Nrf2 Activation in the Glomeruli and Podocytes: Deciphering the Renal Mechanisms of Nrf2

Reactive oxygen species (ROS) are one of the end products of cellular metabolism during physiologic and pathophysiologic processes. These are highly reactive electrophiles with critical biomolecules, including DNA; low levels are important in modulating cellular function; however, when their levels increase, they become progressively more toxic and damage cells in the form of oxidative stress. Excessive oxidative stress in turn is a key contributor to the pathogenesis and progression of numerous disease processes, including kidney diseases.1 Thus, the maintenance of cellular redox homeostasis is of vital importance. This is largely accomplished via endogenous defense mechanisms, particularly the Kelch-like ECH-associated protein 1 (KEAP1) and its target, the nuclear factor erythroid two–related factor 2 (NRF2) system.2 This system acts as a key cellular sensor and first responder to oxidative stress conditions. Under physiologic conditions, KEAP1 actively binds to Nrf2, a master transcriptional regulator of oxidative stress responses. The attachment of KEAP1 to Nrf2 keeps Nrf2 in the cytoplasm and marks it for degradation via the ubiquitin–proteasome pathway, thus keeping its levels low. However, under conditions of increased oxidative stress, electrophiles attach to specific cysteine residues of KEAP1, causing conformational changes that inhibit its binding to Nrf2, and consequently its degradation. This results in an increased number of unbound Nrf2 molecules available for translocation to the nucleus where they activate the transcription of its target genes, which increase the generation of a comprehensive set of cytoprotective enzymes (over 250) with antioxidant and anti-inflammatory properties (e.g., heme-oxygenase 1, superoxide dismutase, catalase, among others), making Nrf2 one of the key regulators of cellular and organ function, as well as a vital defense mechanism against injury.3 Consequently, failure to upregulate Nrf2 during oxidative stress may initiate and/or aggravate injury, whereas potentiating its upregulation may confer organ protection, making it a potential therapeutic target. This contention was supported by early preclinical and experimental studies that showed that blocking Nrf2 exacerbates renal injury, whereas increasing its activity confers protection.4 The plethora of cytoprotective enzymes that are stimulated by Nrf2 together with the supportive preclinical studies led to an unprecedented hype in its possible clinical benefits and consequently the rapid progression from preclinical studies to clinical trials evaluating the effect of a Nrf2 agonist (bardoxolone methyl) in diabetic nephropathy with varying degrees of CKD. Although the initial trials delivered promising results regarding eGFR, the Phase 3 Bardoxolone Methyl in Type 2 Diabetes and Stage 4 Chronic Kidney Disease (BEACON) trial5 was terminated early because of striking adverse safety signals, particularly heart failure. This radically altered the perception Nrf2 agonists in clinical medicine, which nearly halted further development of these agents. Moreover, scathing critiques of the BEACON trial argued that the trial was premature and the toxicity of bardoxolone may have been predictable. Several subsequent experimental studies could not reproduce the beneficial effects of bardoxolone methyl analogs6,7; they in fact found that it worsened diabetic kidney disease, which added fuel to the fire. Whether the adverse effects of bardoxolone methyl or its analogs is due to the upregulation of Nrf2 (and its multitude of enzymes) or an off-target effect is unknown. The one thing that is certain is that to fully understand the therapeutic potential and toxicity of Nrf2 modulators, much more insight is needed regarding the physiologic and pathophysiologic mechanisms of the KEAP1/Nrf2 pathway. In this issue of Kidney360, Kidokoro et al.8 advance our understanding of the mechanisms by which Nrf2 may regulate GFR in healthy mice. This was accomplished via a very comprehensive set of animal experiments wherein Nrf2 was genetically upregulated or knocked down (thus avoiding the potential toxicity and off-target effects of pharmacologic agents) as well as by administering an Nrf2 activator. Three different methodologies were used to verify that NRF2 activation increased GFR. This increase in GFR was associated with increased renal blood flow (RBF) and glomerular volume. However, no changes were observed in the ratio of afferent to efferent arteriolar diameters, leading the authors to surmise that the increase in GFR was likely due to increased ultrafiltration coefficient (Kf), rather than a change in glomerular capillary pressure (GCP). Therefore, they investigated a potential mechanism by which this may occur. Because calcium influx into podocytes can cause conformational changes in actin, leading to a rearrangement of the cytoskeleton, with consequent changes in the glomerular permeability, they tested whether Nrf2 activation altered calcium signaling in the podocytes. The authors demonstrated that oxidative stress-induced calcium influx through the Transient Receptor Potential Canonical (TRPC) 5/6 channels was blunted by Nrf2 activation. Blocking TRPC 6 channels, which mimics the effects of Nrf2 activation on podocyte calcium, increased RBF and GFR. They concluded that the Keap1-Nrf2 pathway regulates GFR by inhibiting ROS-dependent activation of the TRPC 5/6 calcium channels in podocytes, thus increasing Kf. Previous studies have shown that Nrf2 activation increases RBF and/or GFR.9 However, the mechanisms by which GFR is increased remains unknown. In the healthy mammalian kidney, the fundamental mechanisms by which GFR can rise are via an increase in GCP or Kf. The difference between the two is critical because increases in GCP, if sustained, are likely to lead to glomerular damage, whereas changes in Kf are not necessarily deleterious. Nrf2 agonists have the potential to affect either parameter. They can potentially increase GCP by (1) directly inhibiting calcium influx in vascular smooth muscle cells or (2) decreasing ROS thereby improving nitric oxide bioavailability, both of which result in vasodilation of the preglomerular arterioles. Previous studies have suggested that they can affect Kf because of their inhibitory effect calcium influx into the mesangial cells, thus which suppresses mesangial cell contraction.10 This study suggests that the Nrf2 activation also decreases calcium influx into the podocytes, thus potentially altering the morphology of the foot processes, and hence Kf and GFR. Overall, this is an interesting and provocative study. It adds to our knowledge base regarding the mechanisms by which Nrf2 activators exert their renal effects and raises important questions. However, several limitations prevent us from drawing strong conclusions, especially when trying to extrapolate this to CKD and other forms of kidney disease. First, all the studies were performed in healthy rodents without kidney or vascular disease; responses during these disorders may be substantially different. Second, the contribution of each mechanism is not established as they were only indirectly assessed. In this respect, GCP was not measured, rather surmised from the lack of changes in the afferent-to-efferent arteriolar ratios. However, this is not definitive proof of unchanged GCP. In fact, because RBF was increased and afferent arterioles are larger than efferent arterioles, maintenance of the afferent-to-efferent ratios may be expected to result in increased GCP depending on overall changes in absolute diameters. Indeed, this provides an alternative explanation to the increased glomerular volume observed during Nrf2. Finally, there is a discrepancy between the calculated single-nephron GFRs and overall GFRs. This may have been due to the preparation used (e.g., anesthesia) during the single-nephron GFR measurements; thus, independent confirmation of the renal parameters measured will be necessary. However, these limitations do not detract from the major findings of the study and raise important avenues for further exploration. In summary, the KEAP1/Nrf2 pathway remains an important area of research and therapeutic potential. The major setback instigated by the BEACON trial highlights the complexity of these systems. Further studies are needed (1) to elucidate the physiologic effects of NRF2 and its many effector proteins, not only in the renal vasculature and glomerulus but also along the renal tubules, (2) to determine whether these actions are altered in the different stages of diverse disease processes in a manner that would alter when the drug may be administered to maximize the risk-to-benefit ratio and (3) whether attacking more specific downstream targets of the KEAP1/Nrf2 pathway (e.g., heme-oxygenase) may provide more effective therapeutic targets.