RIPK3 plays a key role in the execution of necroptosis. Indeed, the Nomenclature Committee on Cell Death (NCCD) defined in 2018 necroptosis as a type of regulated cell death (RCD) triggered by perturbations of extracellular or intracellular homeostasis that depends on RIPK3 activation and its downstream effector MLKL.11 Autophosphorylated RIPK3 recruits and phosphorylates mixed lineage kinase domain-like (MLKL),10,12,13 which then oligomerizes and translocates to intracellular and plasma membranes, causing membrane rupture.12

Tumor necrosis factor (TNF) receptor super family members promote binding of RIPK1 to RIPK3, which results in the assembly of different context-specific protein complexes. Complex I is activated upon TNFRSF stimulation and subsequent recruitment of TNFR-associated death domain adaptor protein (TRADD) and RIPK1, among others, and results in NF-κB activation.14 Linear ubiquitination of RIPK1 serves as a central checkpoint to regulate NFκB activation, cell proliferation and cell death.15 Under certain conditions, deubiquitylation of RIPK1 induces the formation of cytosolic complex IIa, which includes pro-caspase 8, and triggers apoptosis.14 In this context, caspase inhibitors, as seen for certain viruses, lead to the formation of the necrosome or complex IIb through the interaction of RIPK1 and RIPK3, thus inducing necroptosis.16 TNFSF members TNFα, TWEAK, FasL and TRAIL promote RIPK1/RIPK3-dependent necroptosis that contributes to various conditions, including kidney diseases.17–20

RIPK3 may also be activated by TRIF in response to Toll like receptors (TLRs) activation by stimuli such as polyinosinepolycytidylic acid (poly(I:C) and lipopolysaccharide (LPS).21 TLR4-induced necroptosis has been observed in experimental colitis and in nervous system cells.22–24

RIPK3 activation by viral RNA-DAI or cellular RNA-DAI also triggers necroptosis.14,21 When viral proteins inhibit caspases and prevent the clearance by apoptosis of virus-infected cells, DAI activation of necroptosis eliminates infected cells.25 The key role of RIPK3 in this host defense function was demonstrated in preclinical herpes simplex virus type 1 (HSV-1), influenza virus, or vaccinia virus infections.26–28

Cytosol alkalinization induced by activation of the Na+/H+ exchanger SLC9A1 also triggers RIPK3-dependent necroptosis through a non-canonical pathway, which is independent of RHIM domain, but still requires MLKL phosphorylation.29,30 Since the only described trigger of this pathway is a sudden increase in osmolarity to levels only found in the mammalian kidney medulla, this pathway would initially be expected to have little relevance in vivo, except, potentially, for kidney disease.31 In this regard, its role in kidney disease should be explored. Additionally, its potential role in any condition characterized by increased intracellular pH, independently of extracellular osmolarity, should be studied.

Cells dying by necroptosis release their intracellular contents promoting an inflammatory response and potentially, an immunogenic response. However, RIPK3 also triggers inflammation independent of necroptosis through activation of NFκB or the inflammasome.32

Inhibition of RIPK3 with GSK’872 reduced NLRP3 activation and inflammation in murine LPS-induced acute lung injury and in rat spinal cord injury, supporting the proinflammatory role of RIPK3.33,34 In preclinical cecal ligation and puncture (CLP)-induced sepsis, in which inflammation promotes tissue injury, RIPK3-KO mice survived longer, and had milder systemic inflammation and decreased lung, liver, and kidney injury.35–37 NFκB activation by RIPK3 is cell-type specific. RIPK3 deficient (RIPK3-KO) mouse embryonic fibroblasts (MEFs) and bone marrow derived macrophages (BMDMs) display normal NFκB activation and cytokine expression following LPS stimulation, while RIPK3 was required for NFκB activation and cytokine expression in LPS-stimulated bone marrow dendritic cells (BMDCs) and in TNFα-stimulated vascular smooth muscle cells (VSMCs).38–40 RIPK3-induced activation of NFκB was also described in pancreatic β-cells under metabolic stress where it mediates IL-1β expression, in cadmium-stimulated RAW macrophages where RIPK3 promotes M1 polarization and during osteoclastogenesis where it contributes to ovariectomy-induced bone loss in mice.41–43 The RIPK1 inhibitor necrostatin-1 prevented RIPK3-induced NFκB activation and inflammation in some experimental systems,41,43 but the precise role of RIPK1 in RIPK3-induced NFκB activation requires further in-depth characterization. In this regard, RIPK3-mediated inflammation in experimental shock induced by high dose (25μg) TNFα was independent of RIPK1, as RIPK3-KO mice were protected but necrostatin-1 accelerated death.44 However, necrostatin-1 was as protective as RIPK3 deficiency from low dose (5 or 9μg) TNFα, suggesting that RIPK1 may contribute to mild but not to severe TNF-induced shock, while RIPK3 is involved in both.35

RIPK3 also activates the NLRP3-inflammasome independently of cell death but the molecular pathway is unclear.45–48 The role of caspase-8 in RIPK3-induced activation of NLRP3 is controversial.

RIPK3 activated caspase-8 and pro-IL-1β processing in BMDCs. Conversely, caspase-8 may negatively regulate RIPK3-induced inflammasome activation, as caspase-8 deficiency increased the RIPK3-induced inflammasome activation in LPS-stimulated BMDCs. Furthermore, caspase-8 deficiency in myeloid cells increased RIPK3-induced inflammasome activation in preclinical autoimmune encephalomyelitis.45,47

However, in BMDMs, RIPK3 activated NLRP3 independently of MLKL in presence of caspase-8, while it activated NLRP3 through MLKL in caspase-8 deficient cells.46,48 Thus, the interaction between caspase-8 and RIPK3 is complex.

In humans, plasma RIPK3 levels were independently associated with acute respiratory distress syndrome among critical patients with sepsis or trauma, and they were also increased in patients exposed to mechanical ventilation and in those with moderate or severe COVID-19.49–52 However, the cellular source of plasma RIPK3 remains unclear, as does the mechanism of RIPK3 release from cells: release from living cells, membrane disruption of dying cells or contained in exosomes. It also remains unclear to what extent plasma RIPK3 may guide RIPK3-targeted therapies in the clinic.

Nevertheless, RIPK3 is critical for host defense against Streptococcus pneumoniae infection, since RIPK3-KO mice presented reduced bacterial clearance and severe lung inflammation.53 RIPK3 binding to RIPK1 and MLKL promoted mitochondrial ROS, NLRP3 inflammasome activation and necroptosis in macrophages during Streptococcus pneumoniae infection.53

Additionally, RIPK3 has been associated with fibrosis in heart, liver, and muscle. The profibrotic response induced by high glucose or the air pollutant PM2.5 in cultured cardiomyocytes is prevented by GSK‘872.54,55 In preclinical liver fibrosis induced by CCl4 or bile duct ligation, RIPK3 deficiency in macrophages reduced fibrosis and inflammation.56 Similarly, in murine Duchenne muscular dystrophy, RIPK3 deficiency reduced myofiber degeneration, inflammatory infiltrates, and muscle fibrosis.57 Since RIPK3-induced fibrosis usually occurs in parallel to an inflammatory response, further studies should discern whether RIPK3-promoted fibrosis is a consequence of the proinflammatory role of RIPK3, an independent effect, or both.

PANoptosis refers to a pathway of inflammatory regulated cell death that activates simultaneously apoptosis, the NLRP3/inflammasome/pyroptosis, and necroptosis. PANoptosis is activated by bacterial, viral, or fungal infection in BMDMs, and by cytokines (e.g., TNFα/IFNgamma) in colon tumor cells and in BMDMs, thus it may play a key role during infection, and in the immunotherapy of cancer.58–62 PANoptosis is triggered by a protein complex called PANoptosome composed of apoptosis, pyroptosis and necroptosis proteins, including RIPK3. However, further studies are needed to fully characterize the molecular mechanisms of PANoptosis and PANoptosome assembly and to better understand its relevance in health and disease for different cell types.

The first evidence for the role of necroptosis in AKI was the observation that the RIPK1 inhibitor necrostatin-1 protected from preclinical AKI induced by ischemia/reperfusion injury (IRI-AKI), cisplatin nephrotoxicity (Cisplatin-AKI), contrast-induced AKI (CAKI), oxalate nephropathy, and folic acid nephropathy (FA-AKI).20,63–66 The contribution of RIPK3 to necroptosis in IRI-AKI was demonstrated in RIPK3-KO mice, which were protected, even though other pathways of regulated necrosis, such as ferroptosis and mitochondrial permeability transition-regulated necrosis (MPT-RN), also contribute to IRI-AKI.65,67–69 Kidney protection appeared to depend on RIPK3 expression by kidney cells since RIPK3-KO kidney graft recipients were protected from IRI-AKI associated to the procedure.70 In this regard, freshly isolated renal tubules from RIPK3-KO mice were protected from hypoxia/reoxygenation-induced cell death.65

Necrostatin-1 and genetic deficiency of RIPK3 or MLKL improved renal function, reduced tubular injury, and increased survival in murine cisplatin-AKI.64,65 As in IRI-AKI, additional cell death pathways contribute to cisplatin-AKI since the survival of RIPK3-deficient mice increased when zVAD inhibited caspases or when caspase-8 was also deficient, suggesting that combined necroptosis and apoptosis blockade could increase protection.65 In this regard, despite kidney TUNEL-positive cells not being reduced in RIPK3-KO mice, cultured RIPK3 deficient primary tubular cells were protected from cisplatin-induced cell death.64,65

In murine FA-AKI there is a sequential activation of ferroptosis and necroptosis; an initial ferroptosis wave of cell death promotes an inflammatory response, the upregulation of RIPK3 and MLKL, and a second wave of inflammation-dependent necroptotic cell death. Indeed, RIPK3-KO mice displayed improved renal function, reduced tubular cell death, and reduced translocation of MLKL to plasma membranes in the late FA-AKI stages.20 Moreover, TWEAK, which contributes to FA-AKI, induced RIPK3-dependent necroptosis in an inflammatory milieu in cultured tubular cells, thus identifying one of the inflammatory mediators of the second necroptotic wave in FA-AKI.20

RIPK1, RIPK3 and MLKL are also overexpressed in murine oxalate nephropathy, and Necrostatin-1 or RIPK3 or MLKL deficiency reduced tubular injury and tubular cell death. MPT-RN is also involved in oxalate nephropathy, and dual inhibition of both necroptosis and MPT-RN increased protection.71 In cultured tubular cells, crystals triggered RIPK3-dependent necroptosis and MPT-RN.72

These data suggest that RIPK3 is a potential therapeutic target for different forms of AKI and in kidney transplantation, although in some cases the dual blockade of RIPK3 and other cell forms of death pathways, such as apoptosis or MPT-RN, will be more effective.

The precise role of RIPK3-mediated inflammation on overall kidney injury and dysfunction appears to be context-dependent. Thus, in AKI driven by systemic inflammation, targeting RIPK3-induced inflammation is kidney protective, but this is not the case for the early stages of ferroptosis-driven kidney injury.

RIPK3 has a pro-inflammatory role in the kidney independent of necroptosis. The main evidence that RIPK3 can mediate kidney inflammation derives from inflammatory models such as CLP-induced sepsis or LPS-induced endotoxemia, in which RIPK3 targeting was protective. Specifically, RIPK3 deficiency preserved kidney function and reduced tissue injury in CLP-induced sepsis, while MLKL deficiency was not protective, suggesting that kidney protection was not dependent on inhibit necroptosis.35,37 In CLP-induced sepsis, RIPK3 mediated kidney injury through NOX4 overexpression, production of mitochondrial ROS, mitochondrial disfunction, and cell death. However inflammatory hallmarks such as cytokine expression or inflammatory cell infiltration were not evaluated.37 In LPS-induced endotoxemia (i.e., sterile cytokine storm) GSK’872 improved renal function and reduced tubular injury and cell death.73 In human tubular cells stimulated with LPS, GSK’872 or RIPK3 downregulation by siRNA reduced cell death and apoptosis markers, but necroptotic cell death was not analyzed.73 In line with these results, patients with sepsis had higher urinary RIPK3 levels than non-sepsis patients, and sepsis patients with AKI had higher urinary and plasma RIPK3 than patients without AKI.37 These results suggest that RIPK3 may contribute to kidney injury during cytokine storms, but further studies are needed to discern the precise mechanisms involved as well as the potential role of urinary or plasma RIPK3 as a biomarker that contributes to clinical decision-making.

RIPK3 also play a role in lupus nephritis. RIPK3 inhibition with GSK’872 in NZM2328 or MLR/lpr mice reduced kidney phosphorylation of MLKL and RIPK3, inflammasome activation and IgG and C3 deposition, and improved kidney function, however as cell death was not assessed, it is unknown whether RIPK3-mediated injury in lupus nephritis is dependent on cell death or, as suggested by the reduced immune deposits, it is dependent on reduced immune cell activity.74

In murine FA-AKI, RIPK3 deficiency reduced the early inflammatory response without improving kidney function and this effect was independent of necroptosis since Necrostatin-1 or MLKL deficiency did not modify this early inflammatory response.75,76 As discussed above, ferroptosis was the primary driver of kidney injury in the first 24h of FA-AKI75 while early stages of endotoxemic or sepsis AKI are driven by systemic inflammation. The precise mechanism by which RIPK3 mediates inflammation in the early stages of FA-AKI is poorly characterized. It is dependent on RIPK3 actions in bone marrow cells, since RIPK3 is overexpressed by infiltrating inflammatory cells such as macrophages and lymphocytes in FA-AKI and bone marrow RIPK3 deficiency decreased kidney inflammation.76 NFκB activation is a prime candidate mediator, since RIPK3 deficiency in FA-AKI decreased interstitial and tubular nuclear translocation of NFκB p65, and NFκB is a well-known mediator of kidney inflammation.15,76,77 Consistent with this hypothesis, RIPK3-deficient mice had lower expression of NLRP3 and cleaved caspase 1 in FA-AKI, but NLRP3-deficient mice were not protected, ruling out a major role of the inflammasome.76

RIPK3 also modulates TWEAK-induced kidney inflammation, as this was milder in RIPK3-deficient mice.76 TWEAK is a pro-inflammatory cytokine that activates kidney NFκB without inducing cell death78 and illustrates the complex role of RIPK3 in promoting inflammation.76 RIPK3 deficiency decreased the TWEAK-induced NFκB-dependent inflammatory responses such as MCP-1, IL-1β and IL-6 expression in bone marrow derived cells, while it only inhibited the NFκB-independent production of IL-6 in tubular cells.76 However, there is a RIPK3-dependent crosstalk between both cell types, as conditioned medium from TWEAK-stimulated BMDMs activated tubular cells in a RIPK3-dependent manner.76

Kidney fibrosis plays a key role in the AKI-to-CKD transition and in CKD progression.79,80 RIPK3 also contributes to kidney fibrosis in preclinical AKI-to-CKD transition and CKD.69,81–83

In IRI-AKI, RIPK3-deficient mice are protected from both early stage (2 and 7 days) necroptosis and from later (14 days, 1 month and 3 months) kidney inflammation and fibrosis.69 MLKL-KO mice displayed similar protection, suggesting that protection depends on interference with necroptosis. The initial stages of IRI-AKI depended on kidney expression of RIPK3, while RIPK3 from bone marrow-derived cells contributed to later stage fibrosis, tubular injury and inflammasome activation. In this regard, combined oxygen-glucose, and fetal bovine serum deprivation (OGFD), as might be expected in IRI, caused RIPK3-dependent necroptosis in cultured tubular cells, resulting in release of cellular contents that enhanced inflammasome activation in macrophages.69 Thus, RIPK3 from both tubular cells and bone marrow-derived cells contribute to CKD resulting from IRI-AKI through a loop of necroinflammation, in which initial RIPK3-dependent tubular cell necroptosis recruits RIPK3-dependent inflammatory responses from bone marrow cells.69

FA-AKI also causes AKI-to-CKD transition and kidney fibrosis is evident at 28 days. RIPK3-KO mice displayed less inflammation and fibrosis at this time-point. However, since RIPK3 also contributes to tubular cell death and/or inflammation in the acute phase (24–96h) of FA-AKI,20,75 it remains unclear whether the long-term protection derived exclusively from a milder initial FA-AKI or whether RIPK3 also actively participates in molecular events driving fibrosis in the chronic phase. To clarify this, RIPK3 should be targeted therapeutically, rather than prophylactically, i.e., once kidney injury is already established, for example by starting RIPK3 targeting 72–96h after induction of FA-AKI or IRI-AKI.

Kidney RIPK3 expression was also upregulated in other models of CKD, such as streptozotocin induced-diabetic nephropathy, ureteral obstruction-induced kidney fibrosis and adenine diet-induced CKD, and in human diabetic nephropathy or kidney fibrosis.81,82 In diabetic mice, RIPK3 deficiency decreased tubulointerstitial inflammation, TGF-β1 expression and fibrosis, but did not decrease glomerular injury as assessed by albuminuria or glomerulosclerosis.82 RIPK3 deficiency also improved kidney function and decreased fibrosis in murine adenine diet-CKD, but the mechanism was not studied.81 RIPK3 deficiency also decreased unilateral ureteral obstruction (UUO)-induced kidney fibrosis in mice but had no impact on kidney inflammation or TGF-β1 expression,81 suggesting the presence of shared and specific targets for diverse cause of CKD. RIPK3-induced fibrosis following UUO likely involved AKT-dependent activation of ATP citrate lyase (ACL), since kidney phospho-AKT and phospho-ACL were reduced in RIPK3-deficient mice. Indeed, in cultured fibroblasts, RIPK3 regulated fibrogenesis downstream of TGF-β1 through activation of the AKT-ACL pathway.81 The profibrotic effect of RIPK3 in UUO was independent of MLKL, as MLKL-KO mice were not protected, and may be dependent on RIPK1, as necrostatin-1 prevented AKT-ACL activation and reduced fibrotic gene expression in cultured fibroblasts and necrostatin-1s reduced fibrosis and tubular injury in UUO kidneys.81,84 In chronic oxalate nephropathy, intrarenal injection of CRISPR gRNAs for either RIPK3 or MLKL suppressed fibrosis and leukocyte infiltrates and improved renal function.84 In fact, in chronic oxalate nephropathy TGFβ1 induced fibrosis and leukocyte recruitment through RIPK1/RIPK3/MLKL necrosome formation, mitochondrial ROS production, calcium/calmodulin-dependent protein kinases-II (CaMKII) and Smad2/3.84