RIP 1 kinase inhibition for acute ischemic kidney injury
Item statusRestricted Access
Embargo end date30/11/2021
Gallagher, Kevin Michael
Background: Acute ischemic kidney injury occurs in many clinical settings such as transplantation, major surgery, haemorrhage, dehydration and accounts for major mortality and morbidity in these settings. Acute kidney injury can lead to permanent loss of kidney function (chronic kidney disease, CKD) which is a major cause of long-term morbidity. In acute ischemic kidney injury, renal tubular epithelial cells are injured and necrose. Regulated necrosis may be important in renal tubular cell death in acute ischemic kidney injury. Receptor interacting serine/threonine protein kinase 1 (RIPK1) is a crucial mediator of a form of regulated necrosis called necroptosis. Project aims: -To determine if specific RIPK1 inhibition is beneficial in acute ischemic kidney injury. -To determine if there is evidence of necroptosis in kidney in vitro and in vivo after ischemic injury. -To determine if RIPK1 inhibition could reduce the development of CKD after AKI. Methods: Novel, specific RIPK1 inhibitors (GSK’547 and GSK’963) and a RIPK1 kinase inactive transgenic mouse were used in the following models of ischemia reperfusion injury: In vitro: HK2 proximal tubular epithelial cell (PTEC) line injury with antimycin A, tert-butyl hydroperoxide and mineral oil oxygen exclusion. In vivo in mice: Ischemia reperfusion injury (IRI) for acute kidney injury (AKI) assessment (at 24 and 48 hours), IRI for chronic kidney disease and fibrosis (28 days) and unilateral ureteric obstruction (7 days). Normal human tissue sections were obtained from tumour nephrectomy specimens and analysis of a publicly available transplant organ sequencing dataset was undertaken to provide human validation. Specific RIPK1 inhibition (targeting necroptosis) was compared against inhibition of another form of regulated necrosis (ferroptosis) with ferrostatin-1 (Fer-1) or liproxstatin-1 (LPX-1) in all experiments. In vitro: Quantitative cell death assays were performed. GSK’963 was compared against Fer-1 and other inhibitors of cell death. Flow cytometry, microscopy and caspase activation assays were used to differentiate apoptosis from necrosis in vitro. The effect of RIPK1 inhibition on mitochondrial membrane integrity in in vitro ischemic injury was assessed using MitoTracker Red fluorescent microscopy. Necroptosis was identified in vitro by immunocytochemical staining for RIPK3, total MLKL and phosphorylated MLKL. Ferroptosis was identified in vitro using Bodipy lipid peroxidation staining. In vivo: Kidney injury was assessed using serum creatinine, urea, and histological tubular necrosis scoring. Necroptosis was detected by real-time quantitative PCR (qPCR) for RIPK1, RIPK3 and MLKL and immunohistochemical (IHC) staining for phosphorylation of the necroptosis effector MLKL. Kidney function was assessed at 28 days by glomerular filtration rate measurement using sinistrin excretion kinetics. Fibrosis was assessed by picrosirius red staining and qPCR for fibrotic genes. Inflammation was assessed by qPCR and multi-plex assay for inflammatory proteins. Differential gene expression analysis was performed on a publicly available human kidney transplant dataset and subjected to KEGG pathway analysis for the necroptotic pathway. Results: RIPK1 inhibition reduced necrosis in in vitro models of PTEC injury. RIPK1 inhibition did not affect apoptosis. Evidence of co-existing necroptosis and ferroptosis was found in ischemic injured PTECs in vitro. RIPK1 inhibition improved mitochondrial membrane integrity in PTEC ischemic injury in vitro. Specific GSK’547 and LPX-1 significantly reduced biochemical injury and histological necrosis at 24 and 48 hours after IRI in mice. The effect was similar when drugs were given 15 minutes before or 4 hours after the injury. RIPK1 kinase inactive mice were protected against IRI compared to wild type controls. Treating IRI 4 hours after injury with either GSK’547 or LPX-1 significantly improved kidney function (GFR) and reduced fibrosis at 28 days. However, when drug treatment was not started until 48 hours after IRI only GSK’547 and not LPX-1 improved kidney function and reduced fibrosis and inflammation at 28 days. GSK’547 but not LPX-1 significantly reduced fibrosis in the unilateral ureteric obstruction model. GSK’547 but not LPX-1 was shown to have specific suppressive effects on inflammatory proteins in acute IRI, CKD and ureteric obstruction models. Timing and cellular location of phosphorylation of the necroptosis effector MLKL was identified in mouse kidney after IRI and detailed histological analysis is presented. The cellular location of necroptotic proteins was demonstrated in human kidney tissue by IHC. Significant regulation of the necroptotic pathway after IRI in humans was demonstrated through pathway analysis of pre and post ischemia reperfusion injury transplant biopsy dataset. Conclusion: Specific RIPK1 inhibition is beneficial in acute ischemic kidney injury in human cells in vitro and in IRI in mice. Specific RIPK1 inhibition can reduce AKI to CKD transition when given 2 days after AKI onset. RIPK1 inhibition has cell death independent effects on inflammation and fibrosis. Necroptosis occurs in human PTECs in vitro and in tubular epithelial cells in vivo. This thesis therefore presents the first convincing data that specific RIPK1 inhibition is beneficial and that necroptosis occurs in IRI. This work suggests that RIPK1 inhibition is a promising therapeutic strategy for the treatment of AKI and of AKI to CKD transition, common disease processes that occur in multiple clinical settings.