DHMEQ treatment significantly inhibited the nuclear translocation of p65 in rat kidney tissue
The major form of NF-kB is a heterodimer (p65/p50) that is inactivated when bound to IkB in the cytoplasm; this heterodimer is translocated to the nucleus after the phosphorylation and degradation of IkB via activation signals from the cell surface membrane [30]. DHMEQ has been shown to inhibit nuclear translocation of the activated NF-kB heterodimer (p65/p50) [17, 20]. Therefore, we investigated whether DHMEQ treatment inhibits the nuclear translocation of p65 in a CsA nephropathy model. We did not observe any adverse events (e.g. phenotypical of behavioral abnormalities) on animals in each group due to drug administration.
We separated the nuclear and cytoplasmic proteins from digested kidney samples and evaluated the activity of NF-kB protein in the nuclear and cytoplasmic fraction by ELISA. As suggested in several previous reports [11, 12], NF-kB activation and the nuclear translocation of p65 in the kidneys of rats treated with CsA were significantly increased compared with those in the control rats (control vs CsA, 0.83 ± 0.11-fold vs 4.33 ± 0.84-fold increase, relative ratio of p65 DNA-binding activity in the nucleus to that in the cytoplasm, respectively, p = 0.0005, Fig. 2A). However, the nuclear translocation of p65 in the rat kidney was significantly inhibited by cotreatment with DHMEQ compared with CsA monotherapy (CsA + DHMEQ vs CsA, 1.34 ± 0.23-fold vs 4.33 ± 0.84-fold increase, relative ratio of p65 DNA-binding activity in the nucleus to that in the cytoplasm, respectively, p = 0.0022, Fig. 2A). There was no significant difference of p65 DNA-binding activity between the control and CsA + DHMEQ (control vs CsA + DHMEQ, 0.83 ± 0.11-fold vs 1.34 ± 0.23-fold, respectively, p = 0.7623, Fig. 2A).
We also evaluated the effect of NF-kB activation on the histology by immunohistochemical staining. In accordance with the results obtained by ELISA, the nuclear translocation of p65 was increased in rats treated with CsA compared with control untreated rats (control vs CsA, 9.5 ± 1.8 vs 56.7 ± 7.7 nuclear counts/field, respectively, p < 0.0001, Fig. 2B, C, E). The affected area was mostly in the tubular epithelial cells (Fig. 2C). However, DHMEQ treatment effectively inhibited the nuclear translocation of p65 due to the administration of CsA (CsA + DHMEQ vs CsA, 18.3 ± 2.7 vs 56.7 ± 7.7 nuclear counts/field, respectively, p = 0.0001, Fig. 2C, D, E). There was no significant difference of the nuclear translocation of p65 between the control and CsA + DHMEQ (control vs CsA + DHMEQ, 9.5 ± 1.8 vs 18.3 ± 2.7, respectively, p = 0.4198, Fig. 2B, D, E).
DHMEQ treatment ameliorated renal function deterioration by high-dose CsA
The growth of the rats in each group that was assumed from weight increases from the baseline and to the day of euthanasia was not statistically different in each group (D weight in control vs CsA vs CsA + DHMEQ, 61.7 ± 38.9 vs 26.2 ± 41.9 vs 15.8 ± 21.8 g, p = 0.0931 by ANOVA, supplementary table 1). Repeated administration of CsA (15 mg/kg/day for 28 days) and low-sodium conditions caused the renal function in a 5/6 nephrectomized rat model to deteriorate. Serum UN levels in the CsA nephropathy group were significantly increased compared with those in the control group (control vs CsA, 43.1 ± 1.1 vs 113.5 ± 8.8 mg/dL, respectively, p < 0.0001, Fig. 3A). The serum Cr level was also increased in the CsA nephropathy group compared with the control group (control vs CsA, 0.49 ± 0.02 vs 0.91 ± 0.02 mg/dL, respectively, p < 0.0001, Fig. 3B). We calculated the CCr and normalized the results by body weight (kg). Normalized CCr in the CsA nephropathy group was decreased compared with the control group (control vs CsA, 4.61 ± 0.18 vs 1.94 ± 0.12 ml/min/kg, respectively, p < 0.0001, Fig. 3C).
However, DHMEQ treatment significantly ameliorated renal function deterioration caused by repeated high-dose CsA administration. Serum UN levels in the DHMEQ-treated group were significantly decreased compared with those in the CsA nephropathy group (CsA + DHMEQ vs CsA, 69 ± 6.4 vs 113.5 ± 8.8 mg/dL, respectively, p = 0.0004, Fig. 3A). The serum Cr level in the DHMEQ-treated group was also significantly decreased compared with that in the CsA nephropathy group (CsA + DHMEQ vs CsA, 0.75 ± 0.02 vs 0.91 ± 0.02 mg/dL, respectively, p = 0.0003, Fig. 3B). In addition, CCr was significantly increased in the DHMEQ-treated group compared with the CsA nephropathy group (CsA + DHMEQ vs CsA, 2.57 ± 0.09 vs 1.94 ± 0.12 ml/min/kg, respectively, p = 0.013, Fig. 3C). However, DHMEQ treatment did not completely restore renal function to the control level (serum UN, Cr, and CCr in control vs CsA + DHMEQ; 43.1 ± 1.1 vs 69 ± 6.4 mg/dL, p = 0.0275; 0.49 ± 0.02 vs 0.75 ± 0.02 mg/dL, p < 0.0001; 4.61 ± 0.18 vs 2.57 ± 0.09 ml/min/kg, p < 0.0001; respectively, Fig 3A, B, and C).
In contrast, the urine volume in each group was not significantly different (Control vs CsA vs CsA + DHMEQ, 28.3 ± 1.5 vs 30.6 ± 3.6 vs 27.6 ± 3.1 ml, Fig. 3D). Interestingly, urinary protein extraction was significantly decreased in the CsA nephropathy group compared with the control group (control vs CsA, 17.7 ± 2.6 vs 10.6 ± 1.8 mg/24 hours, respectively, p = 0.0328, Fig. 3E). DHMEQ treatment did not offset the inhibitory effect of urinary protein extraction due to CsA (CsA + DHMEQ vs CsA and control vs CsA + DHMEQ; 9.7 ± 1.0 vs 10.6 ± 1.8 mg/24 hours, p = 0.9255; 17.7 ± 2.6 vs 9.7 ± 1.0 mg/24 hours, p = 0.0237; respectively, Fig. 3E).
DHMEQ treatment significantly inhibited the development of renal fibrosis due to CsA
Next, we investigated whether kidney function was related to renal tissue fibrosis among the three groups. Surgical treatment with 5/6 nephrectomy (control), which was intended to reduce the number of nephrons, did not affect renal fibrosis formation (Fig. 4A, B). In contrast, renal fibrosis developed in the kidneys of rats treated with CsA (Fig. 4C, D). Typical striped renal fibrosis from the corticomedullary boundary to the surface of the cortex was observed (Fig. 4C). The renal fibrosis area was significantly increased in the CsA nephropathy group compared with the control group (control vs CsA, 9.4 ± 5.4 vs 35.6 ± 18.4%, respectively, p < 0.0001, Fig. 4H). However, renal fibrosis formation was remarkably inhibited by DHMEQ treatment (Fig. 4E, F). The renal fibrosis area was significantly decreased in the DHMEQ-treated group compared with the CsA nephropathy group (CsA + DHMEQ vs CsA, 13.4 ± 7.1 vs 35.6 ± 18.4%, respectively, p < 0.0001, Fig. 4H). There was no significant difference of the renal fibrosis area between the control and CsA + DHMEQ (control vs CsA + DHMEQ, 9.4 ± 5.4 vs13.4 ± 7.1%, respectively, p = 0.157, Fig. 4H).
DHMEQ treatment significantly inhibited inflammatory cell infiltration
We further evaluated inflammatory cell infiltration in the kidneys of rats in the three groups. First, we evaluated the transcription of chemokines, MCP-1 and CCL5 in each group. MCP-1 mRNA expression levels in the CsA nephropathy group were higher than those in the control group (control vs CsA, 1.00 ± 0.13 vs 1.82 ± 0.35, Fig. 5A). However, MCP-1 mRNA expression levels in the DHMEQ-treated group were lower than those in the CsA nephropathy group, although this difference was not statistically significant (CsA + DHMEQ vs CsA, 1.14 ± 0.24 vs 1.82 ± 0.35, Fig. 5A). The same tendency was observed for CCL5 (control vs CsA vs CsA + DHMEQ, 1.00 ± 0.10 vs 1.98 ± 0.42 vs 1.28 ± 0.17, Fig. 5B).
Next, we investigated whether these changes in chemokine expression were associated with inflammatory cell infiltration in the renal tissue. Macrophage (ED1-positive cells) infiltration in the CsA nephropathy group was significantly increased compared with that in the control group (control vs CsA, 1.1 ± 0.26 vs 25.1 ± 1.65 positive cells/field, respectively, p < 0.0001, Fig. 6A, B, D). However, macrophage infiltration in the DHMEQ-treated group was significantly decreased compared with that in the CsA nephropathy group (CsA + DHMEQ vs CsA, 4.2 ± 0.48 vs 25.1 ± 1.65 positive cells/field, respectively, p < 0.0001, Fig. 6B, C, D), and there was no significant difference of macrophage infiltration between the control and CsA + DHMEQ (control vs CsA + DHMEQ, 1.1 ± 0.26 vs 4.2 ± 0.48 positive cells/field, respectively, p = 0.0751, Fig. 6A, C, D). These findings were in accordance with the changes in MCP-1 expression.
We subsequently evaluated granulocyte infiltration in the renal tissue. Granulocyte (HIS48 positive cells) infiltration in the CsA nephropathy group was significantly increased compared with that in the control group (control vs CsA, 6.3 ± 0.68 vs 41.8 ± 4.1 positive cells/field, respectively, p < 0.0001, Fig. 6E, F, H). In contrast, granulocyte infiltration was significantly decreased in the DHMEQ-treated group compared with the CsA nephropathy group (CsA + DHMEQ vs CsA, 18.4 ± 1.01 vs 41.8 ± 4.1 positive cells/field, respectively, p < 0.0001, Fig. 6F, G, H), although DHMEQ treatment did not completely inhibited granulocyte infiltration to the control level (control s CsA + DHMEQ, 6.3 ± 0.68 vs 18.4 ± 1.01 positive cells/field, respectively, p = 0.0025, Fig. 6E, G, H).