PFKFB3-mediated glycolysis boosts fibroblast activation and subsequent kidney fibrosis

Renal fibrosis, a hallmark of chronic kidney diseases, is driven by the activation of renal fibroblasts. Recent studies have highlighted the role of glycolysis in this process. Nevertheless, one critical glycolytic activator, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), remains unexplored in renal fibrosis. Upon reanalyzing the single-cell sequencing data from Dr. Humphreys' lab, we noticed an upregulation of glycolysis, gluconeogenesis, and TGFβ signaling pathway in myofibroblasts from fibrotic kidneys after unilateral ureter obstruction (UUO) or kidney ischemia/reperfusion. Furthermore, our experiments showed significant induction of PFKFB3 in mouse kidneys following UUO or kidney ischemia/reperfusion. To delve deeper into the role of PFKFB3, we generated mice with Pfkfb3 deficiency specifically in myofibroblasts (Pfkfb3f/fPostnMCM). Following UUO or kidney ischemia/reperfusion, a substantial decrease of fibrosis in injured kidneys of Pfkfb3f/fPostnMCM mice was identified compared to their wild-type littermates. Additionally, in cultured renal fibroblast NRK-49F cells, PFKFB3 was elevated upon exposure to TGFβ1, accompanied by the increase of α-SMA and fibronectin. Notably, this upregulation was significantly diminished with PFKFB3 knockdown, correlated with a glycolysis suppression. Mechanistically, the glycolytic metabolite lactate promoted the fibrotic activation of NRK-49F. In conclusion, our study demonstrates the critical role of PFKFB3 in driving fibroblast activation and subsequent renal fibrosis.

肾纤维化是慢性肾脏病的标志性病理特征，其发生由肾成纤维细胞的激活所介导。近期已有研究指出糖酵解在该病理过程中发挥重要作用。然而，作为关键糖酵解激活因子的6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶3（6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3，PFKFB3），其在肾纤维化中的功能仍未被阐明。我们重新分析汉弗莱斯实验室的单细胞测序数据集后发现，在单侧输尿管梗阻（unilateral ureter obstruction，UUO）或肾脏缺血再灌注损伤后，纤维化肾脏的肌成纤维细胞中糖酵解、糖异生及转化生长因子β（transforming growth factor β，TGFβ）信号通路均出现显著上调。此外，我们的实验证实，经UUO或肾脏缺血再灌注处理的小鼠肾脏中，PFKFB3的表达水平显著升高。为深入探究PFKFB3的具体功能，我们构建了肌成纤维细胞特异性敲除Pfkfb3的小鼠模型（Pfkfb3f/fPostnMCM）。与同窝野生型小鼠相比，经UUO或肾脏缺血再灌注处理后，Pfkfb3f/fPostnMCM小鼠损伤肾脏的纤维化程度显著降低。另外，在培养的肾成纤维细胞系NRK-49F中，经转化生长因子β1（TGFβ1）刺激后，PFKFB3的表达水平升高，同时α-平滑肌肌动蛋白（α-SMA）与纤连蛋白（fibronectin）的表达也随之增加。值得注意的是，敲低PFKFB3可显著抑制上述蛋白的上调，同时伴随糖酵解通路的活性抑制。机制研究表明，糖酵解代谢产物乳酸可促进NRK-49F细胞的纤维化激活。综上，本研究证实了PFKFB3在介导成纤维细胞激活及后续肾纤维化进程中的关键作用。