NRF2 Deficiency Attenuates Diabetic Kidney Disease in Db/Db Mice via Down-Regulation of Angiotensinogen, SGLT2, CD36, and FABP4 Expression and Lipid Accumulation in Renal Proximal Tubular Cells

The role(s) of nuclear factor erythroid 2-related factor 2 (NRF2) in diabetic kidney disease (DKD) is/are controversial. We hypothesized that Nrf2 deficiency in type 2 diabetes (T2D) db/db mice (db/dbNrf2 knockout (KO)) attenuates DKD progression through the down-regulation of angiotensinogen (AGT), sodium-glucose cotransporter-2 (SGLT2), scavenger receptor CD36, and fatty -acid-binding protein 4 (FABP4), and lipid accumulation in renal proximal tubular cells (RPTCs). Db/dbNrf2 KO mice were studied at 16 weeks of age. Human RPTCs (HK2) with NRF2 KO via CRISPR-Cas9 genome editing and kidneys from patients with or without T2D were examined. Compared with db/db mice, db/dbNrf2 KO mice had lower systolic blood pressure, fasting blood glucose, kidney hypertrophy, glomerular filtration rate, urinary albumin/creatinine ratio, tubular lipid droplet accumulation, and decreased expression of AGT, SGLT2, CD36, and FABP4 in RPTCs. Male and female mice had similar results. NRF2 KO attenuated the stimulatory effect of the Nrf2 activator, oltipraz, on AGT, SGLT2, and CD36 expression and high-glucose/free fatty acid (FFA)-stimulated lipid accumulation in HK2. Kidneys from T2D patients exhibited markedly higher levels of CD36 and FABP4 in RPTCs than kidneys from non-diabetic patients. These data suggest that NRF2 exacerbates DKD through the stimulation of AGT, SGLT2, CD36, and FABP4 expression and lipid accumulation in RPTCs of T2D.


Introduction
Increased plasma-free fatty acid (FFA) levels and disturbances in lipid metabolism, including increased fatty acid oxidation and accumulation of renal lipid droplets contribute to nephropathy progression in diabetes [1][2][3][4][5].Thus, understanding the mechanisms underlying lipid accumulation within the kidney is essential in order to prevent the development of diabetic kidney disease (DKD).
CD36, an 88 kiloDalton (kDa) single-chain membrane glycoprotein and a member of the class B scavenger receptor family, has been identified as a long-chain FFA transporter and signal transduction molecule and plays a role in fatty acid oxidation in numerous organs including the kidney [6].Increased CD36 expression was detected in the kidney cells of patients with diabetes and has been shown to contribute to DKD progression via palmitate-induced tubular apoptosis and tubulo-interstitial fibrosis [3,[7][8][9].
FABP4 (fatty-acid-binding protein 4), a small cytoplasmic protein (~14-15 kDa), is a member of the fatty-acid-binding protein family.FABP4 binds with high affinity to longchain fatty acids and eicosanoids and transports them to cellular components including lipid droplets.Studies have shown a close association between elevated FABP4 and the pathogenesis of metabolic and vascular disease and DKD [10,11].
In the present study, we hypothesized that Nrf2 deficiency in T2D db/db mice (db/dbNrf2 KO) would attenuate DKD progression, attenuating hypertension, hyperglycemia, lipid accumulation, and renal morphological changes/dysfunction through the decreased expression of AGT, SGLT2, CD36, and FABP4 in renal proximal tubular cells (RPTCs).We also examined human RPTCs (HK2) with or without NRF2 KO via CRISPR-Cas9 genome editing, cultured in normal-glucose or high-glucose milieu ± FFA added, and the kidneys of humans with or without diabetes.

Generation of db/dbNrf2 Knockout (KO) Mice
We cross-bred lean male db/m mice (C57BLKS/J strain) with female homozygous Nrf2 −/− KO mice (B6.129X1-Nfe2l2 tm1Ywk /J) (Jackson Laboratories, Bar Harbor, ME, USA (http://jaxmice.jax.org) to generate female db/m heterozygous Nrf2 KO mice, then backcrossed them with male db/m (C57BLKS/J) for at least 6 generations to obtain db/db homozygous Nrf2KO mice (>90% in C57BLKS).(Of note, homozygous Nrf2 KO mice are viable and fertile, whereas homozygous db/db mice are infertile.)Both sexes of db/db and db/dbNrf2 KO mice were studied at age 16 weeks.Age-and sex-matched db/m littermates served as controls.All mice had free access to standard mouse chow and water.Animal care and experimental procedures were approved by the Animal Care Committee of the Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM) and followed the National Institutes of Health (NIH)'s Principles of Laboratory Animal Care (Publication No. 85-23, revised 1985: http://grants1.nih.gov/grants/olaw/references/phspol.htm,accessed on 29 June 2023).

Pathophysiology
Systolic blood pressure (SBP) was measured via a BP-2000 tail-cuff pressure monitor (Visitech Systems, Apex, NC, USA) at least 2 to 3 times per week [17,33].Each mouse was accustomed to the procedure for at least 15 to 20 min per day for 5 days before the first SBP measurement at the age of 16 weeks.SBP values are presented as means ± SEM of 2-3 determinations/mouse/group.
Fasting blood glucose (FBG) levels were measured in mice with the Accu-Chek Performa System (Roche Diagnostics, Laval, QC, Canada) after fasting 4 to 6 h or with a glucose colorimetric detection kit (Cayman Chemical, Ann Habor, MI, USA) at the age of 16 weeks, as described previously [17,33].
The mice were housed individually in metabolic cages for 6 h during the daytime for urine collection prior to euthanasia at 16 weeks.Urine samples were assayed for albumin and creatinine via albumin enzyme-linked immunosorbent assay (ELISA, Albuwell and Creatinine Companion, Exocell, Inc., Philadelphia, PA, USA) [17,33], respectively.
Following euthanasia, the kidneys were removed, decapsulated, and weighed.Left kidneys were used for histology and immunostaining and the right kidneys were processed immediately for isolation of renal proximal tubules (RPTs) via Percoll gradient [17,33,36].Aliquots of freshly isolated RPTs were assessed for total RNA and protein.

Histology
A total of 4-5 sections per kidney and 6 mouse kidneys per group were immunostained using the standard avidin-biotin-peroxidase complex method (ABC Staining, Santa Cruz Biotechnology, Santa Cruz, CA, USA) [17,33].Kidney sections were counterstained with hematoxylin and then analyzed via light microscopy by 2 investigators blinded to treatment groups.
Periodic acid-Schiff (PAS) staining was also performed to assess kidney morphology.Histological changes including glomerular tuft volume, RPTC volume, and tubular luminal area were determined via the methods of Weibel and Gundersen [37,38] by using an image analysis software (Motics Images Plus 2.0; Motic, Richmond, BC, Canada).Tubular injury score was determined as described by Chen et al. [39].
Immunofluorescence (IF) staining for SGLT2 and FABP4 was performed on 4-µm tissue sections from mouse kidneys fixed in formalin and embedded in paraffin followed by staining with ALEXA FLUOR-594-labeled secondary antibody (Invitrogen).Proximal tubules were identified via fluorescein-labeled lotus tetragonolobus lectin (LTL, a marker of RPT [40]) (Vector Labs, Burlingame, CA, USA).Image quantification and merging were assessed via Image J software (http://rsb.info.nih.gov/ij/).The pixel intensity of SGLT2 or FABP4 was divided by LTL intensity to quantify the amount of SGLT2 or FABP4 expression.Six mice per group were analyzed to calculate the average ratio.
WB and RT-qPCR were used to quantify expression of AGT, SGLT2, and CD36 protein and mRNA, respectively.Oil Red O staining was used to assess lipid droplets in HK2 ± NRF2 KO cultured in normal or high glucose ± 200 µM palmitate/oleate-BSA added, respectively.

Immunostaining of CD36 and FABP4 on Human Kidney Specimens
Nephrectomy specimens (paraffin sections) from patients with or without diabetes obtained from the Department of Pathology, CHUM were immunostained for CD36 and FABP4.The CHUM Clinical Research Ethics Committee approved the study.All patients had undergone nephrectomy for kidney cancer and gave signed written informed consent for the use of their kidney tissue for research.Patients' clinical characteristics have been published previously [17] (Figure S4).

Statistical Analysis
The data are expressed as means ±SEM.Statistical comparisons were made via the Student's t-test or 1-way analysis of variance and the Bonferroni correction as appropriate.p < 0.05 values were considered to be statistically significant.

NRF2 Expression in db/db Nrf2KO Mouse Ear and Kidney Tissue
PCR analysis confirmed the presence of Nrf2 gene in the ear tissue of db/m and db/db mice but not in db/mNrf2 −/− (KO) and db/dbNrf2 KO mice (Figure 1a (panel i)).Db/db mice are identified by the presence of the Dock7 gene, which was introgressed into a mutant leptin receptor (Figure 1a (panel ii)).Immunostaining for NRF2 was more pronounced in nuclei of RPTCs from db/db mice than in db/m mice and was not detectable in db/mNrf2 KO and db/dbNrf2 KO mice of both sexes (Figure 1b and Supplementary Figure S1a).In contrast, KEAP1 immunostaining results were similar in the groups studied (Figure 1c).NRF2 and KEAP1 expression in RPTs assessed via respective WB (Figure 1d,e and Supplementary Figure S1b) and RT-qPCR of Nrf2 and Keap1 (Figure 1f,g and Supplementary Figure S1c) were consistent with these findings.

NRF2 Expression in db/db Nrf2KO Mouse Ear and Kidney Tissue
PCR analysis confirmed the presence of Nrf2 gene in the ear tissue of db/m and db/db mice but not in db/mNrf2 −/− (KO) and db/dbNrf2 KO mice (Figure 1a (panel i)).Db/db mice are identified by the presence of the Dock7 gene, which was introgressed into a mutant leptin receptor (Figure 1a (panel ii)).Immunostaining for NRF2 was more pronounced in nuclei of RPTCs from db/db mice than in db/m mice and was not detectable in db/mNrf2 KO and db/dbNrf2 KO mice of both sexes (Figure 1b and Supplementary Figure S1a).In contrast, KEAP1 immunostaining results were similar in the groups studied (Figure 1c).NRF2 and KEAP1 expression in RPTs assessed via respective WB (Figure 1d,e and Supplementary Figure S1b) and RT-qPCR of Nrf2 and Keap1 (Figure 1f,g and Supplementary Figure S1c) were consistent with these findings.

Physiological Measurements in Mice
Genetic deletion of Nrf2 significantly decreased SBP (on average, SBP was ~10 mm Hg lower) in db/mNrf2 KO and db/dbNrf2 KO mice than respective db/m and db/db mice at 16 weeks in both sexes (Figure 2a,b).However, SBP did not differ significantly in db/m and db/db mice despite slight increases in db/db as compared to db/m mice.FBG levels were significantly higher in db/db than in db/m mice in both sexes (Figure 2c,d).Deletion of Nrf2 resulted in significantly lower FBG levels in db/dbNrf2 KO than in db/db mice, whereas FBG levels were similar in both male and female db/m and db/mNrf2 KO mice (Figure 2c,d).
Both male and female db/db and db/dbNrf2 KO mice had significantly higher body weights (BWs) than respective db/m and db/mNrf2 KO mice (Supplementary Figure S2a and Figure 2b).While Nrf2 KO had no detectable effects on BW in db/db mice, it decreased the kidney weight (KW)/tibial length (TL) ratio (Figure 2e,f), glomerular filtration rate (GFR)/BW ratio (Figure 2g,h), and urinary albumin-creatinine ratio (ACR) (Figure 2i,j) in db/dbNrf2 KO vs. db/db mice of both sexes.These parameters did not differ between db/m and db/mNrf2 KO mice (Figure 2e-j).
Fat content/BW (Supplementary Figure S2c and Figure 2d), lean mass/BW (Supplementary Figure S2e and Figure 2f), and total water content/BW (Supplementary Figure S2g and Figure 2h) were similar in db/m and db/mNrf2 KO mice, whereas significant increases in fat content/BW and decreases in lean mass/BW and total water content/BW were detected in db/db and db/dbNrf2 KO mice vs. respective db/m and db/mNrf2 KO mice of both sexes.

Histology and Immunostaining
The kidneys of db/db mice exhibited structural damage vs. db/m mice (Figure 3a), which is consistent with our earlier observations [44].Histological changes including enlarged glomerular tuft volume, tubular luminal area, and RPTC volume in db/db mice, were attenuated in db/dbNrf2 KO mice (Figure 3(ai,aii,aiii), respectively).Tubular injury scores confirmed these observations (Figure 3(aiv)).
Significantly stronger staining for 8-OHdG (Figure 3b) and DHE (Supplementary Figure S3) was detected in RPTs of db/db vs. db/m and db/mNrf2 KO mice, but it did not differ from that in db/dbNrf2 KO mice.In contrast, immunostaining for HO-1 (Figure 3c) and NOX-4 (Figure 3d) in RPTs were significantly increased in RPTs from db/db mice compared to db/m and db/mNrf2 KO mice, whereas these changes were attenuated in db/dbNrf2 KO mice.In contrast, CAT immunostaining was significantly higher in RPTs of db/m than in db/mNrf2 KO, db/db, or db/dbNrf2 KO mice (Figure 3e).These findings were confirmed via semi-quantification of 8-OHdG (Figure 3b) and DHE (Supplementary Figure S3) staining and immunostaining for HO-1 (Figure 3c), NOX-4 (Figure 3d), and CAT (Figure 3e) as well as via RT-qPCR for HO-1 (Figure 3f), Nox-4 (Figure 3g), and Cat (Figure 3h) from isolated mouse RPTs.

Nrf2 Deletion on AGT and SGLT2 Expression in db/db Mice
AGT immunostaining in RPTs of db/db mice was more pronounced vs. db/dbNrf2 KO mice (Figure 4a and Supplementary Figure S1d).Co-immunofluorescence (IF) staining for SGLT2 and LTL revealed higher SGLT2/LTL ratios in RPTs from db/db vs. db/m mice of both sexes (Figure 4b and Supplementary Figure S1e, respectively).Db/dbNrf2 KO mice exhibited lower SGLT2/LTL ratios than db/db mice.No significant changes were detected between db/m and db/mNrf2 KO mouse kidneys.Semi-quantitation of immunostaining images of AGT (Figure 4c) and SGLT2/LTL (Figure 4d and Supplementary Figure S1f) as well as RT-qPCR of their respective mRNAs in isolated RPTs (Figure 4e,f and Supplementary Figure S1g,h) confirmed these observations.

Nrf2 Deletion on AGT and SGLT2 Expression in db/db Mice
AGT immunostaining in RPTs of db/db mice was more pronounced vs. db/dbNrf2 KO mice (Figure 4a and Supplementary Figure S1d).Co-immunofluorescence (IF) staining for SGLT2 and LTL revealed higher SGLT2/LTL ratios in RPTs from db/db vs. db/m mice of both sexes (Figure 4b and Supplementary Figure S1e, respectively).Db/dbNrf2 KO mice exhibited lower SGLT2/LTL ratios than db/db mice.No significant chan tected between db/m and db/mNrf2 KO mouse kidneys.Semi-quantit munostaining images of AGT (Figure 4c) and SGLT2/LTL (Figure 4d and Su Figure S1f) as well as RT-qPCR of their respective mRNAs in isolated RPT and Supplementary Figure S1g,h) confirmed these observations.pressed as mean ± SEM; n = 6 per group for staining; n = 8 per group for mRNA expre were obtained via one-way ANOVA followed by Bonferroni post hoc test.* p < 0.05 p < 0.005 vs. db/m; ns, not significant.

Nrf2 Deficiency on Lipid Accumulation and Expression of CD36 and FABP4 i
Oil Red O staining increased in renal tubules of db/db vs. db/m mice (Fi this was markedly decreased in the renal tubules of db/dbNrf2 KO mice.Imm

Nrf2 Deficiency on Lipid Accumulation and Expression of CD36 and FABP4 in db/db Mice
Oil Red O staining increased in renal tubules of db/db vs. db/m mice (Figure 5a), and this was markedly decreased in the renal tubules of db/dbNrf2 KO mice.Immunostaining of CD36 revealed higher expression in RPTs from db/db vs. db/m and db/mNrf2KO mice (Figure 5b), whereas db/dbNrf2KO mice exhibited lower CD36 expression than db/db mice.Similar changes were detected for FABP4 immunostaining, with significant increases in db/db vs. db/m mice (Figure 5c), whereas FABP4 immunostaining was lower in db/dbNrf2KO mice than in db/db mice.Semi-quantitation of Oil Red O staining (Figure 5d), CD36 (Figure 5e), and FABP4 (Figure 5f) immunostaining, and RT-qPCR of CD36 (Figure 5g) and Fabp4 (Figure 5h) confirmed these observations.

Oil Red O Staining in HK-2 with or without NRF2 KO
While BSA did not increase oil droplet accumulation, FFA enhanced the accumulation of oil droplets in HK2 cells in NG in a dose-dependent manner, which was attenuated in NRF2 KO HK2 cells (Figure 7a).Moreover, HG did not increase oil droplet accumulation but FFA enhanced accumulation of oil droplets in HK2 cells in NG and HG, which was attenuated in NRF2 KO HK2 cells (Figure 7b) and confirmed by the quantitation of oil droplets accumulation (Figure 7(di,dii)).

CD36 and FABP4 Immunostaining in Human Kidney Sections
Increased CD36 and FABP4 immunostaining were detected in kidney specimens from diabetic patients vs. those without diabetes (Figures 8a and 8b, respectively) and confirmed via quantitation of immunostaining (Figures 8c and 8d), respectively).These observations are consistent with CD36 and FABP4 changes observed in the RPTs of db/db mice.

Oil Red O Staining in HK-2 with or without NRF2 KO
While BSA did not increase oil droplet accumulation, FFA enhanced the accumulation of oil droplets in HK2 cells in NG in a dose-dependent manner, which was attenuated in NRF2 KO HK2 cells (Figure 7a).Moreover, HG did not increase oil droplet accumulation but FFA enhanced accumulation of oil droplets in HK2 cells in NG and HG, which was attenuated in NRF2 KO HK2 cells (Figure 7b) and confirmed by the quantitation of oil droplets accumulation (Figure 7(di,dii)).

Discussion
Our results document that the genetic deletion of Nrf2 in db/db mice was associated with attenuated DKD features (SBP, FBG, GFR, urinary ACR, tubular lipid accumulation, kidney injury) in tandem with decreased expression of AGT, SGLT2, and CD36 protein and mRNA in RPTCs.HK2 cells with NRF2 KO also exhibited lower expression of AGT, SGLT2, and CD36 and did not respond to the stimulatory effect of oltipraz or FFA on the expression of these proteins/genes.These data would appear to link NRF2 activation, intrarenal RAS and SGLT2 expression and tubular lipid accumulation, to the exacerbation of hypertension, dysglycemia, and kidney injury in T2D.
The db/db mouse (LepR db/db in BKS background) is an established T2D murine model [45] (http://www.diacomp.org/).We found elevated NRF2 and HO-1 expression in RPTs of 16-week-old db/db mice, with no detectable differences in KEAP1 expression.In contrast, HO-1 expression was decreased in the RPTs of db/mNrf2 KO and db/dbNrf2 KO mice vs. respective db/m and db/db mice.These data confirmed that HO-1 is a downstream target of NRF2 and confirmed the usefulness of db/dbNrf2 KO mice as a preclinical model to study the role of NRF2 in T2D.
Significantly lower SBP was found in both male and female db/mNrf2 KO and db/dbNrf2 KO mice vs. respective db/m and db/db mice.Although SBP was slightly higher in db/db mice vs. db/m mice, it did not reach statistical significance.These might be due to the short duration (15-20 min per day) of BP measurement via the tail-cuff pressure monitor.More sensitive BP measurements such as telemetry for continuous 24-48 h monitoring are required to detect the differences in BP between these mice.
Significantly lower FBG levels, KW/TL, urinary ACR, and GFR were found in db/dbNrf2 KO mice vs. db/db mice of both sexes.However, these parameters were similar in db/m mice and db/mNrf2 KO mice at 16 weeks of age, implying that Nrf2 deficiency may improve these parameters in db/db mice via lowering blood glucose.At present, the exact reason for the decline in the GFR following Nrf2 deletion is not clear.However, since Nrf2 deletion down-regulated SGLT2 and AGT/Ang II expression in the RPTs, we speculate that the reduction in GFR and SBP could be attributed, at least in part, to lowering FBG levels and SGLT2 expression in RPTs.This would lead to vasoconstriction in the afferent arteriole induced by the restoration of the tubulo-glomerular feedback (TGF) [46,47] and the down-regulation of intrarenal AGT/Ang II expression, causing vasodilation in the efferent arteriole to lower intraglomerular pressure [48,49].Cumulatively, all these factors could lead to decreases in GFR, SBP, and urinary ACR.No significant changes of fat mass/BW, lean mass/BW, and total water content/BW were observed between db/dbNrf2 KO and db/db mice as well as between db/m and db/mNrf2 KO mice.These findings lend further support to the idea that the beneficial effects of Nrf2 deficiency on BG level might be predominately due to the loss of NRF2 action in the kidney rather than in fat or muscle tissue.It remains to be determined whether Nrf2 deletion in db/db mice could affect insulin sensitivity or production, food consumption, circulating levels of triglycerides, and fatty acids.Works are ongoing to address these issues.
Increased 8-OHdG staining and DHE (markers of oxidative stress) were found in RPTs of db/db mice vs. db/m, but the staining was lower than in db/mNrf2 KO mice and db/dbNrf2 KO mice.Increased NOX4 expression was also observed in db/db mice vs. db/m and db/mNrf2 KO mice and attenuated in db/dbNrf2 KO mice.We do not presently understand why Nrf2 deletion exhibited higher oxidative stress in RPTs of db/mNrf2 KO and db/dbNrf2 KO mice vs. db/db mice.We have observed, however, that Cat mRNA expression was lower in RPTs of db/db vs. db/m mice, and deletion of Nrf2 did not change Cat expression in db/dbNrf2 KO mice vs. db/db mice.These data indicate that hyperglycemia/hyperlipidemia and Nrf2 deletion would alter relative expression and activity of Nox4 and Cat, thereby enhancing oxidative stress and likely contributing to renal injury in db/db mice.
The mechanism(s) by which Nrf2 deficiency leads to the downregulation of renal AGT expression in diabetes remain(s) undefined.By employing gene deletion analysis and site-directed mutagenesis, we have shown that NRF2 binds to two NRF2-responsive elements (REs) in the 5 -flanking region of the Agt promoter and that deletion of both NRF2-REs is more effective in inhibiting the stimulatory effect of oltipraz than deletion of either NRF2-RE alone [33,50].These findings would indicate that NRF2 regulates Agt gene expression at the transcriptional level.
Our present results demonstrate that Nrf2 deficiency lowers SGLT2 expression in db/dbNrf2 KO vs. db/db mice.These findings are consistent with our previous studies in that genetic deletion of Nrf2 decreases the expression of SGLT2 in AkitaNrf2 KO mice and NRF2 stimulates Sglt2 transcription via the NRF2-RE in the Sglt2 gene promoter, as shown by gene deletion, site-directed mutagenesis, gel mobility shift, and chromatinimmunoprecipitation (ChIP) assays [17], further supporting the pathophysiological role of NRF2-mediated up-regulation of Sglt2 expression in diabetic mice.
Studies on the protective role of NRF2 in renal diseases yielded conflicting results [51].In contrast to our findings that Nrf2 deficiency improves kidney injury in db/db mice, Jiang et al. [30] and Yok et al. [52] reported that streptozotocin (STZ)-induced diabetic Nrf2 −/− mice displayed increased glomerular fibrosis and lower creatinine clearance vs. non-diabetic mice.More recently, Liu et al. [53] reported that Nrf2 deficiency led to deterioration of DKD with no change of SGLT2 expression in AkitaNrf −/− mice vs. Akita mice.The authors also found that blood glucose levels were elevated and fractional excretion of glucose was lower in Akita Nrf2 −/− mice vs. Akita mice, indicating that SGLT2 expression is not under NRF2 regulation.At present, we do not have a clear explanation for these discrepancies.However, Liu et al. [53] utilized a unique Nrf2 KO Nfe212 tm1Mym mouse line established by their laboratory [54], whereas we used Nrf2 −/− KO mice (B6.129X1-Nfe2l2 tm1Ywk /J) procured from The Jackson Laboratory (http://jaxmice.jax.org).Thus, it remains to be investigated whether differences in mutation of Nrf2 gene in these Nrf2 −/− mice could explain these discrepancies.
Consistently, our in vitro results in HK2 with NRF2 KO confirm Nrf2 regulation of endogenous AGT, SGLT2, and CD36 expression and that NRF2 deletion renders HK2 unresponsive to oltipraz stimulation of AGT, SGLT2, and CD36 protein and mRNA expression.Intriguingly, we found that HG could stimulate CD36 expression but not AGT and SGLT2 expression in HK2 with NRF2 KO.These results suggest that additional signaling pathway(s) might mediate HG stimulation of CD36 expression which is independent of NRF2/KEAP1 signaling.This possibility is supported by the recent study of Huang et al. [22] that SGLT2i ameliorates FFA-induced lipotoxicity in RPTCs via the peroxisome proliferator-activated receptor-gamma (PPARγ)/CD36 pathway in obese mice, suggesting that SGLT2 up-regulation by HG may stimulate CD36 expression via PPARγ.
Our data revealed that db/dbNrf2 KO mice exhibit decreased tubular lipid droplets and CD36 and FABP4 expression vs. db/db mice, implying a role for NRF2 in the regulation of tubular lipid accumulation via increased CD36 and FABP4 expression.However, the mechanisms of CD36 and FABP4 regulation in RPTCs remain to be defined.Studies by Maruyama et al. [55] show that NRF2 stimulates CD36 gene transcription in macrophages, and Sussan et al. [56] reported decreased CD36 expression in macrophages and in atherosclerotic plaques from Apol −/− Nrf2 −/− mice, indicating that NRF2 regulates CD36 expression at the transcriptional level.We have found that Fabp4 protein and mRNA expression is also down-regulated in the RPTs of db/dbNrf2 KO mice.It remains to be investigated whether NRF2 regulates Fabp4 expression at the transcriptional level.Our results may help explain the potentially harmful effects of NRF2 activation (with bardoxolone methyl (bardoxolone)) reported in T2D patients with advanced CKD (BEACON trial) [29,32].The BEACON trial was discontinued after 9 months due to increased mortality and heart failure.Moreover, bardoxolone resulted in increased estimated GFR and urine ACR (29,32), which was also confirmed in a recent trial of this drug in non-diabetic patients with Alport syndrome (CARDINAL trial) [57].There has been a concern of increased intraglomerular pressure induced by bardoxolone, but its mechanisms remain unclear [58,59].We speculate that bardoxolone upregulation of SGLT2 expression in RPTs might have played a role in these events, ultimately leading to glomerular hyperfiltration via tubulo-glomerular feedback and aggravated kidney injury.Indeed, the recent studies of Rush et al. [60] have reported that genetic activation of Nrf2 in Keap1 FA/FA mice or pharmacologic Nrf2 activation via bardoxolone analog increase proteinuria in several mouse models of chronic kidney disease.However, the question whether NRF2 activation may be harmful in T2D patients with CKD warrants further investigation.

Conclusions
Our present findings have demonstrated that genetic deletion of Nrf2 can lead to the down-regulation of renal AGT, SGLT2, CD36, and FABP4 expression with subsequent decreases in SBP, FBG, and tubular lipid accumulation, which ultimately resulted in the attenuation of kidney injury in db/db Nrf2 KO mice.Our results imply an important role for NRF2-mediated stimulation of AGT, SGLT2, CD36, and FABP4 expression in exacerbating renal injury in T2D.Our findings would indicate that selective targeting of renal NRF2 might be a novel approach for the prevention or treatment of diabetic kidney disease.

Figure 4 .
Figure 4. AGT and SGLT2 expression in male db/db mouse kidneys at week 16 imm ical staining of AGT (a), immunofluorescent staining of SGLT2 and LTL (b), semi-qu AGT immunostaining (c), and SGLT2/LTL ratio (d) in mouse kidneys.RT-qPCR ana and Sglt2 (f) levels in RPTs of db/m, db/mNrf2 KO, db/db, and db/dbNrf2 KO mice.pressed as mean ± SEM; n = 6 per group for staining; n = 8 per group for mRNA expre were obtained via one-way ANOVA followed by Bonferroni post hoc test.* p < 0.05 p < 0.005 vs. db/m; ns, not significant.

Figure 4 .
Figure 4. AGT and SGLT2 expression in male db/db mouse kidneys at week 16 immunohistochemical staining of AGT (a), immunofluorescent staining of SGLT2 and LTL (b), semi-quantification of AGT immunostaining (c), and SGLT2/LTL ratio (d) in mouse kidneys.RT-qPCR analysis of Agt (e) and Sglt2 (f) levels in RPTs of db/m, db/mNrf2 KO, db/db, and db/dbNrf2 KO mice.Values are expressed as mean ± SEM; n = 6 per group for staining; n = 8 per group for mRNA expression.Statistics were obtained via one-way ANOVA followed by Bonferroni post hoc test.* p < 0.05; ** p < 0.01; *** p < 0.005 vs. db/m; ns, not significant.

Figure 7 .
Figure 7. Oil Red O Staining in HK-2 with or without NRF2 KO.(a) Oil Red O staining in HK2 with or without NRF2 KO cultured in NG in FFA-depleted BSA or different concentrations of FFA (palmitate/oleate).(b) Oil Red O staining in HK2 with or without NRF2 KO cultured in NG or HG ± FFA-depleted BSA (400 μM) or palmitate (200 μM)/oleate (200 μM).(c) and (d) Semi-quantification of Oil Red O staining in (i) HK2 control or (ii) HK2 with NRF2 KO cultured in NG or HG ± FFAdepleted BSA or palmitate/oleate.Values are mean ± SEM; n = 3. Statistics were obtained via one-

Figure 7 .
Figure 7. Oil Red O Staining in HK-2 with or without NRF2 KO.(a) Oil Red O staining in HK2 with or without NRF2 KO cultured in NG in FFA-depleted BSA or different concentrations of FFA (palmitate/oleate).(b) Oil Red O staining in HK2 with or without NRF2 KO cultured in NG or HG ± FFA-depleted BSA (400 µM) or palmitate (200 µM)/oleate (200 µM).(c) and (d) Semiquantification of Oil Red O staining in (i) HK2 control or (ii) HK2 with NRF2 KO cultured in NG or HG ± FFA-depleted BSA or palmitate/oleate.Values are mean ± SEM; n = 3. Statistics were obtained via one-way ANOVA followed by Bonferroni post hoc test.*** p < 0.005; ns, not significant.HK2NRF2 KO versus HK2 control (Ctrl).