The impact of the cytoplasmic ubiquitin ligase TNFAIP3 gene variation on transcription factor NF-κB activation in acute kidney injury.

Nuclear factor κB (NF-κB) activation is a deleterious molecular mechanism that drives acute kidney injury (AKI) and manifests in transplanted kidneys as delayed graft function. The TNFAIP3 gene encodes A20, a cytoplasmic ubiquitin ligase and a master negative regulator of the NF- κB signaling pathway. Common population-specific TNFAIP3 coding variants that reduce A20's enzyme function and increase NF- κB activation have been linked to heightened protective immunity and autoimmune disease, but have not been investigated in AKI. Here, we functionally identified a series of unique human TNFAIP3 coding variants linked to the autoimmune genome-wide association studies single nucleotide polymorphisms of F127C; namely F127C;R22Q, F127C;G281E, F127C;W448C and F127C;N449K that reduce A20's anti-inflammatory function in an NF- κB reporter assay. To investigate the impact of TNFAIP3 hypomorphic coding variants in AKI we tested a mouse Tnfaip3 hypomorph in a model of ischemia reperfusion injury (IRI). The mouse Tnfaip3 coding variant I325N increases NF- κB activation without overt inflammatory disease, providing an immune boost as I325N mice exhibit enhanced innate immunity to a bacterial challenge. Surprisingly, despite exhibiting increased intra-kidney NF- κB activation with inflammation in IRI, the kidney of I325N mice was protected. The I325N variant influenced the outcome of IRI by changing the dynamic expression of multiple cytoprotective mechanisms, particularly by increasing NF- κB-dependent anti-apoptotic factors BCL-2, BCL-XL, c-FLIP and A20, altering the active redox state of the kidney with a reduction of superoxide levels and the enzyme super oxide dismutase-1, and enhancing cellular protective mechanisms including increased Foxp3+ T cells. Thus, TNFAIP3 gene variants represent a kidney and population-specific molecular factor that can dictate the course of IRI.

Repurposing of metformin and colchicine reveals differential modulation of acute and chronic kidney injury.

Acute kidney injury (AKI) is a major health problem affecting millions of patients globally. There is no effective treatment for AKI and new therapies are urgently needed. Novel drug development, testing and progression to clinical trials is overwhelmingly expensive. Drug repurposing is a more cost-effective measure. We identified 2 commonly used drugs (colchicine and metformin) that alter inflammatory cell function and signalling pathways characteristic of AKI, and tested them in models of acute and chronic kidney injury to assess therapeutic benefit. We assessed the renoprotective effects of colchicine or metformin in C57BL/6 mice challenged with renal ischemia reperfusion injury (IRI), treated before or after injury. All animals underwent analysis of renal function and biomolecular phenotyping at 24 h, 48 h and 4 weeks after injury. Murine renal tubular epithelial cells were studied in response to in vitro mimics of IRI. Pre-emptive treatment with colchicine or metformin protected against AKI, with lower serum creatinine, improved histological changes and decreased TUNEL staining. Pro-inflammatory cytokine profile and multiple markers of oxidative stress were not substantially different between groups. Metformin augmented expression of multiple autophagic proteins which was reversed by the addition of hydroxychloroquine. Colchicine led to an increase in inflammatory cells within the renal parenchyma. Chronic exposure after acute injury to either therapeutic agent in the context of reduced renal mass did not mitigate the development of fibrosis, with colchicine significantly worsening an ischemic phenotype. These data indicate that colchicine and metformin affect acute and chronic kidney injury differently. This has significant implications for potential drug repurposing, as baseline renal disease must be considered when selecting medication.

CD47 limits autophagy to promote acute kidney injury.

Acute kidney injury (AKI) initiates a complex pathophysiological cascade leading to epithelial cell death. Recent studies identify autophagy, a key intracellular process that degrades cytoplasmic constituents, as protective against AKI. We have previously reported that the protein thrombospondin-1 and its receptor CD47 are induced in AKI; however, the mechanism underlying their regulation of injury is unknown. Here, we investigated whether CD47 signaling affects autophagy to regulate AKI. Wild-type (WT) and CD47-/- mice were challenged with renal ischemia-reperfusion injury. All animals underwent analysis of renal function and biomolecular phenotyping. CD47-/- mice were resistant to AKI, with decreased serum creatinine and ameliorated histologic changes compared with WT animals. These mice also displayed increased abundance of key autophagy genes, including autophagy-related gene (Atg)5, Atg7, beclin-1, and microtubule-associated proteins 1A/1B light chain 3 (LC3) at baseline and post-AKI, which were significantly reduced in WT mice. Changes in protein expression correlated with increased autophagosome and autolysosome formation in renal tubular epithelial cells (RTECs). In mouse kidney transplantation, treatment with a CD47-blocking antibody that improved function was associated with increased autophagy compared with control mice. Primary isolated RTECs from CD47-/- mice demonstrated increased basal expression of several autophagy components that was preserved under hypoxic stress. These data suggest that activated CD47 promotes AKI through inhibition of autophagy and point to CD47 as a target to preserve renal function following injury.-El-Rashid, M., Ghimire, K., Sanganeria, B., Lu, B., Rogers, N. M. CD47 limits autophagy to promote acute kidney injury.