The role of Src family kinases in chronic kidney disease

Abstract

Chronic kidney disease (CKD) is emerging worldwide, and its high prevalence implies that the current therapeutic approach cannot adequately prevent the progression of CKD. Therefore, identifying and validating novel therapeutic targets are paramount to develop novel agents that can halt the progression of CKD. Growing evidence suggests that Src family kinases (SFKs) might be a promising target for treatment of CKD. The role of Src kinase has been vastly elucidated in the development of kidney fibrosis. Src and Fyn kinase have also been shown to modulate important cellular homeostasis signaling, such as ER stress, as well as nutrient sensing pathway, i.e., AMPK and mTOR in skeletal muscle and adipose tissues. However, the mechanism of SFKs in mediating the pathogenesis of CKD remains unclear. It also remains interesting to develop a relatively safe pan-Src kinase inhibitor (SKI) and to evaluate the efficacy of chronic pan-SKI treatment in CKD. A preliminary study with dataset from human diabetic kidney suggested that unfolded protein response-related genes were elevated along with Fyn gene, and ER stress was one the most enriched pathways in the diabetic kidney. Fyn knockdown demonstrated inhibition on P70S6 kinase and JNK/CHOP signaling pathway in mouse proximal tubular cells exposed to high glucose and palmitate (HG-Pal). This eventually suppressed tubular cell apoptosis, indicated by a decrease in cleaved caspase 3 protein expression. Fyn knockdown also modulated the AMPKα protein expression that might affect mTORC1 activity. The modulation in P70S6 kinase, JNK/CHOP, as well as apoptosis were interestingly not found in Lyn knockdown in HG-Pal exposed mouse proximal tubular cells. Thus, Fyn kinase might mediate the progression of diabetic kidney disease (DKD), in part through exacerbation of ER stress signaling. Next, a new scaffold of SKI was created by maintaining the backbone of ponatinib. KF-1607 is a new orally active compound and a potent inhibitor of multiple member of SFKs. It has a relatively lower toxicity compared to ponatinib, indicated by in vitro kinetic cytotoxicity assay, and a low potency of cardiotoxicity from hERG safety assay. The efficacy of KF-1607 was then verified in the CKD experimental model, including DKD and unilateral ureteral obstruction (UUO) injury, in which elevated expressions of multiple SFK members were demonstrated. Streptozotocin-induced diabetic rats received 8-week-treatment of either KF-1607 or losartan, a standard treatment for patients with DKD. Diabetic rats treated with this SKI showed attenuation in inflammation, oxidative stress, as well as structural and functional evidences of progressive DKD, in a similar trend as those treated with losartan. The renoprotective effects of SFKs inhibition are in part through attenuation of ER stress signaling. Furthermore, the pan SKI treatment attenuated kidney fibrosis, a final common outcome of kidney injuries, in both diabetic rats and mice with UUO. The progression of renal tubular injury, inflammation, and tubulointerstitial fibrosis was significantly prevented in UUO mice treated with KF-1607, as much as those with PP2 treatment. In conclusion, SFKs are demonstrated to mediate the progression of DKD, in part through exacerbation of ER stress. Fyn knockdown in the in vitro system suggests that this specific member of SFKs activates mTORC1 and ER stress signaling leading to proximal tubular injury under diabetic milieu. Furthermore, KF-1607 is a newly synthesized pan-SKI that effectively prevents the progression of CKD in rodent models with diabetic and obstructed kidneys. Hence, this present study emphasizes that SFKs is a viable avenue for developing therapeutic agents against CKD.;만성콩팥병(chronic kidney disease: CKD)은 현대 고령화사회에서 유행병으로 대두되고 있어, CKD의 발생과 진행 기전을 밝히고 이에 근거한 치료전략을 개발하는 것이 시기적으로 중요한 숙제이다. CKD의 최종 병변인 콩팥 섬유화의 진행에 Src family kinases (SFKs)가 관여한다는 보고는 SFKs가 CKD 치료의 신규 타겟일 수 있음을 시사한다. 한편, Src과 Fyn kinase는 골격근과 지방조직에서 소포체(ER) 스트레스와 AMPK/mTOR 경로 변화에 관여한다. 그러나 SFKs에 의한 CKD 발병기전을 보다 명확하게 이해하고, CKD와 같은 만성질환을 타겟한, 보다 안전한, SFK 저해제 개발이 필요하다. 당뇨병성콩팥병(diabetic kidney disease: DKD) 환자의 빅데이터 분석 결과, DKD 콩팥내 Fyn 유전자는 unfolded protein response (UPR)에 관여하는 유전자와 함께 그 발현이 상향조절되어 있었고 ER 스트레스는 가장 활성화된 경로 중 하나였다. Fyn siRNA는 mProx 세포(근위세뇨관 상피세포주)에서 고농도의 포도당과 팔미틴산에 의한 P70S6 kinase와 JNK/CHOP 활성화를 효과적으로 억제하였고, 나아가 caspase 3 단백의 절단을 억제하여 apoptosis를 억제하였다. Fyn siRNA는 AMPKα 활성화와 mTORC1억제도 유발하였다. 이상의 배양세포 실험결과는 SFKs중 Fyn kinase가 ER 스트레스를 악화시킴으로써 DKD의 발생과 진행에 관여함을 시사하였다. 본 연구에서는 ponatinib을 기반한 구조변경을 통하여 새로운 골격의 경구투여가 가능한 SFKs 저해제를 개발하였다. KF-1607는 세포독성이 상대적으로 낮았고 hERG 저해 작용이 매우 낮았다. 나아가, KF-1607는 DKD의 발생을 억제하였고, 한쪽요관폐쇄(unilateral ureteral obstruction: UUO)에 의한 콩팥섬유화를 효과적으로 억제하였디. Streptozotocin으로 제1형 당뇨병을 유발한 흰쥐에 8주동안 KF-1607이나 DKD 환자 치료약인 losartan을 투여하였을 때, KF-1607은 당뇨 콩팥에서 증가한 염증반응, 산화성스트레스 및 콩팥의 구조적 기능적 변화를 losartan 만큼 억제하였다. KF-1607은 당뇨 콩팥의 ER 스트레스 역시 억제하였고, CKD의 최종병변인 섬유화 역시 DKD와 UUO 콩팥에서 억제하였다. KF-1607은 요관폐쇄에 의한 콩팥 세뇨관 손상, 염증반응, 세뇨관간질 섬유화를 pan-SFK 저해제인 PP2 만큼 효과적으로 억제하였다. 종할할 때 , SFKs 중 특히 Fyn kinase는 ER 스트레스를 경유하여 DKD를 유발하고 이 과정에 mTORC1 활성화가 관여하는 듯 하다. 새로운 SFK 저해제인 KF-1607는 경구투여시 설치류에서의 DKD와 UUO를 효과적으로 억제하였다. 즉, 본 연구 결과는 Fyn kinase를 비롯한 SFKs가 CKD 치료를 위한 새로운 타겟이 될 수 있음을 시사하였다.