Differential gene expression in renal cortex resulting from SPAK gene disruption

Poorly defined adaptive processes maintain salt balance when the renal thiazide-sensitive sodium-chloride cotransporter is inhibited, limiting diuretic efficacy. Here, we identify underlying mechanisms in SPAK kinase null mice, which are unable to phospho-activate NCC. Global transcriptional profiling, combined with biochemical, cell biological and physiological phenotyping, identified the gene expression signature of the response, and revealed how it establishes a new adaptive physiology. Salt reabsorption pathways are created by the coordinate induction of a multi-gene transport system, involving solute carriers (Slc26a; Slc4a8; Slc4a9), carbonic anhydrase isoforms, and V-type H+-ATPase subunits in pendrin-positive intercalated cells (PP-IC), and ENaC subunits in principal cells. A distal nephron remodeling process and induction of Jagged 1-Notch signaling, which expands the cortical connecting tubule with principal cells and replaces acid-secreting α- intercalated cells with PP-IC, is partly responsible. Salt reabsorption is also activated by induction of an alpha-ketoglutarate (α-KG) paracrine signaling system. Coordinate regulation of a multigene α-KG synthesis and transport pathway cause α-KG to be secreted into the pro-urine as the α-KG activated GPCR (Oxgr1) increases on the PP-IC apical surface, allowing paracrine delivery of α-KG to stimulate salt transport. Identification of the integrated compensatory NaCl reabsorption mechanisms provides new insights into thiazide diuretic efficacy. Gene transcrtipt isolated form the renal cortex of 4 SPAK KO and 4 litter-mate WT animals was compared to evaluate differential gene expression with the goal of identifing a nextwork of genes that function cooperatively compensatory to mitigate renal salt loss when the SPAK function is lost.

当肾脏噻嗪类敏感性钠氯协同转运蛋白（renal thiazide-sensitive sodium-chloride cotransporter，NCC）受到抑制时，机体通过调控机制尚不明确的适应性代偿过程维持盐平衡，这一现象限制了噻嗪类利尿剂的临床效用。本研究以无法磷酸化激活NCC的SPAK激酶（SPAK kinase）敲除小鼠为模型，解析了该适应性代偿反应的潜在分子机制。我们通过全局转录组分析结合生化、细胞生物学与生理表型鉴定，明确了该适应性反应的基因表达特征谱，并揭示了其如何构建全新的适应性生理状态。在pendrin阳性闰细胞（PP-IC）中，通过协同诱导多基因转运系统（涵盖溶质转运蛋白家族成员Slc26a、Slc4a8、Slc4a9、碳酸酐酶同工型以及V型H+-ATP酶亚基）的表达，同时在主细胞中诱导上皮钠通道（ENaC）亚基的表达，从而构建了盐重吸收通路。远端肾单位重塑过程与Jagged1-Notch信号通路的激活同样发挥了部分代偿作用：该通路可扩增皮质连接小管的主细胞群体，并将泌酸α闰细胞替换为PP-IC。盐重吸收还可通过激活α-酮戊二酸（α-KG）旁分泌信号系统得以增强：多基因α-KG合成与转运通路的协同调控，使得α-KG被分泌至原尿中；与此同时，PP-IC顶膜表面的α-KG激活型G蛋白偶联受体（Oxgr1）表达上调，从而介导α-KG的旁分泌递送以刺激盐转运过程。本研究鉴定出的整合性代偿性NaCl重吸收机制，为理解噻嗪类利尿剂的临床疗效提供了全新视角。为鉴定SPAK功能缺失时协同发挥代偿作用以减轻肾脏盐丢失的基因调控网络，我们对4只SPAK敲除（SPAK KO）小鼠与4只同窝野生型（WT）小鼠的肾皮质分离得到的转录本进行了差异基因表达分析。