The protective effect of Phellinus linteus decoction on podocyte injury in the kidney of FSGS rats

The powder of P. linteus (Mesima) was prepared and provided by professor You-gui Li, Zhejiang Academy of Agricultural Science. The P. linteus used in this study was authenticated by the Institute of Microbiology of Chinese Academy of Sciences. The specimen of P. linteus has been kept at the herbarium of Zhejiang Academy of Agricultural Science.

According to the ancient books (Sheng ji zong lu and Pu ji Fang), the recommended clinical dosage of P. linteus is about 10 g/d for adults. The appropriate dosage for each rat is calculated on the basis of body surface area [13]. In our experiment, in combination with some preliminary experiments, we finally chose the dosage of PLD-LD, PLD-MD, and PLD-HD is 4 g /d, 8 g/d, and 16 g/d, respectively. The decoction was prepared as follows: the powder was initially soaked in distilled water for half an hour, and then decocted for 30 min two times, in accordance with conventional method. Finally, the decoctions were combined and filtered using a double-layer gauze, and concentrated to the required volume for spare.

The rats were allowed to acclimatize for a week prior to stating the experiments, and then weighed and numbered according to their body weight (from light to heavy). At first, six rats were randomly selected as the control group by the random number table. The remaining 24 rats were used to establish the FSGS model (n = 6 rats/group). Based on the proteinuria difference between the control and model group in our preliminary experimental results, the total rat numbers in our study were determined. Thereafter, the FSGS rat model was established as previously described [4, 13, 14]. Briefly, the rats were firstly subjected to uninephrectomy (left side) on day 1, followed by the administration of doxorubicin through caudal vein, 5 mg/kg (on day 7) and 3 mg/kg (on day 28), respectively. For the control rats, they were correspondingly injected equivalent saline on day 7 and day 28 following the sham operation.

The FSGS rats were allocated randomly to four groups: model group, PLD-LD, PLD-MD, and PLD-HD (n = 6/each group). The intervention with different concentrations of P. linteus decoction by gavage was initiated on day 2. After administration for eight consecutive weeks, the serum and whole right kidneys were harvested for biochemical, histological, and molecular analyses, and followed by the euthanisation of the animals by dislocation of the cervical spine. Urine samples were collected for 24 h by using the metabolic cages. The urinary protein level in the rats was quantified by a biochemical analyser (HITACHI 7180).

A portion of kidney was fixed with 4% paraformaldehyde and embedded in paraffin. 3 μm-thick sections were cut and stained with haematoxylin and eosin (H&E) and Masson’s trichrome stain for examination of kidney histology. The degree of sclerosis in each glomerulus was subjectively graded on a scale of 0 to 4 as described previously [13]. The glomerular sclerosis index (GSI) was calculated by using the following formula as previously reported:

\( \mathrm{GSI}=\frac{\left(1\times \mathrm{N}1\right)+\left(2\times \mathrm{N}2\right)+\left(3\times \mathrm{N}3\right)+\left(4\times \mathrm{N}4\right)}{\mathrm{N}0+\mathrm{N}1+\mathrm{N}2+\mathrm{N}3+\mathrm{N}4} \), where N is the number of glomeruli at each grade of sclerosis.

After fixation in 2.5% glutaraldehyde overnight, the kidney tissue (~ 1 mm³ in size) was rinsed in 0.1 M PBS thrice. Then the specimens were post-fixed with 1% osmium tetroxide for 1 h, dehydrated in graded series of acetone and embedded in graded Epon 812. Ultrathin sections (80–100 nm) were cut and stained with uranyl acetate (2%) and lead citrate [15], and observed with a JEM-1400 transmission electron microscope (JEOL, Japan).

Total RNA was isolated from kidneys of individual rats using the TRIzol reagent (Invitrogen, USA) and then cDNA was synthesized using a Primescript™ RT reagent kit (TaKaRa, Japan) according to the manufacturer’s instructions. Thereafter, expression levels of nephrin, podocin, and GAPDH were quantified via real-time PCR using Applied Biosystems® 7500 Fast real-time PCR system (Thermo Fisher Scientific, USA). The sequences of primers used are shown in Table 1. Relative mRNA expression levels were normalized to those of GAPDH. Each PCR experiment was performed in triplicate and repeated independently at least thrice.

The rat kidney tissues were incubated with RIPA lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and 1 mM EDTA] containing a proteinase inhibitor cocktail (Beyotime) at 4 °C for 30 min. Cell lysates were centrifuged at 10,000 g for 10 min at 4 °C. After quantification using the BCA assay, the protein extracts (80 μg) were separated by 10% SDS-PAGE gels under reducing conditions. After the proteins were transferred onto a PVDF membrane, 5% skimmed milk was used as the blocking agent, and then the membranes were next incubated with the primary antibodies. Antibodies used were as follows: anti-nephrin (Abcam, ab58968); anti-podocin (Abcam, ab93650); and anti-GAPDH (ProteinTech, 60,004–1-Ig). After hybridization, the blots were washed and incubated with infrared labelled anti-rabbit/mouse IgG Ab (1:15000). Finally, the signal was detected using an Odyssey CLx image system (LI-COR).

To establish a model for rat FSGS, we subjected rats to uninephrectomy followed by repeated injection of doxorubicin. The urinary protein levels for FSGS rats were significantly higher than that of control rats (p < 0.01, Fig. 1a), indicating that the FSGS model had been successfully established. No rat mortalities were recorded during the course of the whole experiment. Besides, the serum albumin (ALB) level in the FSGS model decreased significantly compared with the control group (p < 0.01, Fig. 1b). After the intervention with the P. linteus decoction, the 24 h urinary protein levels in PLD-LD and PLD-HD groups were much lower than that in the model group (Fig. 1a). Meanwhile, the ALB in the PLD-LD group showed obvious improvement (Fig. 1b). No significant differences in the ALB levels between the model group and PLD-MD and PLD-HD group were observed. Collectively, these results indicate that the PLD-LD can attenuate urinary protein excretion and improve the serum ALB levels in the FSGS rat.

To further evaluate the effects of the P. linteus decoction on kidney function, the serum markers in different groups were measured. Serum creatinine (Scr), urea nitrogen (BUN), triglycerides (TG), and cholesterol (TC) were significantly increased in the FSGS group compared with the control group (Fig. 2). Additionally, the Scr, BUN, and TG were significantly reduced in the PLD-LD group compared with the model group. However, there were no significant differences in TC levels between the model group and the PLD-LD, PLD-MD and the PLD-HD groups (Fig. 2). These results are basically in line with the data from urinary protein, providing further evidence that PLD-LD could ameliorate kidney damage.

The glomerular pathomorphology was further examined by histological analysis. H&E and Masson’s staining demonstrated that there was obvious focal glomerular sclerosis, interstitial lesions, and inflammatory cell infiltration in the model rats, whereas the control rats had no pathological changes (Fig. 3a). Furthermore, the GSI in the model group was much higher than that in the control group (Fig. 3b). After the P. linteus decoction intervention, such pathological damages were much improved compared with that in the model group, especially the PLD-LD group (Fig. 3a). Similarly, the GSI in the PLD-LD group decreased significantly compared to that in the model group (Fig. 3b).

Next, TEM was used to observe the degree of damage in the podocyte foot process. The results showed clear processes were observed on the surface of the glomerular epithelial cells in the control group (Fig. 4a). However, the foot processes diffusely effaced, became flat and fused and even disappeared in the model group. Compared with the control group, the foot process rate in the model group increased from 2.67 ± 1.00 to 80% ± 6.12% (Fig. 4b, p < 0.01). In the PLD-LD group, the lesions were significantly alleviated and foot fusion rate (35.33% ± 5.03%) reduced compared with the model group. However, the lesions in PLD-MD and PLD-HD groups were much more serious than that in the PLD-LD group (Fig. 4a). Taken together, these results indicated that the PLD-LD treatment can alleviate the renal pathological damage.

To further assess the effect of the P. linteus decoction on FSGS progression, the podocyte slit diaphragm proteins, nephrin and podocin were assessed. RT-PCR results showed the expression levels of nephrin and podocin were markedly decreased in the model group (Fig. 5a). In the PLD-LD intervention group, the expression of nephrin and podocin were increased significantly compared with that in the model group. However, there was no significant difference between the model and PLD-MD and PLD-HD groups (Fig. 5a). Similar results were observed by western blotting (Fig. 5b and c). These results suggested that PLD-LD can protect the podocyte slit diaphragm proteins nephrin and podocin.