Tacrolimus ameliorates tubulointerstitial inflammation in diabetic nephropathy via inhibiting the NFATc1/TRPC6 pathway

Abstract

Tubulointerstitial inflammation is crucial for the progression of diabetic nephropathy (DN), and tubular cells act as a driving force in the inflammatory cascade. Emerging data suggested that tacrolimus (TAC) ameliorates podocyte injury and macrophage infiltration in streptozotocin (STZ) mice. However, the effect of TAC on tubulointerstitial inflammation remains unknown. We found that albuminuria and tubulointerstitial damage improved in db/db mice treated with TAC. Macrophage infiltration and expression of IL-6, TNF-α, fibronectin, collagen 1 and cleaved caspase 3 were inhibited as well. In addition, the expression of nuclear factor of activated T cell 1 (NFATc1) and transient receptor potential channel 6 (TRPC6) was up-regulated in the kidneys of DN patients and correlated with tubular injury and inflammation. The expression of NFATc1 and TRPC6 also increased in the kidneys of db/db mice and HK-2 cells with high glucose (HG), while TAC inhibited these effects. HG-induced inflammatory markers and apoptosis were reversed by TAC and NFATc1 siRNA in HK-2 cells, which was abolished by TRPC6 plasmid. Furthermore, HG-induced TRPC6 expression was inhibited by NFATc1 siRNA, while NFATc1 nuclear translocation was inhibited by TAC, but was restored by TRPC6 plasmid in HK-2 cells under HG conditions. These findings suggest that TAC ameliorates tubulointerstitial inflammation in DN through NFATc1/TRPC6 feedback loop.

Keywords: NFATc1; TRPC6; diabetic nephropathy; inflammation; tacrolimus; tubular cell.

Figure 1

Tacrolimus (TAC) ameliorated tubulointerstitial injury in db/db mice. A, Bodyweight changes in db/db mice with or without TAC (0.5 or 1.0 mg/kg) for 12‐20 wk. B, Blood glucose concentrations in each group. C, Serum creatinine level. D, Urinary albumin excretion (ACR). E, Haematoxylin‐eosin (HE) (a‐d), periodic acid‐Schiff (PAS) (e‐h) and Masson's staining (i‐l) of renal tissues from the mice. F, G, Quantitative analysis of interstitial fibrosis and tubular atrophy (IFTA) scores and glomerular damage in the kidneys in each group. H, I, Transmission electron microscopy (TEM) showing thickening of the basement membrane and mitochondrial fragmentation in the kidneys of db/db mice compared with those of db/m mice, which were ameliorated by TAC in a dose‐dependent manner. The values are the mean ± SD, *P < .05 vs db/m; ^# P < .05 vs db/db; ^@ P < .05 vs db/db + 0.5 mg/kg TAC mice. n = 6

Figure 2

Tacrolimus (TAC) reduced tubulointerstitial fibrosis and apoptosis in db/db mice. A, Fibronectin (FN) (a‐d) and collagen 1 (Col‐1) (e‐h) expression levels were assessed by IHC (magnification × 400). Apoptosis in kidney tubular cells was detected with TUNEL staining (i‐l). B, C, Semiquantification of FN and Col‐1 intensity. n = 6 per group. D, Statistical analysis of TUNEL‐positive cells. n = 6 per group. E, Western blot analysis of FN, Col‐1 and cleaved caspase 3 (C‐CAS3) expression; β‐actin was used as a control. F, G, Relative band density of FN, Col‐1 and C‐CAS3. n = 3 per group. The values are the mean ± SD, *P < .05 vs db/m; ^# P < .05 vs db/db; ^@ P < .05 vs db/db + 0.5 mg/kg TAC mice. IHC, immunohistochemistry; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end‐labelling

Figure 3

Tacrolimus (TAC) inhibited macrophage infiltration and pro‐inflammatory cytokine protein expression in the kidneys of db/db mice. A, Representative immunohistochemistry (IHC) images of TNF‐α (a‐d) and IL‐6 (e‐h) in the mouse kidney sections (magnification × 400). A1, A2, Semiquantification of IHC staining of TNF‐α and IL‐6. n = 6 per group. B, Western blot analysis of IL‐6 expression in the kidney tissue. The expression of β‐actin was used as a control. n = 3 per group. B1, Relative band density of IL‐6. C, Representative IHC staining of F4/80 to show macrophages. n = 6 per group. C1, Quantitative analysis of macrophages. D, D1, Flow cytometric analysis of CD11b⁺F4/80⁺ macrophages in the renal tissues from the different groups. n = 3 per group. The values are the mean ± SD, *P < .05 vs db/m; ^# P < .05 vs db/db; ^@ P < .05 vs db/db + 0.5 mg/kg TAC mice

Figure 4

Up‐regulated expression of nuclear factor of activated T cell 1 (NFATc1) and transient receptor potential channel 6 (TRPC6) in the kidney sections of db/db mice was attenuated with tacrolimus (TAC) treatment. A, Representative IHC images of NFATc1 and TRPC6 in the kidneys of mice. B, C, Semiquantification analysis of NFATc1 and TRPC6 expression determined by IHC. n = 6 per group. D, E, Real‐time PCR showed the relative mRNA expression of NFATc1 and TRPC6. n = 6 per group. F, Western blot analysis of NFATc1 and TRPC6. n = 3 per group. G, Relative band density of NFATc1 and TRPC6. The values are normalized by β‐actin. Values are the mean ± SD, *P < .05 vs db/m; ^# P < .05 vs db/db; ^@ P < .05 vs db/db + 0.5 mg/kg TAC mice. Real‐time PCR, real‐time polymerase chain reaction; IHC, immunohistochemistry

Figure 5

Increased nuclear factor of activated T cell 1 (NFATc1) and transient receptor potential channel 6 (TRPC6) expression positively correlated with tubulointerstitial inflammation in the kidney biopsy sections of type 2 diabetic nephropathy (DN) patients. A, Histological changes as shown by Haematoxylin‐eosin (HE), periodic acid‐Schiff (PAS) and Masson's staining. B, Immunohistochemistry (IHC) staining of IL‐6, NFATc1 and TRPC6. C1, C2, Quantitative analysis of interstitial fibrosis and tubular atrophy (IFTA) scores and glomerular injury. D1‐D3, Relative intensity of IL‐6, NFATc1 and TRPC6 in the kidney tissues of type 2 DN patients. E1‐E2, The correlations of NFATc1 and TRPC6 expression levels with IFTA scores. E3‐E4, The correlations between NFATc1, TRPC6 and IL‐6 expression in the kidney of type 2 DN patients. The values are the mean ± SD. *P < .05 vs control group. r: correlation coefficient. n = 10

Figure 6

Tacrolimus (TAC) suppressed nuclear factor of activated T cell 1 (NFATc1), transient receptor potential channel 6 (TRPC6) and extracellular matrix protein in HK‐2 cells incubated with high glucose (HG). A, Western blot analysis of TRPC6, fibronectin (FN), collagen 1 (Col‐1) and nuclear NFATc1 expression in HK‐2 cells with 30 mmol/L HG exposure for 0‐48 h. A1‐A4, Quantification of the average Western blot band intensity. B, The expression of TRPC6, FN, Col‐1 and nuclear NFATc1 in HK‐2 cells incubated with 30 mmol/L HG and 50‐400 nmol/L TAC was detected by Western blot. B1‐B4, Quantification of the average Western blot band intensity. The values are presented as the mean ± SD, *P < .05 vs LG; ^# P < .05 vs HG. n = 3

Figure 7

NFATc1 (nuclear factor of activated T cell 1)/TRPC6 (transient receptor potential channel 6) contributed to high glucose (HG)‐induced inflammation and apoptosis in HK‐2 cells treated with tacrolimus (TAC). A, The expression of IL‐6, fibronectin (FN), collagen 1 (Col‐1) and cleaved caspase 3 (C‐CAS3) in HK‐2 cells incubated with TAC or NFATc1 siRNA and treated with or without the TRPC6 overexpression plasmid under HG conditions was detected by Western blot. A1‐A3, Quantification of the average Western blot band intensity. B, C, The expression of NFATc1 and TRPC6 by real‐time PCR. D, Western blot analysis of TRPC6 and nuclear NFATc1 expression. D1‐D2, Quantification of the average intensity of NFATc1 and TRPC6 by Western blotting. E, Immunofluorescence staining of NFATc1 and TRPC6. The values are presented as the mean ± SD, *P < .05 vs low‐glucose (LG); ^# P < .05 vs HG; ^@ P < .05 vs HG + TAC. n = 3. Real‐time PCR, real‐time polymerase chain reaction

Figure 8

Diagram of the potential mechanism by which tacrolimus (TAC) protects against tubulointerstitial inflammation and fibrosis in diabetic nephropathy (DN). Nuclear factor of activated T cell 1 (NFATc1) is dephosphorylated and translocates to the nucleus in tubular cells in hyperglycaemia conditions. NFAT targets downstream genes, such as transient receptor potential channel 6 (TRPC6), and causes macrophage aggregation and inflammatory cytokine release, eventually leading to tubulointerstitial inflammation and fibrosis. Encouragingly, treatment with TAC significantly inhibits NFATc1 dephosphorylation and nuclear translocation and reduces the transcriptional activation of TRPC6, which ameliorates macrophage infiltration and inflammatory cytokine expression. On the other hand, TRPC6 also induces NFATc1 dephosphorylation and nuclear translocation. The positive feedback loop between NFATc1 and TRPC6 contributes to tubulointerstitial inflammation and injury in DN with TAC treatment