1. Endogenous Nrf2 aActivation in hHuman CKD

The transcription factor Nrf2 regulates the gene expression of about 250 target genes involved in redox regulation and antioxidant response, inflammation, heme and iron metabolism, intermediary lipid and carbohydrate metabolism, and reactions of detoxification and biotransformation ^[1]. An overwhelmingly broad spectrum of factors and cellular conditions that can regulate Nrf2 abundance and activity is known from preclinical studies [2,3,4]^[2][3][4]. On the contrary, the clinical data on Nrf2 abundance and activity in human CKD are patchy.

2. Oxidative sStress

Oxidative stress refers to an imbalance between oxidants and antioxidant mechanisms that leads to deviations from “steady state” redox signaling, and may finally result in oxidative damage of molecules. Pathogenic mechanisms of oxidative stress in CKD have extensively been reported and discussed [5-10]^{[5][6][7][8][9][10]}. Specific factors, both positive and negative, that influence the overall extent of oxidative stress in human CKD, are for example related to treatment modality or medication [11,12]^[11][12] and uremic toxin accumulation [13-15]^[13][14][15]. The differential expression of antioxidant and mitochondrial enzymes like superoxide dismutase 1/2 (SOD1/2), thiosulfate sulfurtransferase (rhodanese), or GPx in dependence of CKD stage or severity is well described [16-19]^{[16][17][18][19]}. Activation of Nrf2 in CKD in response or relation to oxidative stress has been reported. In a pharmacological “stress test” study in patients with CKD stage 3 and 4 (CKD3/4), effects of intravenous tin protoporphyrin application, which transiently induces oxidative stress, were investigated. Around half of those patients suffered from diabetes and three-fourths were hypertensive. The injections resulted in significant increases in plasma concentrations of the Nrf2 target proteins NQO1, HO-1, and ferritin ^[20]. Interestingly, this study tested the hypothesis that tin protoporphyrin application could be used to assess the antioxidant reserve in CKD [115]. The response of the three Nrf2 targets was for the most part comparable between CKD3/4 and healthy subjects. This points to a persisting capability in this patient population with CKD3/4 to react to acute oxidative stress via Nrf2 activation, although the cells or tissues responsible for the increased NQO1, HO-1, and ferritin plasma concentrations in this context are not known. In diabetic kidney disease (DKD), an increase of different ROS species has been shown in kidney tissue, and mitochondrial fragmentation seems to be important for hyperglycemia-induced increases in mitochondrial ROS production ^[7]. Compared to normal functioning kidney transplant tissue, activation of Nrf2 was shown in kidney tissue from patients with DKD and proteinuria ^[21]. In these diabetic glomeruli, Nrf2 and NQO1 protein abundance was increased and glomeruli showed immunohistochemical signs of oxidative damage. Presence or extent of GFR reduction in these patients was not reported. Likewise, in kidney tissue from patients with Lupus nephritis, who presented with pronounced proteinuria but apparently normal GFR, the glomerular protein amount of Nrf2 and NQO1 was increased compared to healthy kidney tissue, and signs of oxidative damage of the glomeruli were present ^[22]. Furthermore, in patients with CKD5 and hemodialysis therapy, synovial tissue showed increased HO-1 protein staining together with increased malondialdehyde, the latter an indicator of sustained oxidative stress in this tissue ^[23].

3. Uremic tToxins

With advancing CKD, an increasing amount of substances that normally are eliminated by the kidney, accumulate in the body. These uremic toxins contribute to CKD-related complications such as cardiovascular disease and impairment of the immune system. The specific effects of a uremic toxin depend on its nature and concentration.

Indoxyl sulfate, a metabolite of the tryptophan pathway, is one of the very important uremic toxins ^[24]. Indoxyl sulfate (43 mg/L) induced the production of ROS in proximal tubule epithelial cells ^[14]. In addition, indoxyl sulfate activates the arylhydrocarbon receptor (AhR). Indoxyl sulfate concentrations of 11 mg/L and 53 mg/L significantly increased TNF-α protein concentration in macrophages through AhR activation ^[25]. A bi-directional cross-talk between Nrf2 and the AhR exists and AhR can induce Nrf2 gene transcription ^[3]. The AhR also induces the expression of the Nrf2 target NQO1 ^[26]. Therefore, indoxyl sulfate could lead to Nrf2 activation through ROS production and AhR activation. On the other hand, indoxyl sulfate at a concentration of 53 mg/L decreased Nrf2 gene and protein expression in a human proximal tubular cell line ^[27], and AhR protein was decreased in monocytes of patients with advanced CKD (CKD5 with hemodialysis treatment) compared to healthy control subjects ^[25]. In human CKD, the relation between indoxyl sulfate and Nrf2 has only sparsely been investigated. The mean indoxyl sulfate concentration lies around 0.5 mg/L in healthy subjects and ranges from around 5 mg/L in CKD3 to 38 mg/L in CKD5 with hemodialysis treatment (The European Uremic Toxins (EUTox) Database, available online at www.uremic-toxins.org, accessed on 25 February 2022). At indoxyl sulfate concentrations between 1 mg/L and 11 mg/L in patients with CKD3 and 4, indoxyl sulfate correlated positively with Nrf2 gene expression in peripheral blood polymorphonuclear cells (PBMCs) ^[28]. In the light of a mainly decreased Nrf2 gene expression in advanced CKD ^[29], a bi-phasic, concentration-dependent effect of indoxyl sulfate in human CKD seems plausible, with Nrf2 activation in the lower concentration range and Nrf2 repression at high concentrations.

Methylglyoxal is a uremic toxin that shows around 2.4 times higher concentrations in uremic serum compared to normal serum concentrations (The European Uremic Toxins (EUTox) Database, available online at www.uremic-toxins.org, accessed on 30 March 2022). In human physiology, cellular methylglyoxal is mainly formed through spontaneous degradation of intermediates of glycolysis. In the cytosol, methylglyoxal is metabolized by glyoxalase 1 ^[30]. Methylglyoxal that is not metabolized can non-enzymatically modify DNA and proteins. Modification of proteins by methylglyoxal results in protein misfolding and subsequent activation of the unfolded protein response that has been linked for example to the development of DKD ^[30]. Methylglyoxal has been shown to modify Keap1. This results in crosslinking, with Keap1 dimer formation and resulting Nrf2 accumulation, and increased expression of Nrf2 target genes NQO1 and HO-1 ^[31]. As intracellular elevation of glucose leads to accumulation of methylglyoxal, it is likely that methylglyoxal contributes to the increase of Nrf2 and Nrf2 targets NQO1 and HO-1 that has been observed in diabetes without [32,33]^[32][33] and with DKD [21,32-36]^{[21][32][33][34][35][36]}.

4. Nuclear fFactor κ-light-chain eLight-Chain Enhancer of aActivated B cCells (NF-κB)

NF-κB refers to a transcription factor family that forms protein complexes that regulate DNA transcription in response to diverse cellular stressors, such as inflammation, infection, cytokines and ROS. The spectrum of NF-κB targets is wide. It includes the inflammasome components NLRP3 and caspase-1, the inflammasome substrates pro-interleukin-1 and pro-interleukin-18 ^[37], adhesion molecules like intercellular adhesion molecule 1 (ICAM), and tumour necrosis factor-alpha (TNF-α) ^[38]. The relation between Nrf2 and NF-κB is complex and bi-directional (for review see [1,3]^[1][3]). The Nrf2 gene contains several NF-κB binding sites, which enable Nrf2 induction by inflammatory stimuli through NF-κB. On the other hand, Nrf2 is able to suppress NF-κB transcriptional activity. The relation between Nrf2 and NF-κB in human CKD has rarely been investigated. The complexity of this relationship is illustrated in a study on Nrf2 and NF-κB gene expression in PBMCs from patients with CKD ^[39]. Herein, in patients with CKD3/4 higher NF-κB gene expression was associated with higher Nrf2 gene expression, likely reflecting NF-κB driven pro-inflammatory responses and subsequent Nrf2 activation. On the other hand, in the patient group with CKD5 and hemodialysis therapy the decrease of Nrf2, which has frequently been observed in advanced CKD ^[29], is present. In this group, decreased Nrf2 gene expression was associated with significantly increased NF-κB, finally amounting to a doubling of NF-κB gene expression ^[39] that may reflect a lack of suppressive Nrf2 action on NF-κB. Table 1 gives an overview over factors involved in endogenous NRF2 activation in human CKD.

5. Nrf2 aActivation in pPatients with CKD

The assessment of Nrf2 activation and activity in human disease is not straightforward. Nrf2 abundance is regulated on the transcriptional and posttranslational levels. Therefore, Nrf2 gene expression, Nrf2 protein amount, Nrf2 protein structure, and Nrf2 transcriptional activity are of relevance [1-3,40]^[1][3][40]. Nrf2 activation state can be deduced from gene expression analyses of Nrf2 target genes. Still, also with this approach, some challenges remain. Firstly, Nrf2 transcriptional activity is modified by Nrf2 acetylation, by availability of MafG, through interaction with the retinoid X receptor (RXR), and by NRF2’s interaction with CBP [83], which complicates inference about specific Nrf2 activation modes in cells from a patient. Secondly, for Nrf2 targets like NQO1 and HO-1, Nrf2-independent regulation of gene transcription has been reported [41-46]^{[41][42][43][44][45][46]}.

6. Nrf2 aActivation in renal cRenal Cells of hHuman CKD

A majority of the analyses in human kidney tissue that are reported below have been performed in kidney biopsy material. A kidney biopsy in a patient is performed for diagnostic reasons, often in the case of thitherto undiagnosed kidney disease or in the case of unexplained worsening of established CKD. It should therefore be noted that the findings for the Nrf2 system in human kidney tissue may frequently reflect a more rapidly progressing disease state.

7. Acute Kidney Injury (AKI)-to-CKD pProgression

An interesting study investigated kidney biopsies from acute, subacute and chronic tubulointerstitial nephritis ^[47]. Nrf2 protein was significantly increased in all types of tubulointerstitial nephritis (TIN), including chronic interstitial nephritis (~CKD3), compared to healthy kidney tissue. But, the highest nuclear and cytoplasmic Nrf2 protein amount was found in acute TIN, thereafter Nrf2 protein decreased gradually to subacute and chronic TIN. Interesting complementary data are provided by a study that compared successful and non-successful renal coping with an AKI event. Successful renal coping was presented by histologically normal kidney transplant tissue, while non-successful renal coping was represented by kidney tissue from patients with progressive CKD after diverse AKI causes ^[48]. Time span between AKI event and kidney biopsy, comorbidities, or CKD stages were not reported. Compared to healthy kidney tissue, the normal transplant biopsies showed an increase in Nrf2 protein, nuclear Nrf2 accumulation, increased gene expression of Nrf2 target genes NQO1, HO-1 and thioredoxin (Trx1), and concomitantly a slight increase of oxidative damage. In the example tissues for non-successful renal coping, Nrf2 protein in the cytoplasm showed a pronounced increase compared to healthy and kidney transplant kidney tissue, but nuclear Nrf2 accumulation seemed diminished, Nrf2 target gene expression was decreased, and oxidative damage increased. The discrepancy between high Nrf2 protein abundance and decreased Nrf2 target induction was suggested to result from high concomitant tubular GSK-3β protein abundance. While different GSK-3β–mediated mechanisms of Nrf2 repression are known ^[2], implications of these mechanisms in human kidney disease have not been sufficiently investigated so far.

8. Diabetes mMellitus and DKD

In an analysis of nephrectomy specimens from patients with diabetes, Nrf2 protein was increased, while Keap1 protein was equal compared to samples from patients without diabetes and normal kidney function ^[36]. The diabetic population sample size was small and contained patients with no CKD and CKD2-3b with varying degrees of proteinuria. Another study included patients with DKD and proteinuria, CKD stage was not reported. In this study, Nrf2 and NQO1 protein abundance was increased in diabetic glomeruli [54]. One research group investigated the role of tubular iron deposition in human CKD ^[21]. Patients, who showed tubular iron deposition also showed increased protein amounts of ferritin and HO-1, which both are Nrf2 targets ^[40]. Increased amounts of HO-1 were found in diabetic kidney disease with and without tubular iron deposition in this study, the extend of GFR reduction was not reported ^[35]. A recent publication investigated Nrf2 protein in kidney tissue from different kidney diseases. Diabetic kidney disease samples showed increased Nrf2 protein abundance in podocytes and increased Nrf2 nuclear translocation; details on CKD status were not reported ^[34]. In another small study including patients diagnosed with DKD, on the contrary, a decrease in Nrf2 protein compared to healthy kidney tissue was found ^[49]. Patient characteristics, CKD stage or extent of proteinuria were not reported. In view of partially conflicting results on Nrf2, it is interesting to have a look at one more Nrf2 target protein. Glyoxalase 1 (GLO1) metabolizes cytosolic methylglyoxal, and Nrf2 increases GLO1 expression ^[50]. In patients with long duration of diabetes (≥50 years) without development of DKD, GLO1 protein was significantly increased compared to both, age-matched non-diabetic controls without kidney disease and T2D patients (patients with diabetes mellitus typ 2) who had developed DKD (CKD3/4) ^[51]. This could point to a protective role of this Nrf2 target in those patients, who successfully sustained GLO1 upregulation over time.

9. Nrf2 rRepression in renal cRenal Cells of hHuman CKD

Repression of the Nrf2 system in renal cells in human CKD for some etiologies has been described. As noted for several of the publications in the earlier sections, also publications on Nrf2 repression partially lack detailed reporting of patient characteristics like CKD stage, proteinuria, or comorbidities. For patients with obesity-related nephropathy (~CKD3) a reduction of renal Nrf2 gene and protein expression was reported in comparison to kidney tissue samples from patients with nephritis and without GFR reduction ^[52]. An investigation about autosomal dominant polycystic kidney disease (ADPKD) reported a significant reduction of Nrf2 protein in ADPKD kidney tissue (~CKD1-3b) compared to healthy controls. Herein, lower Nrf2 protein amount correlated with higher total kidney volume and lower GFR, both signs of ADPKD severity ^[53]. A study on differential gene expression compared calcineurin inhibitor nephrotoxicity (CNIT) in kidney transplant tissue to normal kidney allograft tissue. This study identified a large number of Nrf2 targets with decreased gene expression in CNIT ^[54]. These included 1.6 to 2.0 fold decreases in gene expression of Trx1, peroxiredoxin, ATP binding cassette/subfamily C, glutathione S-transferase class M4, microsomal glutathione S-transferase 2, aldehyde dehydrogenase 1 family/ member A1, and transaldolase. In this respect, it is interesting that another study in kidney allograft tissue reported increased GSK-3β protein in chronic kidney allograft dysfunction compared to healthy kidney tissue. Furthermore, GSK-3β expression increased with severity of tubulointerstitial damage in the transplanted kidneys ^[55].

Finally, it should be noted that a study that otherwise reported increased Nrf2 protein in membranous nephropathy, fibrillary glomerulonephritis, focal segmental glomerulosclerosis, and diabetic nephropathy did not observe such an increase in renal amyloidosis ^[34].

Taken together, the state of the Nrf2 system in human CKD is far from homogenous. It varies among others with cause of kidney disease, comorbidities, stage of CKD, duration of CKD, and severity of uremic toxin accumulation and inflammation. Overall, earlier CKD stage or rapid progression of kidney disease, and inflammatory nature of the underlying kidney disease or comorbidities, were associated with more robust Nrf2 system activation. More advanced CKD on the other hand, was associated with Nrf2 system repression either in comparison to the healthy condition or to the upregulated state in matched controls. It should be noted that the responsible factors for both the endogenous activation and repression of the Nrf2 system in human CKD are insufficiently investigated.

The knowledge about consequences of the state of the Nrf2 system in a certain CKD context is likewise fragmentary. The endogenous activation of the Nrf2 system as described in DKD or LN was accompanied by signs of oxidative damage [21-23]^[21][22][23], therefore seems to have been only partially effective. On the other hand, effective endogenous upregulation of the Nrf2 system has been reported to prevent DKD development in diabetes ^[51].

10. Pharmacological Nrf2 aActivation in hHuman CKD

Pharmacological Nrf2 activators that were tested in human CKD to date for therapeutic purposes are electrophilic compounds that target Keap1. This group comprises bardoxolone methyl, curcumin, resveratrol and sulforaphane. They modify Keap1-cysteine-151 and thereby act as Keap1 inhibitors, resulting in the escape of newly synthesized Nrf2 from ubiquitination and degradation ^[56]. As chronic inflammatory processes and oxidative stress are characteristics of CKD, the hope is that Nrf2 activation could alleviate those features, thereby slowing CKD progression and/or reducing CKD-attributable morbidity. As discussed earlier, varies the activity state of the Nrf2 system in CKD between increased and repressed, dependent on CKD stage, cause of kidney disease, and comorbidities.

Besides the careful analysis of adverse effects, two aspects are of importance with respect to the general prospects of any type of future pharmacological Nrf2 activation therapy in CKD. First, can the respective substance elicit an Nrf2 activation that results in an appropriate functional response of Nrf2 targets in the respective CKD setting? Second, is this response coupled with a desirable effect on CKD progression or a reduction of CKD-attributable morbidity?

We report data on those two aspects for bardoxolone methyl, curcumin, resveratrol and sulforaphane below.

11. Bardoxolone mMethyl

A short history: From 2006 to 2008 bardoxolone methyl was advanced into the first phase I clinical trial as a potential anticancer drug ^[57]. In this study, an improvement of eGFR was observed that seemed more pronounced in patients with reduced baseline eGFR. A phase II trial in patients with CKD3b-4 and type 2 diabetes (BEAM study) found an increase in eGFR of ~6 to 11 ml/min/1.73m2 within 52 weeks of treatment, and an improvement of ~0.7 to 2.5 ml/min/1.73m2 was maintained during 4 weeks after completion of dosing ^[58]. A phase III trial in patients with CKD4 and type 2 diabetes (BEACON study) was terminated prematurely due to an increased rate of cardiovascular events ^[59]. Post hoc analyses suggested a possible delay of the onset of the end-stage of CKD ^[60]. One more phase II trial was performed in patients with CKD3-4 and type II diabetes (TSUBAKI study). This trial evaluated GFR directly by measuring inulin clearance and found an ~6 ml/min/1.73m2 increase in GFR during the 4 months study period ^[61]. Subsequently, a phase III trial in patients with CKD3-4 caused by DKD was initiated (NCT03550443, AYAME study) with an estimated study completion date in 2024. Also, a phase II/III trial was performed in CKD patients with Alport syndrome (NTC03019185, CARDINAL study), whose results have not been published peer-reviewed yet.

The first question, concerning effective activation of Nrf2 system components by bardoxolone methyl in CKD has insufficiently been investigated to date. The initial phase I trial reported an ~2.6fold increase in NQO1 gene expression on day 2 and a ~5.6fold increase on day 22 at different bardoxolone methyl dose levels in PBMCs ^[57]. Although some of the participants in this analysis might have had a reduced kidney function, the exact number is not known.

Serum analyses in the BEACON study included the determination of gamma-glutamyl transferase (GGT) ^[62]. The enzyme GGT, which is a biomarker for liver pathologies, increases in response to oxidative stress and glutathione depletion. It is a biomarker for cardiovascular and metabolic risk ^[63]. For GGT1, a function as Nrf2 target has been shown [83], with increased cellular GGT1 gene expression induced by sulforaphane treatment or Keap1 knockdown ^[64]. Although it needs to be emphasized that the relation between cellular and circulating GGT is not known, the serum GGT data from the BEACON study are interesting. The GGT concentration increased steeply after bardoxolone treatment initiation reaching a ~2.7fold increase at the first (day 28) measurement. It stayed around this concentration until week 12 and declined slowly thereafter, seemingly converging to a 1.5fold increase at the last blood sampling in the treatment period at week 48 ^[62]. The temporal pattern of GGT concentration in these patients with CKD4 could indicate a strong initial Nrf2 activation with subsequent decline of the effect and convergence to a new “higher-than-baseline” steady-state of Nrf2 activation.

The second question, if Nrf2 response to bardoxolone methyl was coupled with a desirable effect on CKD progression or a reduction of CKD-attributable morbidity is speculative as long as the molecular Nrf2 responses to bardoxolone in human CKD are not defined. The increase of GFR during and after bardoxolone treatment, its effect size and temporal pattern, possible underlying mechanisms, and if it indicates a reduction in CKD progression, have extensively been discussed [60^{[60][65][66][67]},65-67], and shall be discussed further when the data from currently finalized or ongoing trials get published. Other effects that have been observed in CKD patients during bardoxolone treatment included worsening of preexisting heart failure, increase in blood pressure/pulse pressure, hypomagnesemia, increased proteinuria, and weight loss. The TSUBAKI study also reported an increase in viral upper respiratory tract infections. The broad effect spectrum is not surprising, given the central role of Nrf2 in redox, protein and metabolic homeostasis as well as immune responses, and considering the again broad functional spectrum of Nrf2 targets like NQO1 or HO-1 [1,4,36,68]^{[1][4][36][68]}. The more urgent is the comprehensive investigation of molecular and physiological responses that are/were elicited in this patient population during bardoxolone treatment.

12. Sulforaphane

Sulforaphane was first isolated from red cabbage and later from broccoli [69]. Glucoraphanin, the biogenic precursor of sulforaphane is found in market-stage broccoli, but most abundant in broccoli sprouts and seeds. A comprehensive review recently discussed issues of sulforaphane source and dosing requirements ^[69].

Clinical studies that investigated the regulation of molecular Nrf2 targets in response to sulforaphane/glucoraphanin treatment are sparse. One study in healthy subjects, reported an increase in the serum enzyme activity of two Nrf2 targets 24 hours after glucoraphanin (NQO1 ~1.3fold, GST ~1.9fold). Our review did not identify sulforaphane/glucoraphanin intervention studies in patients with CKD.

13. Resveratrol

Resveratrol is a polyphenol found for example in grapes and raspberries. Like the aforementioned substances it targets Keap1 thereby resulting in increased Nrf2 protein. To date, just one study investigated resveratrol effects on the Nrf2 system in human CKD. Patients with CKD3/4 received resveratrol in a randomized controlled trial. The Nrf2 gene expression was analyzed and no significant effects were observed ^[70]. With respect to the anticipated effect of resveratrol on Nrf2 protein and activity, an additional investigation of Nrf2 target genes would be of importance. Nevertheless, as Nrf2 can target the Keap1 and the Nrf2 gene itself ^[40], and since resveratrol has a broad spectrum of further molecular effects ^[71], also Nrf2 gene expression is a potential target parameter for resveratrol.

14. Curcumin

Curcumin is a polyphenol compound found in the rhizomes of turmeric (Curcuma longa). Curcuminoids account for only around 1 to 6 percent of Curcuma longa extracts, and curcumin is one of those curcuminoids ^[72].

Our first question, concerning effective activation of Nrf2 system components by curcumin in CKD, was analyzed in a few studies. One study investigated a mixed population of patients with T2D with and without DKD in an uncontrolled intervention study ^[73]. After 15 days of curcumin treatment, the study showed in PBMCs a significant increase of Nrf2 (~1.8fold) and NQO1 (~2.3fold) protein. The effect occurred in non-DKD and DKD (~CKD1-3A) patients, although the effect was numerically lower in DKD. Two further studies with turmeric in CKD5-HD ^[74] and CKD3 with and without diabetes ^[75], did not report significant changes in Nrf2 although opposing directions for Nrf2 changes were observed between intervention and placebo groups in both studies. The number of patients in both studies was small, well-powered studies are necessary to obtain a meaningful evaluation of effects on the NRF2 system. If the NRF2 repression in advanced CKD, as in CKD5-HD, can be overcome by electrophilic compounds is unclear, since endogenous stimulation is already pronounced in this patient group.

The answer to the second question, if Nrf2 response to curcumin was coupled with a desirable effect on CKD progression or a reduction of CKD-attributable morbidity is unclear, partially due to very low number of included CKD patients, partially because observed salutary effects were not paralleled by respective Nrf2 system changes. The above-named study in CKD5-HD observed a reduction of inflammatory parameters in the curcumin group (decreased NF-κB gene expression in PBMCs and serum high-sensitivity C-reactive protein (hsCRP)). Another controlled trial in CKD5-HD reported a significant reduction of hsCRP with turmeric treatment for 2 months ^[76]. In the abovementioned study in early DKD (CKD1-3A), the increases in Nrf2 and NQO1 were seemingly paralleled by a reduction of albuminuria ^[73]. Also, a controlled study in patients with T2D and early DKD with non-reduced GFR and proteinuria ≥500 mg/d, reported decreased urinary protein excretion in patients treated with turmeric for 2 months ^[77].

As for sulforaphane, the issue of source and dosing in curcumin therapy is not resolved. The discussed studies used curcumin doses between 65 and 500 mg/d. High doses of curcumin (4000-6000 mg/d) given perioperatively in aortic aneurysm repair increased the risk for acute kidney injury ^[78].

The issue of curcumin excretion and thereby the possibility for accumulation in human CKD is not clear. Generally, methylation, sulfation and glucuronidation of polyphenols have been described in humans ^[79]. While it is accepted, that urinary excretion of curcumin occurs through glucuronide and sulfate conjugates, the overall proportion of renal excretion in humans is not known ^[72]. If turmeric is used, the contribution of curcumin to a certain molecular effect is unclear, as turmeric contains a multitude of potentially bioactive compounds ^[80]. Furthermore, it should be noted, that turmeric contains a high percentage of water-soluble oxalate, and turmeric doses comparable to those used in some of the studies (~3g/d) result in a significantly increased urinary oxalate excretion in healthy subjects ^[81]. This should raise some caution, as oxalate accumulates in CKD5-HD and high oxalate concentrations in those patients were associated with cardiovascular events ^[82].

15. Future dDirections in Nrf2-tTargeted tTherapies in hHuman CKD

Successful Nrf2-targeted therapies in CKD require a diversified approach. The Nrf2 system is a complex network that works to ensure adequate responses to redox perturbations, varying energy and metabolic demands, inflammation, and other cellular stresses. This includes dynamic increases and decreases of Nrf2 activity according to demand. The following strategies can be considered:

16. Targeting Nrf2 sSystem dDisturbances in CKD more sMore Specifically

The antioxidant effectiveness through the Nrf2 system in CKD seems to be insufficient in those instances where oxidative damage through ROS develops. The underlying disturbances can occur on all levels of the Nrf2 system, like inadequate Nrf2 gene expression, Nrf2 protein amount and structure, modulation of Nrf2 transcriptional activity by other factors, and disturbances of Nrf2 targets’ protein amount, structure and activity. Therefore, knowledge about such disturbances in a specific CKD setting is inevitable. If for example, as reported for chronic kidney allograft dysfunction an increase of GSK-3β protein exists ^[55], it might be useful to target this kinase using a GSK-3 inhibitor to prevent undue β-TrCP-GSK-3β-mediated Nrf2 degradation. If, as reported in patients with liver cirrhosis, the E3 ubiquitin ligase synoviolin (HRD1) is increased leading to Nrf2 ubiquitylation and Keap1-independent Nrf2 degradation, then targeting HRD1 rather than Keap1 might be promising ^[83]. Finally, if already significantly increased NQO1 gene expression as shown in some CKD stages does not translate into a correspondingly large increase in NQO1 protein amount ^[84], then it may be promising to find and target the mechanism responsible for this discrepancy. Altogether, strategies to remove CKD-specific disturbances of the Nrf2 system could result in an improved Nrf2 response with the ability to react adequate to demand.

17. Pharmacological Keap1 iInhibition

Respective substances, like bardoxolone methyl, mimic the effects of endogenous electrophiles and oxidants on Nrf2 by inhibiting Nrf2 protein degradation. Thereby, they activate Nrf2-dependent responses of both positively and negatively regulated Nrf2 target genes. Several aspects require attention in this respect. First, is the responsiveness of the Nrf2 system to concentration changes of electrophiles/oxidants in CKD per se preserved? The answer seems to be yes at least in some patient populations, based on the GGT increase following bardoxolone methyl in CKD4 ^[62] and the NQO1, HO-1 and ferritin increase following tin protoporphyrin in CKD3/4 ^[20]. Second, is pharmacological Keap1 inhibition more effective than endogenous Nrf2 activation according to antioxidant requirements? It could be more effective if exhaustive Nrf2 activation is exerted. Third, if the antioxidant demand is permanently increased in CKD, this would require a permanent pronounced Nrf2 activation. Can this be beneficial? The answer is unclear. Endogenous Nrf2 activation correlated with proteinuria ^[32]; and a higher CVD prevalence was observed in patients with higher NQO1 gene expression ^[84]. Of course, this does not prove causality. Furthermore, pharmacological Nrf2 activation by bardoxolone methyl led to weight loss at all doses of the BEAM study, in the BEACON study, and also in the TSUBAKI study [85,58,61]^[58][61][85]. Weight loss is not necessarily negative and was reported to associate with improved glycemic control in obese patients with T2D ^[85]. Nevertheless, this effect needs careful observation with respect to patient subgroups, like normal- or underweight patients or children, and a close observation of the long-term course. This is the more important, as there is a high probability that this is a genuine Nrf2-mediated effect. Enzymes that upregulate beta-oxidation, such as carnitine palmitoyltransferase, CD36, and the downregulation of enzymes that induce lipid biosynthesis, such as acetyl-CoA carboxylase, the enzyme that catalyzes lipid biosynthesis, SCD1 and SREBP-1 are regulated by Nrf2 in the respective directions ^[40]. Finally, based on the reported decline of GGT concentration after the initially strong response to bardoxolone treatment, a restrain of Nrf2 activation through feedback regulation seems likely. Therefore, intermittent dosing schemes, maybe with lower doses, could be worth considering.

18. Reduction of fFactors rResponsible for eEndogenous Nrf2 aActivation

As outlined earlier, a multitude of factors can result in endogenous activation of the Nrf2 system, some of them, as discussed for indoxyl sulfate may in high concentrations also contribute to Nrf2 repression. To reduce such repression, and to avoid a permanent overactivation of the Nrf2 system, a reduction of Nrf2 stimulating factors in addition to the above proposed strategies is desirable. Those measures will vary according to CKD cause, stage, comorbidities or treatment modality. Indoxyl sulfate reducing strategies for example comprise oral adsorbents, synbiotics, or special hemodialysis cartridges ^[86]. Methylglyoxal reduction can be achieved by increasing removal through extended hemodialysis or hemodiafiltration ^[87], but also through Nrf2 activation by trans-resveratrol-hesperitin (tRES-HESP) which induces significant glyoxalase-1 activity and reduction of methylglyoxal [88]^[30].

19. Conclusions

Alterations in redox signaling, oxidative stress and disturbed activity of the Nrf2 system have a central role in CKD progression and CKD-related morbidity. Activation of the Nrf2 system in CKD is in multiple ways related to inflammation, kidney fibrosis, and mitochondrial and metabolic effects. Dependent on the actual antioxidant requirements and the disease related state of other signaling pathways, Nrf2 system activation can be beneficial, but also disadvantageous, depending on the disease and patient context. The Nrf2 system comprises a complex network that functions to ensure adequate responses to redox perturbations, varying energy and metabolic demands, and cellular stresses. It must be kept under homeostatic control within a physiologic activity range. A constant overactivation seems not desirable. It is therefore important to realize, that in human CKD both endogenous Nrf2 activation and repression exist. The state of the Nrf2 system varies with cause of kidney disease, comorbidities, stage of CKD, duration of CKD, and severity of uremic toxin accumulation and inflammation. Earlier CKD stage or rapid progression of kidney disease, and inflammatory processes are associated with a more robust Nrf2 system activation. Advanced CKD is associated with stronger Nrf2 system repression. For the development and evaluation of future Nrf2-targeted therapies, it is necessary to answer the following questions: Is the substance able to elicit an Nrf2 activation that results in an appropriate functional response of Nrf2 targets in the respective CKD setting? Is this response coupled with a desirable effect on CKD progression or a reduction of CKD-attributable morbidity? Is the substance overall beneficial for the patient? Therefore, future Nrf2-targeted therapies in CKD should apply a diversified approach that enables dynamic increases and decreases of Nrf2 activity according to homeostatic requirements. Resulting Nrf2 system responses should be sufficient to cope with oxidative stress, inflammatory state, and activated pro-fibrotic mechanisms. Such new approaches need to be fitted to specific CKD-related disturbances in the Nrf2 system, like increases of GSK-3β, CKD-related protein modifications on Nrf2 and Nrf2 target proteins, and accumulation of uremic toxins. While the aim is to support Nrf2 activity, Nrf2 overactivation should be avoided.

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