Klotho and activin A in kidney injury: plasma Klotho is maintained in unilateral obstruction despite no upregulation of Klotho biosynthesis in the contralateral kidney

Abstract

In a new paradigm of etiology related to chronic kidney disease-mineral and bone disorder (CKD-MBD), kidney injury may cause induction of factors in the injured kidney that are released into the circulation and thereby initiate and maintain renal fibrosis and CKD-MBD. Klotho is believed to ameliorate renal fibrosis and CKD-MBD, while activin A might have detrimental effects. The unilateral ureter obstruction (UUO) model is used here to examine this concept by investigating early changes related to renal fibrosis in the obstructed kidney, untouched contralateral kidney, and vasculature which might be affected by secreted factors from the obstructed kidney, and comparing with unilateral nephrectomized controls (UNX). Obstructed kidneys showed early Klotho gene and protein depletion, whereas plasma Klotho increased in both UUO and UNX rats, indicating an altered metabolism of Klotho. Contralateral kidneys had no compensatory upregulation of Klotho and maintained normal expression of the examined fibrosis-related genes, as did remnant UNX kidneys. UUO caused upregulation of transforming growth factor-β and induction of periostin and activin A in obstructed kidneys without changes in the contralateral kidneys. Plasma activin A doubled in UUO rats after 10 days while no changes were seen in UNX rats, suggesting secretion of activin A from the obstructed kidney with potentially systemic effects on CKD-MBD. As such, increased aortic sclerostin was observed in UUO rats compared with UNX and normal controls. The present results are in line with the new paradigm and show very early vascular effects of unilateral kidney fibrosis, supporting the existence of a new kidney-vasculature axis.

Keywords: Klotho; Wnt; activin A; renal fibrosis; unilateral ureter obstruction.

Fig. 1.

Plasma Klotho in unilateral ureter obstruction (UUO), unilateral nephrectomized (UNX), and healthy control rats and kidney Klotho mRNA and protein expression in obstructed and contralateral kidneys from UUO and UNX rats. A: plasma Klotho (p-Klotho). Rats with only one functioning kidney demonstrated an increase in plasma Klotho from day 1 and forward, as all UUO and UNX rats had elevated plasma Klotho levels, P < 0.05 compared with controls. No difference was observed between UUO and UNX rats at any time points; n = 6 in all groups. B–F: Klotho protein expression. Representative Western blot from baseline kidneys compared with obstructed kidneys on days 1 and 3 (B), obstructed kidney on days 4 and 10 (C), contralateral kidney on days 1 and 3 (D), and contralateral kidney on days 4 and 10 (E). Each lane represents individual rats. Protein and molecular size markers are presented at each blot. Lane 4 in B is excluded because of a lane-specific error; rKl, recombinant Klotho; M, protein molecular weight marker. F: quantification of all Western blots depicted as Klotho in relation to GAPDH with baseline normalized to 1; n = 6 in all subgroups. Renal Klotho protein expression decreases continuously over time in obstructed kidneys with an early onset already on day 1 (P < 0.05) and a subsequent more pronounced decline (P < 0.0001). In the contralateral kidneys no difference was found on days 1, 3, 4, and 10. G: Klotho (Kl) mRNA expression. Unilateral ureter obstruction resulted in a rapid and gradual decline in Klotho gene expression in the obstructed kidney with onset already on day 1 (P < 0.0001) and nadir on day 10 (P < 0.0001). No compensatory upregulation was present in the contralateral kidney. The contralateral kidney showed no change in renal gene expression of Klotho at any time points, and no difference could be observed compared with remnant and baseline UNX kidneys; n = 30 in the baseline (Base) group, and n = 6 in all UNX and UUO groups. Data are presented as means ± SE. Significantly different from baseline: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2.

Transforming growth factor (TGF)-β, periostin, and bone morphogenetic protein-7 (BMP7) mRNA expression in obstructed and contralateral kidneys from unilateral ureter obstruction (UUO) rats, remnant kidneys from unilateral nephrectomized (UNX) rats, and normal baseline kidneys. A: TGF-β (Tgfb1) mRNA expression increased in the obstructed kidney on day 1 (P < 0.001) and continued to rise throughout the study with a peak on day 10 (P < 0.0001). No significant differences were present in contralateral kidneys, which had expression of TGF-β similar to that of remnant and baseline kidneys. B: periostin (Postn) was undetectable in healthy baseline kidneys. However, unilateral ureter obstruction caused an extreme induction of renal periostin mRNA expression appearing on day 1 (P < 0.0001), peaking on day 2 (P < 0.0001) and subsequently declining to levels still substantially higher than baseline (P < 0.0001). Contralateral kidneys from UUO rats and remnant kidneys from UNX rats were untraceable, similar to baseline. C: the antifibrotic BMP7 (Bmp7) mRNA expression was downregulated in obstructed kidneys from day 1 (P < 0.001) and forward (P < 0.05) stabilizing around half the expression level of baseline kidneys. Contralateral kidneys maintained its expression of BMP7 within normal range, similar to remnant UNX and baseline kidneys; n = 30 in the baseline (Base) group, and n = 6 in all other subgroups. Data are presented as means ± SE. Significantly different from baseline: *P < 0.05, ***P < 0.001, ****P < 0.0001.

Fig. 3.

Plasma activin A levels in normal, unilateral nephrectomized (UNX), and unilateral ureter obstruction (UUO) rats and kidney activin A mRNA expression in obstructed and contralateral kidneys from UUO rats, remnant kidneys from UNX rats, and baseline normal kidneys. A: plasma activin A (p-activin). After 10 days of obstruction, UUO rats had an almost doubling of activin A plasma levels compared with baseline (P < 0.0001). No increase was present on days 1 and 3 in UUO rats, and UNX rats did not show elevation in plasma activin A at any time points; n = 4–7. B: activin A (Inhba) mRNA expression. Unilateral ureter obstruction resulted in a clear induction of renal activin A mRNA on day 1 (P < 0.0001), which increased continuously with a peak on day 10 (P < 0.0001). Simultaneously, activin A was untraceable in contralateral, remnant, and baseline kidneys; n = 4–6. Data are presented as means ± SE. Significantly different from baseline (Base): ****P < 0.0001.

Fig. 4.

Increased aortic sclerostin mRNA expression in unilateral ureter obstruction (UUO) rats and plasma sclerostin levels in normal, unilateral nephrectomized (UNX), and UUO rats. A: plasma sclerostin (p-sclerostin) increased in both UUO and UNX rats to similar levels (P < 0.05). No differences were present between UUO and UNX at any time points; n = 4–6. B: sclerostin (Sost) mRNA expression in thoracic aorta. After 15 days of unilateral ureter obstruction, sclerostin mRNA expression increased markedly compared with both UNX and Sham rats (P < 0.01). There were no differences in sclerostin expression between UNX and Sham rats; n = 5. Data are presented as means ± SE. Significantly different from baseline (Base): *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5.

Transforming growth factor (TGF)-β, periostin, and activin A mRNA expression in thoracic aorta from unilateral ureter obstruction (UUO), unilateral nephrectomized (UNX), and Sham rats. A: TGF-β (Tgfb1) mRNA expression in thoracic aorta from UUO, UNX, and Sham rats after 15 days. Aortic TGF-β mRNA did not increase significantly in either UUO or UNX rats although the results suggest a tendency toward a rise in both groups. B: periostin (Postn) mRNA expression in thoracic aorta from UUO, UNX, and Sham rats after 15 days. No changes in aortic periostin gene expression were present between UUO, UNX, and Sham rats. C: activin A (Inhba) mRNA expression in thoracic aorta from UUO, UNX, and Sham rats after 15 days. Similar levels of activin A gene expression were present in all groups; n = 4–5 in all groups. Data are presented as means ± SE.