Tetramethylpyrazine alleviates acute kidney injury by activating the Wnt/β-catenin pathway independent of DKK1

Abstract

Acute kidney injury (AKI) is a group of common clinical syndromes characterized by a rapid decline in renal function over a short period of time. At present, the treatment methods are limited, and research is needed to identify drugs that could alleviate renal ischemia-reperfusion (I/R) injury. Tetramethylpyrazine (TMP) is a bioactive alkaloid extracted from the Chinese herbal medicine Chuanxiong. TMP is known to possess various anti-inflammatory and cardiovascular and renal protective effects; however, the therapeutic molecular targets are still unclear. In the present study, using a rat renal I/R model, the effects of TMP on renal injury, dickkopf-1 (DKK1) expression, Wnt/β-catenin signaling and apoptosis were evaluated through morphological examination, renal function tests, western blotting, immunohistochemistry and TUNEL assays. It was determined that TMP ameliorated tubular pathologic injury and improved renal function in rats following renal I/R. In addition, in rats following I/R, TMP promoted the expression of DKK1, an inhibitor of the Wnt/β-catenin signaling pathway, in renal tissues, activated the Wnt/β-catenin signaling pathway in kidney tissues and reduced apoptosis of renal cells. To the best of our knowledge, the present study is the first to investigate the regulatory effects of TMP on DKK1 and the Wnt/β-catenin signaling pathway, revealing that TMP could attenuate AKI by activating Wnt/β-catenin signaling independent of the inhibitory effect of DKK1.

Keywords: Wnt/β-catenin pathway; dickkopf-1; inflammation; renal ischemia-reperfusion injury; tetramethylpyrazine.

Figure 1

TMP protects against acute kidney injury by alleviating renal tubular pathological injury and improving renal function following I/R in a rat model. (A) Serum creatinine and (B) blood urea nitrogen levels of rats in different groups were measured using a biochemical analyzer. n=6. (C) Body weight in different groups. n=6. (D) Representative H&E images showing the renal morphological changes (black arrows) and renal tubular damage scores of rats in different groups. Scale bars, 20 µm, magnification, x400. n=10. ^*P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001. I/R, ischemia-reperfusion; TMP, tetramethylpyrazine; H&E hematoxylin and eosin.

Figure 2

TMP promotes the expression of DKK1 in the renal tissue of rats following I/R. (A) Representative western blots and semi-quantified data showing the expression of DKK1 in the renal tissue of rats in the sham, I/R and I/R + TMP groups. n=6. (B) Representative IHC images of DKK1 in the renal tissue of rats in the sham, I/R and I/R + TMP groups, and semi-quantification. n=3. IHC images: Top row, scale bars, 200 µm, magnification, x40; bottom row, scale bars, 20 µm, magnification, x400. ^*P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001. I/R, ischemia-reperfusion; TMP, tetramethylpyrazine; DKK1, dickkopf-1; IHC, immunohistochemistry.

Figure 3

TMP activates the Wnt/β-catenin signaling pathway in the kidney of rats following I/R. (A) Representative western blots and semi-quantified protein levels showing the expression levels of Wnt1 in the renal tissue of rats in different groups. n=6. (B) Representative IHC images and protein semi-quantification of Wnt1 in the renal tissue of rats in the sham, I/R and I/R + TMP groups. n=3. (C) Representative western blots and semi-quantified protein levels showing the expression levels of β-catenin in the renal tissue of rats in different groups. n=6. (D) Representative IHC images and protein semi-quantification of β-catenin in the renal tissue of rats in the sham, I/R and I/R + TMP groups. n=3. IHC images: Top row, scale bars, 200 µm, magnification, x40; bottom row, scale bars, 20 µm, magnification, x400. ^*P<0.05, ^**P<0.01, ^****P<0.0001. I/R, ischemia-reperfusion; TMP, tetramethylpyrazine; IHC, immunohistochemistry.

Figure 4

TMP ameliorates renal cell apoptosis after I/R in rats. (A) TUNEL staining showing the renal cell apoptosis levels in different groups. Scale bars, 50 µm. n=3. Representative IHC images and protein semi-quantification of (B) Bax and (C) caspase-3 in renal tissue of rats in different groups. n=3. (D) Relative ratio of cleaved caspase-3/caspase-3 determined by western blotting. n=6. (E) Representative images and protein semi-quantification of Bcl-2 in the renal tissue of rats in different groups. n=3. (F) Representative western blots and semi-quantified protein levels showing the expression levels of Bcl-2 in the renal tissue of rats in different groups. n=6. IHC images: Top row, scale bars, 200 µm, magnification, x40; bottom row, scale bars, 20 µm, magnification, x400. ^*P<0.05, ^**P<0.01, ^***P<0.001. I/R, ischemia-reperfusion; TMP, tetramethylpyrazine; IHC, immunohistochemistry.