

Effect of GTPs and Jagged1 on preglomerular arteriopathy. A: GTPs relieved the pathological symptoms caused by OA in RK rats. Representative images of arcuate arteries (×200), interlobular arteries (×200), and afferent arteries (×400). B: Expression of Jagged1 mRNA on the preglomerular arteries of rats in the RK + OA group was analyzed by ISH (representative images). C: The levels of Jagged1 mRNA on the preglomerular arteries in the Sham, RK, and RK + OA groups. D: The M/L ratios of the preglomerular arteries were determined by Pearson’s correlation analysis.

Involvement of the jagged1/notch1 pathway in preglomerular vascular lesions

To investigate the mechanism underlying preglomerular arteriopathy, the expression of Jagged1 was examined using ISH (Figure 2B). In the normal rats, most of the Jagged1 mRNA was found on the arterial intima and media, and only small amounts were found on the arterial adventitia. Rats in the RK and RK + OA groups displayed significantly decreased levels of Jagged1 expression in the arcuate (Sham vs. RK vs. RK + OA, 23.36 ± 4.56 vs. 13.49 ± 2.74 vs. 8.10 ± 3.04, P < 0.01), interlobular (Sham vs. RK vs. RK + OA, 24.99 ± 2.24 vs. 9.29 ± 1.86 vs. 5.83 ± 2.23, P < 0.01), and afferent arteries (Sham vs. RK vs. RK + OA, 23.17 ± 2.43 vs. 11.06 ± 1.48 vs. 7.10 ± 2.43, P < 0.01) (Figure 2C). Moreover, the levels of Jagged1 mRNA were significantly correlated with the M/L ratios of the preglomerular arteries (Figure 2D).

Jagged1/Notch1 signaling is critical for vascular remodeling [19]. Accordingly, we determined the levels of Jagged1, Notch1-ICD, and Hes5 on the preglomerular vessels (Figure 3A). Our results showed that the levels of Jagged1, Notch1-CID, and Hes5 were significantly lower in the RK rats, and were further decreased in the hyperuricemic RK rats when compared to their levels in the control rats (Figure 3B), suggesting that Notch1 signaling is suppressed during the formation preglomerular vascular lesions. Similarly, the preglomerular vascular levels of MnSOD2, a ROS scavenger, and also p-STAT3 expression, were decreased in the RK group and were lowest in the RK + OA group (Figure 3C and ​and3D).3D). In contrast, XO, which promotes ROS production, exhibited higher levels in the RK group and reached its highest level in the RK + OA group (Figure 3C and ​and3D).3D). qPCR and western blot studies indicated that the levels of Jagged1, Notch1-ICD, Hes5, and p-STAT3 were significantly decreased in RK + OA group when compared to those in the RK group (Figure 4A and ​and4B).4B). Importantly, a quantitative analysis revealed positive correlations among the levels of Notch1-ICD, Jagged1, Notch1-ICD, Hes5, p-STAT3, and MnSOD2 in the RK + OA group, (Figure 4C), suggesting that Jagged1/Notch1-STAT3-MnSOD2 signal transduction plays a role in the progression of preglomerular vascular injuries.

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Figure 3

Expression of Jagged1, Notch1-ICD, Hes5, p-STAT3, MnSOD2, and XO on the preglomerular arteries. A: Expression of Jagged1, Notch1-ICD, and Hes5 on the preglomerular arteries was analyzed by IHC, and representative images are shown. B: Quantitative analysis of the Jagged1, Notch1-ICD, and Hes5 levels. C: Representative images of p-STAT3, MnSOD2, and XO staining in the Sham group. D: The levels of p-STAT3, MnSOD2, and XO on the preglomerular arteries in each group were quantified.

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Figure 4

Expression of Jagged1, Notch1-ICD, Hes5, and p-STAT3 and the correlations among the Sham, RK, RK + OA, RK + OA + Tea, and RK + OA + Tea + AP groups. A: mRNA levels were detected by qPCR. The levels of Jagged1, Notch1-ICD, and Hes5 were remarkably decreased in the RK rats and RK rats treated with OA. The administration of Tea or AP reversed the effect of OA. B: Protein levels were measured by western blot methods. Jagged1, Notch1-ICD, Hes5, and p-STAT3 expression was reduced in the RK rats and RK rats treated with OA. The administration of Tea or AP reversed the effect of OA. C: Correlations between the levels of Jagged1, Notch1-ICD, Hes5, and p-STAT3, A Pearson’s correlation analysis showed positive correlations among the levels of Notch1-ICD, Jagged1, Notch1-ICD, Hes5, p-STAT3, and MnSOD2.

This research was supported by National Natural Science Foundation of China (No. 30500546).