The sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that is now appreciated as key regulatory factor for various cellular functions in the kidney, including matrix remodeling. It is generated by two sphingosine kinases (Sphk), Sphk1 and Sphk2, which are ubiquitously expressed, but have distinct enzymatic activities and subcellular localizations. In this study, we have investigated the role of Sphk2 in podocyte function and its contribution to diabetic nephropathy. We show that streptozotocin (STZ)-induced nephropathy and albuminuria in mice is prevented by genetic depletion of Sphk2. This protection correlated with an increased protein expression of the transcription factor Wilm's tumor suppressor gene 1 (WT1) and its target gene nephrin, and a reduced macrophage infiltration in immunohistochemical renal sections of STZ-treated Sphk2^-/- mice compared to STZ-treated wildtype mice.

To investigate changes on the cellular level, we used an immortalized human podocyte cell line and generated a stable knockdown of Sphk2 (Sphk2-kd) by a lentiviral transduction method. These Sphk2-kd cells accumulated sphingosine as a consequence of the knockdown, and showed enhanced nephrin and WT1 mRNA and protein expressions similar to the finding in Sphk2 knockout mice. Treatment of wildtype podocytes with the highly selective Sphk2 inhibitor SLM6031434 caused a similar upregulation of nephrin and WT1 expression. Furthermore, exposing cells to the profibrotic mediator transforming growth factor β (TGFβ) resulted on the one side in reduced nephrin and WT1 expression, but on the other side, in upregulation of various profibrotic marker proteins, including connective tissue growth factor (CTGF), fibronectin (FN) and plasminogen activator inhibitor (PAI) 1. All these effects were reverted by Sphk2-kd and SLM6031434. Mechanistically, the protection by Sphk2-kd may depend on accumulated sphingosine and inhibited PKC activity, since treatment of cells with exogenous sphingosine not only reduced the phosphorylation pattern of PKC substrates, but also increased WT1 protein expression. Moreover, the selective stable knockdown of PKCδ increased WT1 expression, suggesting the involvement of this PKC isoenzyme in WT1 regulation.

The glucocorticoid dexamethasone, which is a treatment option in many glomerular diseases and is known to mediate a nephroprotection, not only downregulated Sphk2 and enhanced cellular sphingosine, but also enhanced WT1 and nephrin expressions, thus, suggesting that parts of the nephroprotective effect of dexamethasone is mediated by Sphk2 downregulation.

Altogether, our data demonstrated that loss of Sphk2 is protective in diabetes-induced podocytopathy and can prevent proteinuria, which is a hallmark of many glomerular diseases. Thus, Sphk2 could serve as a new attractive pharmacological target to treat proteinuric kidney diseases.

1-磷酸鞘氨醇 (S1P) 是一种具有生物活性的鞘脂，现在被认为是肾脏各种细胞功能（包括基质重塑）的关键调节因子。它由两种鞘氨醇激酶 (Sphk)、Sphk1 和 Sphk2 产生，它们普遍表达，但具有不同的酶活性和亚细胞定位。在这项研究中，我们研究了 Sphk2 在足细胞功能中的作用及其对糖尿病肾病的贡献。我们表明，Sphk2 的基因缺失可以防止链脲佐菌素 (STZ) 诱导的小鼠肾病和蛋白尿。这种保护与转录因子 Wilm 的肿瘤抑制基因 1 (WT1) 及其靶基因 nephrin 的蛋白质表达增加以及 STZ 处理的 Sphk2 的免疫组织化学肾切片中巨噬细胞浸润减少相关^-/-小鼠与 STZ 处理的野生型小鼠相比。

为了研究细胞水平的变化，我们使用了一种永生化的人足细胞系，并通过慢病毒转导方法产生了稳定的 Sphk2 (Sphk2-kd) 敲低。由于敲低，这些 Sphk2-kd 细胞积累了鞘氨醇，并显示出增强的 nephrin 和 WT1 mRNA 和蛋白质表达，类似于在 Sphk2 敲除小鼠中的发现。用高选择性 Sphk2 抑制剂 SLM6031434 处理野生型足细胞引起了 nephrin 和 WT1 表达的类似上调。此外，将细胞暴露于促纤维化介质转化生长因子 β (TGFβ) 一方面导致 nephrin 和 WT1 表达降低，另一方面导致各种促纤维化标志蛋白上调，包括结缔组织生长因子 (CTGF)，纤连蛋白 (FN) 和纤溶酶原激活物抑制剂 (PAI) 1. 所有这些作用都被 Sphk2-kd 和 SLM6031434 逆转。从机制上讲，Sphk2-kd 的保护可能取决于积累的鞘氨醇和抑制的 PKC 活性，因为用外源鞘氨醇处理细胞不仅降低了 PKC 底物的磷酸化模式，而且还增加了 WT1 蛋白的表达。此外，PKCδ的选择性稳定敲低增加了WT1的表达，表明该PKC同工酶参与了WT1的调节。但也增加了 WT1 蛋白的表达。此外，PKCδ的选择性稳定敲低增加了WT1的表达，表明该PKC同工酶参与了WT1的调节。但也增加了 WT1 蛋白的表达。此外，PKCδ的选择性稳定敲低增加了WT1的表达，表明该PKC同工酶参与了WT1的调节。

糖皮质激素地塞米松是许多肾小球疾病的治疗选择，已知可介导肾保护作用，不仅下调 Sphk2 和增强细胞鞘氨醇，而且增强 WT1 和 nephrin 表达，因此，表明地塞米松的部分肾保护作用是由 Sphk2 下调介导。

总之，我们的数据表明，Sphk2 的缺失对糖尿病引起的足细胞病具有保护作用，并且可以预防蛋白尿，这是许多肾小球疾病的标志。因此，Sphk2 可作为治疗蛋白尿性肾病的新药理学靶点。