Angiotensin II blockade upregulates the expression of Klotho, the anti-ageing gene, in an experimental model of chronic cyclosporine nephropathy

Abstract

Background: The Klotho gene plays a role in suppressing ageing-related disorders. It is suggested that activation of renin-angiotensin system (RAS) or oxidative stress suppresses Klotho in the kidney. This study evaluated the association between Klotho expression and RAS in cyclosporine (CsA)-induced renal injury.

Methods: Chronic CsA nephropathy was induced by administering CsA (30 mg/kg) to mice on a low-salt diet (LSD) for 4 weeks. A normal-salt diet (NSD) was used as the control. Reverse transcription-polymerase chain reaction, western blot and immunohistochemistry were performed for Klotho and intrarenal RAS activity was measured using immunohistochemistry for angiotensinogen and renin. Oxidative stress was measured with urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG).

Results: CsA treatment decreased Klotho mRNA and protein in mouse kidney in a dose-dependent and time-dependent manner, but a concurrent treatment with losartan, an angiotensin II type 1 (AT1) receptor blocker, reversed the decrease in Klotho expression with histological improvement. This finding was more marked in the LSD than the NSD. Klotho expression was correlated with angiotensinogen and renin expression, tubulointerstitial fibrosis score and urinary 8-OHdG excretion.

Conclusions: Angiotensin II may play a pivotal role in regulating Klotho expression in CsA-induced renal injury. AT1 receptor blocker may inhibit the ageing process by decreasing oxidative stress caused by CsA.

Fig. 1

Localization of Klotho in normal mouse kidneys. (A) Immunohistochemistry for Klotho. Klotho was expressed in the tubules of the renal cortex (a; a-1, a-2, a-3), but not in the outer medulla (b) or the inner medulla (c). (B) Double staining for Klotho and other tubular proteins (a. aquaporin-1, b. calbindin D_28k, c. aquaporin-2). Klotho co-localized with calbindin D_28k (b; b’, arrows) in distal tubules, but not with aquaporin-1 (a; a’, arrowheads) in proximal tubules or aquaporin-2 (c; c’, arrowheads) in collecting ducts.

Fig. 2

Influence of salt intake and angiotensin II blockade on Klotho expression in normal mice kidneys. Western blot for Klotho. Klotho expression was significantly decreased in the low-salt diet (LSD4) group compared with the normal-salt diet (NSD4) group. The administration of losartan (LSRT) tended to increase Klotho expression both in the NSD4 and LSD4 groups. *P = 0.008 vs NSD4; **P = 0.027 vs NSD + LSRT4 (n = 6 for each group). NSD4 = normal-salt diet for 4 weeks, NSD + LSRT4 = normal-salt diet with addition of losartan for 4 weeks, LSD4 = low-salt diet for 4 weeks, LSD + LSRT4 = low-salt diet with addition of losartan for 4 weeks.

Fig. 3

The dose-dependent effect of cyclosporine (CsA) on expression of Klotho and renin in mouse kidneys. (A) Western blot for Klotho in CsA-treated mice with different doses. The dose-dependent decrease in Klotho was significant at 15 and 30 mg/kg of CsA. (B) Representative photomicrographs of immunohistochemical staining for renin. The number of renin-positive glomeruli increased with an increase in CsA doses. (C) The association between Klotho expression and intrarenal renin expression. Klotho expression was negatively correlated with the intrarenal renin immunoreactivity (r = − 0.84, P < 0.01). *P = 0.000 vs VH, **P = 0.001 vs CsA7.5, † P = 0.000 vs CsA7.5, ‡ P = 0.000 vs CsA15 (n = 6 for each group). VH = vehicle for 4 weeks, CsA7.5 = cyclosporine (7.5 mg/kg) for 4 weeks, CsA15 = cyclosporine (15 mg/kg) for 4 weeks, CsA30 = cyclosporine (30 mg/kg) for 4 weeks.

Fig. 4

The time-dependent effect of cyclosporine (CsA) on Klotho mRNA and protein expression in the mouse kidney. (A) RT–PCR for Klotho mRNA in CsA-treated mice on the normal-salt diet (NSD). Klotho mRNA expression was significantly decreased by CsA treatment for 1 week and further decreased by CsA treatment for 4 weeks. (B) RT–PCR for Klotho mRNA in CsA-treated mice on the low-salt diet (LSD). The time-dependent decrease in Klotho mRNA was more apparent in the LSD mice than the NSD mice. (C) Western blot for Klotho in CsA-treated mice on the NSD. CsA treatment for 1 week did not significantly decrease Klotho expression, but CsA treatment for 4 weeks did. (D) Western blot for Klotho in CsA-treated mice on the LSD. The time-dependent decrease in Klotho was significant after CsA treatment for 1 week and was more apparent in the LSD mice than the NSD mice. *P = 0.033 vs VH1, **P = 0.000 vs VH1, #P = 0.000 vs CsA1, † P = 0.000 vs VH4 (n = 6 for each group). VH1 = vehicle for 1 week, CsA1 = cyclosporine for 1 week, VH4 = vehicle for 4 weeks, CsA4 = cyclosporine for 4 weeks.

Fig. 5

Immunohistochemical findings for Klotho expression under cyclosporine (CsA) treatment in relation to salt intake. (A) Immunohistochemistry for Klotho in CsA-treated mice on the normal-salt diet. CsA treatment for 1 week minimally decreased and CsA treatment for 4 weeks markedly decreased Klotho expression. (B) Immunohistochemistry for Klotho in CsA-treated mice on the low-salt diet. CsA treatment for 1 week markedly decreased Klotho expression, while CsA treatment for 4 weeks almost completely abolished expression (n = 6 for each group). VH1 = vehicle for 1 week, CsA1 = cyclosporine for 1 week, VH4 = vehicle for 4 weeks, CsA4 = cyclosporine for 4 weeks.

Fig. 6

The effect of angiotensin II blockade on Klotho expression in cyclosporine (CsA)-treated mice in relation to salt intake. (A) Western blot for Klotho in CsA-treated mice on the normal-salt diet (NSD). Klotho expression was significantly decreased by CsA treatment for 4 weeks compared with the VH4 group and VH + LSRT4 group. With the concurrent administration of losartan (LSRT), Klotho expression was significantly increased compared with the CsA4 group. (B) Western blot for Klotho in CsA-treated mice on the low-salt diet (LSD). The decrease in Klotho expression caused by CsA treatment and the increase caused by concurrent administration of LSRT were more apparent in mice on the LSD than those on the NSD. *P = 0.032 vs VH4, **P = 0.047 vs VH + LSRT4, #P = 0.001 vs CsA4, † P = 0.003 vs VH4, ‡ P = 0.000 vs VH + LSRT4, †† P = 0.000 vs CsA4 (n = 6 for each group). VH4 = vehicle for 4 weeks, VH + LSRT4 = vehicle + losartan for 4 weeks, CsA4 = cyclosporine for 4 weeks, CsA + LSRT4 = cyclosporine + losartan for 4 weeks.

Fig. 7

The association between tubulointerstitial fibrosis (TIF) and Klotho expression in chronic cyclosporine (CsA) nephropathy. (A) Histological findings in CsA- and losartan (LSRT)-treated mice on the low-salt diet. CsA-treated mice at 4 weeks showed diffuse interstitial fibrosis, mononuclear cell infiltrates and tubular atrophy. The administration of LSRT dramatically decreased this damage. Magnification, × 200. (B) Semiquantitative analysis of TIF in CsA- and LSRT-treated mice. The TIF score was significantly increased by CsA treatment and decreased by the concurrent administration of LSRT. (C) The association between Klotho expression and TIF score. Klotho expression was negatively correlated with the TIF score (r = − 0.69, P < 0.01). *P = 0.000 vs VH4, **P = 0.000 vs VH + LSRT4, † P = 0.016 vs VH4, ‡ P = 0.016 vs VH + LSRT4, †† P = 0.001 vs CsA4 (n = 5 for VH4, VH + LSRT4 and CsA4 groups; n = 4 for CsA + LSRT4 group). VH4 = vehicle for 4 weeks, VH + LSRT4 = vehicle + losartan for 4 weeks, CsA4 = cyclosporine for 4 weeks, CsA + LSRT4 = cyclosporine + losartan for 4 weeks.

Fig. 8

The association between angiotensinogen (AGT) expression and Klotho expression in chronic cyclosporine (CsA) nephropathy. (A) Representative photomicrographs of immunohistochemical staining for AGT. In every case, immunoreactive areas were confined to mostly cortical brush border-rich tubules (proximal tubules). In the CsA4 group on the normal-salt diet (NSD), immunoreactivity for AGT was enhanced over the VH4 group. This increase was more prominent in mice on the low-salt diet (LSD) than those on the NSD. In the CsA + LSRT4 group, the concurrent administration of losartan (LSRT) reduced the immunoreactivity for AGT compared with the CsA4 groups, on both the NSD and the LSD. Magnification, × 200. (B) Semiquantitative analysis of the intensity of intrarenal AGT immunoreactivity. On the NSD, CsA treatment increased intrarenal AGT immunoreactivity compared with the VH4 group, and the concurrent administration of LSRT decreased it compared with the CsA4 group, although this was not significant. The CsA4 group on the LSD showed a significantly greater AGT immunoreactivity than all groups on the NSD, as well as the VH and VH + LSRT4 groups on the LSD. Concurrent LSRT administration markedly decreased AGT expression compared with the CsA4 group. (C) The association between Klotho expression and AGT immunoreactivity. Klotho expression was negatively correlated with the AGT immunoreactivity (r = − 0.77, P < 0.01). *P < 0.05 vs NSD, #P = 0.000 vs VH4, ##P = 0.000 vs VH + LSRT4, † P = 0.000 vs CsA4 (n = 4 for each group). VH4 = vehicle for 4 weeks, VH + LSRT4 = vehicle + losartan for 4 weeks, CsA4 = cyclosporine for 4 weeks, CsA + LSRT4 = cyclosporine + losartan for 4 weeks.

Fig. 9

The association between oxidative stress and Klotho expression in chronic cyclosporine (CsA) nephropathy. (A) The urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) excretion of CsA- and losartan (LSRT)-treated mice in relation to salt intake. The urinary 8-OHdG excretion in the CsA4 group was higher than that in the VH4 group, and this was more significant in mice on the low-salt diet (LSD) than those on the normal-salt diet (NSD). The concurrent administration of LSRT significantly decreased urinary 8-OHdG excretion compared with the CsA4 group, in mice on the LSD. (B) The association between Klotho expression and urinary 8-OHdG excretion. Klotho expression was negatively correlated with the urinary 8-OHdG level (r = − 0.64, P < 0.01). *P = 0.003 vs VH4, **P = 0.006 vs VH + LSRT4, #P = 0.026 vs CsA (n = 5 for each group). VH4 = vehicle for 4 weeks, VH + LSRT4 = vehicle + losartan for 4 weeks, CsA4 = cyclosporine for 4 weeks, CsA + LSRT4 = cyclosporine + losartan for 4 weeks.

Fig. 10

Klotho expression in renal insufficiency with or without cyclosporine (CsA). Klotho expression significantly decreased in Sham + CsA and 5/6NX groups compared to Sham group and further decreased in 5/6NX + CsA group compared to the other three groups. *P = 0.000 vs Sham, #P = 0.001 vs Sham + CsA, † P = 0.004 vs 5/6NX (n = 6 for each group). Sham = sham operation and vehicle treatment for 4 weeks, Sham + CsA = sham operation and CsA treatment for 4 weeks, 5/6NX = 5/6 nephrectomy and vehicle treatment for 4 weeks, 5/6NX + CsA = 5/6 nephrectomy and CsA treatment for 4 weeks.