GSTM1 Gene, Diet, and Kidney Disease: Implication for Precision Medicine? Recent Advances in Hypertension

Abstract

In the United States, the prevalence of chronic kidney disease in adults is ~14%. The mainstay of therapy for CKD are angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, but many chronic kidney disease patients still progress to end stage kidney disease. Increased oxidative stress is a major molecular underpinning of chronic kidney disease progression. In humans, a common deletion variant of the glutathione-S-transferase μ−1 (GSTM1) gene, the GSTM1 null allele (GSTM1(0)), results in decreased GSTM1 enzymatic activity and is associated with higher levels of oxidative stress. GSTM1 belongs to the superfamily of GSTs that are phase II antioxidant enzymes and are regulated by nuclear factor erythroid 2-related factor 2 (Nrf2). Cruciferous vegetables in general, and broccoli in particular, are rich in glucoraphanin, a precursor of sulforaphane that has been shown to have protective effects against oxidative damage through the activation of Nrf2. This review will highlight recent human and animal studies implicating the role of GSTM1 deficiency in hypertension and kidney disease, and its impact on the effects of cruciferous vegetables on kidney injury and disease progression, illustrating the significance of gene and environment interaction and a potential for targeted precision medicine in the treatment of kidney disease.

Overview of GSTM1.

GSTM1 belongs to the superfamily of glutathione-S-transferases (GSTs) that catalyze a number of distinct glutathione-dependent reactions. The primary role of GSTs is the metabolism of xenobiotics, and a broad range of reactive oxygen species (ROS) and electrophilic (or electron loving) compounds (ECs) such as reactive aldehydes (RAs). GSTs are phase II enzymes that biotransform drugs and ECs by catalyzing the conjugation of these compounds with the nucleophilic (nucleus loving and electron donor) reduced glutathione (GSH). The importance of these enzymes is highlighted by observations that GSTs can constitute up to 10% of cytosolic protein in some mammalian organs¹. There are 13 distinct classes of soluble GSTs that have been identified thus far according the substrate specificity, chemical affinity, structure and kinetic behavior of the enzyme^2–4. Gstm1 belongs to the Mu (μ) class, and is one of the 7 μ class Gst genes in mice (all 7 are clustered on mouse chromosome 3)^{5, 6}, and one of 5 in humans⁷. Gstm1 is virtually ubiquitously expressed, and is the most abundantly expressed isoform among all Gst genes in the murine kidney^8–10. The murine GSTM1 shares 93% amino acid identity with the rat GSTM1 and ~ 80% with the human GSTM1 (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).

Electrophilic reactive aldehydes as biological mediators.

Biological systems are constantly exposed to a wide range of environmental and endogenously generated ECs. These electron loving compounds are highly reactive with the major cellular nucleophile, GSH, as well as nucleophilic residues of proteins such as cysteine, histidine, and lysine¹¹. Their acute cellular effects are largely related to the depletion of GSH by direct reaction and by a reaction catalyzed by glutathione S-transferases^12–14. Among the most common ECs are the reactive α,β-unsaturated aldehydes such as acrolein (ACR), 4-hydroxy-2-hexenal (4-HHE), and 4-hydroxy-2-nonenal (4-HNE). These reactive aldehydes (RAs) are environmental pollutants but can also be endogenously produced from lipid oxidation¹⁵. At biological/cellular concentrations, these RAs induce various stress responses, and can activate or inactivate signaling pathways that are involved in transcription factor activation and regulation of gene expression. Their effects ultimately determine cellular fates, such as the production of cellular inflammatory mediators, or cell proliferation, survival or apoptosis^{16, 17}. RAs may also indirectly cause an increase in cellular production of ROS by modification of critical target proteins. For example, ACR and 4-HNE have been demonstrated to directly interact with the nucleophilic seleno-cysteine residue of the redox protein thioredoxin reductase (TrxR), and thereby inhibiting its reducing activities, resulting in increased levels of ROS¹⁸. 4-HNE has also been shown to inactivate the redox protein peroxiredoxin (Prx) by direct alkylation¹⁹, thereby inhibiting its ability to metabolize H₂0₂. In the cytosol, exposure to RAs results in the release of Nrf2 from the Nrf2-Keap1 complex; Nrf2 then escapes from Keap-1 mediated ubiquitination and translocates to the nucleus to activate transcription of Phase II oxidative stress response genes^20–23. Deletion of Nrf2 in the mouse results in decreased expression of Gstm1-Gstm4, and Gsta1 and Gsta2²⁴, and increased susceptibility to organ toxicity after acute exposure to environmental agents and drugs²⁵.

GSTM1 enzyme as an important cellular defense against reactive aldehydes.

The most important detoxification mechanism for RAs is thought to involve their conjugation with GSH by GSTs^{11, 12, 26}. Enzyme kinetic assays have demonstrated that GSTM1 has activity against ACR and 4-HNE^{12, 27–29}. However, the extent of its role in regulating intracellular levels and activities of RAs has not been well studied. In the ischemic reduction of renal mass (RRM) mouse model of hypertension and CKD, Gstm1 expression is increased along with increased levels of the RA 4-HNE, a product of lipid peroxidation, suggesting that Gstm1 is normally upregulated in RRM/CKD in response to increased oxidative stress³⁰. In vascular smooth muscle cells (VSCMs), decreased Gstm1 expression through siRNA knockdown resulted in increased in both 4-HNE protein adducts³⁰, and the ROS superoxide (O₂•-) and hydrogen peroxide (H₂O₂)³¹. Since there is no evidence that GSTM1 has superoxide dismutase nor catalase activity to metabolize O₂•- and H₂O₂, respectively, it is likely that the elevation in ROS levels in VSMCs after loss of GSTM1 indirectly results from accumulation of endogenously generated RAs that alter the redox state. It has become increasingly recognized that RAs potentially have more cell damaging effects than free radicals or ROS such as O₂•- or H₂O₂. For example, compared to O₂•- or H₂O₂, 4-HNE is chemically better suited for the role of a signaling molecule because of its longer half-life and thus greater range of diffusion, and a higher selectivity for reaction with specific protein targets^{32, 33}. In recent years, the physiological role of 4-HNE as an important “signaling molecule” has been established³⁴. Importantly, the effect of 4-HNE is concentration dependent, and thus 4-HNE may have disparate effects on cellular homeostasis, including cytotoxicity, cell cycle, and cell proliferation³⁵. However, the precise proteins modified by 4-HNE and other reactive aldehydes are unknown, though their protein adducts have been widely demonstrated in experimental models of cell toxicity. Currently, the use of non-specific antioxidants for treatment of various diseases has yielded disappointing results, likely due to their lack of specificity. Recently, in a mouse model lacking one, two or all three of the GSTP, GSTM, or GSTT gene families, the GSTM family was identified as the primary mediator of glutathione (GSH) conjugation to acrylamide⁹, an environmental electrophile that has been demonstrated to alter renal function in the rat³⁶. Identification of the protein targets of reactive aldehydes and other substrates of GSTM1 and their functions in disease states such as hypertension and CKD may be key in determining the molecular consequences of electrophilic aldehydes. Uncovering these pathways may enable development of more specific and targeted preventive and treatment modalities.

Non-catalytic domain of GSTM1 plays a functional role in the ASK1 signaling pathway.

In addition to its well-known catalytic activity in intracellular glutathione conjugation and metabolism, GSTM1 acts through its non-catalytic domain to inhibit activation of the ASK1-p38 signaling pathway of apoptosis. Under normal conditions, GSTM1, via its non-catalytic site, binds to the apoptosis signaling-regulating kinase-1 (ASK1), thereby inhibiting ASK1 activation³⁷. In conditions of stress such as heat shock or exposure to H₂0₂, GSTM1 is released from ASK1, leading to activation of ASK1 and downstream kinases³⁸. Although the non-catalytic domain of GSTM1 plays a role in the ASK1-signalling cascade, the exact effect of this domain on cell function in health and disease state has not been well studied. In a rat model of 4-vinylcyclohexene diepoxide (VCD) induced ovotoxicity, chronic exposure to VCD resulted in increased ovarian GSTM:ASK1 protein complex³⁹ that was temporally associated with a depletion of primordial and primary follicles⁴⁰.

Supportive evidence for role of GSTM1 gene as modifier of disease susceptibility.

Approximately 30–50% of humans in various populations are homozygous carriers of the GSTM1 null allele (GSTM1(0))⁴¹, and are completely deficient of the GSTM1 enzyme. The null allelic variant arose from a recombination event during evolution between two highly homologous regions flanking this locus, resulting in deletion of a 20 kb segment of the gene^{42, 43}. There is a growing body of evidence suggesting that GSTM1 plays an important role in determining disease susceptibility. The significance of the null variant was first recognized in cancer studies demonstrating that patients carrying the GSTM1(0)/(0) genotype were at increased risks for colon⁴⁴ and lung cancers⁴⁵. In subsequent association studies in cardiovascular diseases, GSTM1(0)/(0) subjects were shown to have significant coronary artery disease/atherosclerosis^46–48.

Role of GSTM1 in Hypertension

In genetic studies of blood pressure in the rat species, Gstm1 is a leading positional and functional candidate gene in a quantitative trait locus (QTL) identified for hypertension^49,50. Gstm1 mRNA expression is reduced in the stroke-prone spontaneously hypertensive rat (SHRSP) compared to the normotensive congenic and Wistar Kyoto rats⁵¹. The differences in mRNA expression levels is reflected at the protein levels in the kidney, and is inversely correlated with renal levels of ROS. Mice with Gstm1 gene deletion have a modest elevation of baseline blood pressure and increased urinary levels of 8-isoprostane, a marker of oxidative stress⁵². Conversely, kidney injury-prone SHRSP rat with transgenic overexpression of Gstm1 display lower level of renal oxidative stress and blood pressure⁵³. These studies suggest that the pathophysiological role of Gstm1 deficiency in hypertension is likely to involve oxidative stress in the kidney⁵¹ and in VSMCs³¹. In humans, studies examining the role of GSTM1 in hypertension have yielded mixed results. While several reports showed increased risks of hypertension in the relative young (average age in the 30’s)⁵⁴, and elderly subjects (> 80 years of age) carrying the GSTM1 null allele^55–57, and an association between the null allele and resistant hypertension⁵⁷, other studies showed no association between the GSTM1 null allele and hypertension^{58, 59}. A meta-analysis of 12 studies revealed a significant association between the GSTM1 null genotype and risk of hypertension (odds ratio of 1.22; 95% CI: 1.08 – 1.39; p = 0.002)⁶⁰. These studies highlight the potential influence of the environment on the observed phenotypic effect of a genetic variant.

Role of GSTM1 in Kidney Disease

Gstm1 is a strong positional candidate gene in the Agtr1a null mouse model of renal vascular remodeling⁶¹. In this model, compared to the 129S6 strain (formerly 129/SvEv), the C57BL/6 strain is more susceptible to development of renal vascular remodeling characterized by VSMC hypertrophy and hyperplasia⁶¹. Additional studies revealed that the susceptible C57BL/6 strain has 50% lower expression of Gstm1 gene and its protein product that could be explained by having half the Gstm1 gene copy numbers. The lower Gstm1 gene expression in the C57BL/6 strain was associated with higher ROS levels in the kidney and VSMCs, and higher VSMC proliferation and migration rates³¹. Cause and effect was established by siRNA knockdown of Gstm1 which showed that reduction of the Gstm1 expression directly results in increased VSMC proliferation in a dose dependent manner, as well as increased VSMC migration and ROS levels³¹. In the renal ischemia reperfusion injury (IRI), compared to the 129/Sv, the C57BL/6 mice also display exaggerated reduction in glomerular filtration rate and renal blood flow⁶². It remains to be determined whether the difference in Gstm1 gene expression between the two strains can explain the difference in susceptibility to renal IRI.

It is worth noting that despite its strong association with various diseases, genome wide association studies (GWAS) have not identified the GSTM1 locus for kidney disease, cancer or CVD in humans. This can be explained by the lack of single nucleotide polymorphisms (SNPs) that capture the GSTM1(0) allele. In this regard, in a large case-control study in subjects of European decent, GWAS for bladder cancer failed to identify a signal at the GSTM1 locus, but use of a deletion assay to genotype for the GSTM1 null allele showed a significant association in the same cohort⁶³. This finding was later confirmed in a case-control analysis of candidate variants in a Chinese cohort⁶⁴.

Chang et al used the trimodal GSTM1 genotyping assay by real time PCR to evaluate the effect of the GSTM1(0) allele on the clinical course of ~ 700 African American patients with chronic kidney disease (CKD) Stage 3 or 4 attributable to hypertensive nephrosclerosis in the African American Study of Kidney Disease (AASK) Trial³⁰. Compared to those homozygous for the active GSTM1 allele (2 copies), those heterozygous (1 copy) or homozygous (0 copies) for the GSTM1(0) allele have a significantly increased risks of the composite outcome of reduction in glomerular filtration rate (GFR) or dialysis, and the composite outcome of reduction in GFR, dialysis, or death³⁰ (Table 1). This finding is supported by other evidence that GSTM1 may modify susceptibility to renal disease. In a case-control study of 184 end stage renal disease (ESRD) patients and 569 age and sex-matched population controls in Northern India, Agrawal et al showed that those individuals with GSTM1(0) allele have increased odds of ESRD relative to those without null alleles (OR for GSTM1(0) allele = 1.45, 95% CI = 1.03–2.02)⁶⁵. In a case-control study of 392 CKD patients and 202 healthy controls in Southern India, GSTM1(0) allele is associated with nearly 3 fold increased risk of CKD development (OR 2.93; 95% CI = 1.89 – 4.53; p < 0.0001) and 2 fold increased risk of progression to ESKD (OR 1.84; 95% CI 1.19 – 2.83; p = 0.006)⁶⁶. In a another case-control study of 110 ESRD and 125 healthy Mexicans, GSTM1(0) allele is associated with susceptibility to ESRD of unknown etiology (OR=2.05, 95% CI = 1.21–3.45, p = 0.007)⁶⁷. In a study of 199 Serbian patients with ESRD on hemodialysis, GSTM1(0) allele is associated with significantly shorter overall (HR 1.6, p = 0.018) and cardiovascular survival (HR 2.1, p = 0.01)⁶⁸. These studies, together, support the notion that GSTM1 may modify clinical outcomes in CKD. The effect could be explained by the role of GSTM1 in endothelial cells. In this regard, human umbilical vein endothelial cells (HUVECs) exposed to uremic serum exhibited enhanced lipid peroxidation and expression of inflammatory cytokines⁶⁹. Knockdown of GSTM1 in HUVECs resulted in increased expression of the cytokine MCP-1, and intracellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1), correlating with higher serum levels of ICAM-1 and VCAM-1 in hemodialysis patients carrying the GSTM1(0) allele compared to those with only the active allele⁶⁹.

Table 1.

Data summarized from Reference # 30.

Summary of Hazard Ratios and 95% confidence interval (CI) for GSTM1 copy number associated with composite outcomes; 2 copy state as reference.

	AASK (n=692)
	Genotypic Model GSTM1 copies
Hazard Ratios (95% CI)	0 vs 2 copies	1 vs 2 copies
GFR Event, Dialysis – 141 events	1.82 (1.03, 3.23)	1.70 (1.0, 2.87)
P-value	0.041	0.049
GFR Event, Dialysis, or Death - 151 events	2.03 (1.17, 3.53)	1.71 (1.03, 2.85)
P-value	0.012	0.039

A follow up study examined the combined effects of GSTM1(0) and APOL1 high risk variants in nearly 700 AASK participants, and showed that the GSTM1(0) and the APOL1 high risk alleles both confer deleterious effects in AASK participants⁷⁰. Moreover, GSTM1(0) has a deleterious effect on CKD independent of APOL1, and perhaps even a modulating effect on APOL1 high risk genotype in CKD progression in AASK. Of interest, those with APOL1 high risk alleles but homozygous for the GSTM1 active allele (GSTM1(1/1)) might be protected from CKD progression⁷⁰. However, interaction analysis failed to refute nor confirm their interaction, likely due to lack of power⁷⁰.

In a study of 5,715 participants in the Atherosclerosis Risk in Communities (ARIC) cohort, a similar survival analysis of GSTM1 with kidney failure was observed⁷¹. Table 2 shows the results for 256 ESRD or kidney failure events in ARIC, where model 1 is minimally adjusted (age, sex, and principal components of ancestry) and model 2 adds adjustment for prevalent diabetes, hypertension, coronary heart disease, smoking status, BMI, baseline eGFR. Race was included as a covariate in the combined analysis. In both African Americans (AA) and European Americans (EA), there is a significant association of time to ESRD event with number of copies of GSTM1, dominant model (0 or 1 copy vs 2 copies)⁷¹. The EA findings in ARIC strongly support the dominant genetic model, (loss of 1 copy has similar effect as loss of 2) and while the AA results in AASK and ARIC suggest that there may be a trend of increasing risk from 2 to 1 to 0 copies, this was not statistically significant in either study. Importantly, the dominant genetic model implicates that even in heterozygotes with one copy of the GSTM1(0) risk allele and one copy of the active allele, increasing the expression of GSTM1 might protect against kidney disease progression.

Table 2.

Modified from Reference # 71.

Summary of Hazard Ratios and 95% confidence interval (CI) for GSTM1 copy number associated with composite outcomes; 2 copy state as reference.

	Atherosclerosis Risk in Communities (ARIC) Study
	Genotypic Model (GSTM1 copies)			Dominant Genetic Model
Hazard Ratios (95% CI)	0 vs 2 copies	1 vs 2 copies	P for Trend	0 or 1 vs 2 copies	P-value
EA (n=3,461) – 99 ESRD events
Model 1	2.52 (1.32, 4.82)	2.54 (1.32, 4.89)	0.04	2.53 (1.34, 4.79)	0.004
Model 2	2.45 (1.26, 4.77)	2.67 (1.36, 5.23)	0.12	2.54 (1.32, 4.88)	0.005
AA (n=2,254) – 157 ESRD events
Model 1	1.62 (1.18, 2.22)	1.38 (1.03, 1.85)	0.003	1.46 (1.11, 1.92)	0.007
Model 2	1.55 (1.12, 2.14)	1.33 (0.99, 1.78)	<0.01	1.40 (1.06, 1.84)	0.018
Overall (n=5,715) – 256 events
Model 1	1.76 (1.35, 2.31)	1.58 (1.22, 2.05)	0.001	1.65 (1.29, 2.12)	<0.001
Model 2	1.76 (1.32, 2.35)	1.59 (1.20, 2.11)	<0.001	1.66 (1.27, 2.17)	<0.001

Model 1: age, sex, center, and ten genetic principal components.

Model 2: Model 1 plus prevalent diabetes, hypertension, coronary heart disease, smoking status, BMI, and eGFR. Race was a covariate in the Overall analysis.

Recently, another community study reported no association between GSTM1 and risk of kidney failure in a large Geisinger cohort⁷². However, the cohort was younger than ARIC, less than 2% of the participants were African Americans, and the event rate for kidney failure in the entire cohort was 1.4% which was significantly lower than kidney event rates in AASK (20.3% for GFR and dialysis) and ARIC (4.5% for kidney failure), suggesting the study was underpowered to detect for association in that population. Furthermore, compared to the ARIC study which followed patients for median of 25 years during which the survival curve did not separate among GSTM1 genotypes until past 10 years of follow up⁷¹, the Geisinger cohort was followed for a mean of only 9 years⁷².

To establish that loss of GSTM1 can directly influence kidney disease, Gigliotti et al. generated a Gstm1 gene knockout mouse line⁵². In both the angiotensin II model of hypertension and the reduction of nephron mass model of CKD, Gstm1 knockout mice have increased renal oxidative stress and kidney inflammation and injury, independent of both the superoxide dismutase pathway and activation of the renin-angiotensin system⁵². This has clinical implications for the inadequacy of angiotensin converting enzyme inhibitors (ACE-Is) and angiotensin receptor blockers (ARBs) in CKD patients, especially those with the GSTM1(0/0) genotype. Furthermore, Gstm1 knockout mice display no difference in gene expression of the NADPH oxidase (Nox) subunits Nox2 and Nox4⁵². Since Gstm1 is expressed in the bone marrow and renal tubular cells, the authors used a bone marrow cross-transplant approach and found that Gstm1 deletion in the parenchyma, and not in the bone marrow, drives renal inflammation⁵². It remains to be determined which GSTM1 deficient cell type(s) in the kidney drives inflammation. Moreover, Gstm1 KO mice, and not wild-type mice, derive a renal protective effect when their diet is supplemented with a powder form of sulforaphane-rich cruciferous vegetable broccoli. While patients with the GSTM1(0) allele are more at risk for CKD progression³⁰ and kidney failure⁷¹, importantly, the authors showed that in the ARIC cohort of nearly 11,000 patients, compared to those carrying one or two active GSTM1 alleles, those carrying the GSTM1(0) allele also derive more benefit from high intake of cruciferous vegetables, similar to their findings in the mouse⁵².

Sulforaphane-Nrf2-GSM1 pathway

Over the past decade, the potential benefit of isothiocyanates (ITCs) has garnered significant attention. These compounds are biologically active metabolites of glucosinolates that are abundant in cruciferous vegetables such as broccoli. When consumed, the cruciferous plant tissue is injured by the process of mastication, glucoraphanin, a chemically inert glucosinolate, is exposed to the released enzyme myrosinase found in the plant tissue, or to the gut microbiome, and becomes hydrolyzed⁷³. The result of this hydrolysis is the release of glucose and the formation of ITC sulforaphane. The reader is referred to the reference⁷⁴ for more detailed discussion on Nrf2 and sulforaphane. Sulforaphane has been shown to be highly potent in inducing phase II detoxification enzymes – GSTs (including GSTM1), UD-glucuronosyltransferases (UGTs), sulfotransferases (SULTs), N-acetyltransferases (NATs) and S- and O-methyltransferases (MTs)^{75, 76}, by activating the Nrf2-Keap1 pathway, enabling the release of Nrf2 for translocation to the nucleus and transcriptional activation of genes with an antioxidant response element (ARE) in their promoters, among which are GST genes⁷⁷. Sulforaphane may be more potent than other antioxidants such as N-acetyl cysteine which is unable to induce Gsts in the rat⁷⁸. Gigliotti et al reported that sulforaphane-rich broccoli powder induced Gstm1 in wild-type mice, but not in Gstm1 knockout mice⁵². Sulforaphane, administered as a concentrated broccoli extract, has been shown to reduce hepatic glucose production and improve glucose control in obese type 2 diabetics⁷⁹. The therapeutic potential of Nrf2 activators has also been demonstrated in a mouse model of diabetic nephropathy⁸⁰.

It is worth noting that another Nrf2 pathway activator, bardoxolone, was tested in 2 clinical trials for advanced type 2 diabetic kidney disease, BEACON⁸¹ and BEAM⁸². Although there was improvement in GFR, there was increased incidence of heart failure among those receiving bardoxolone. The TSUBAKI study was a phase 2 randomized double-blind, placebo controlled study to evaluate whether bardoxolone methyl would increase eGFR in patients with type 2 diabetes and stage 3–4 CKD in whom risk factors for heart failure (elevated baseline B-type natriuretic peptide levels > 200 pg/mL and prior history of hospitalization for heart failure) were absent⁸³. After 16 weeks of follow up, the bardoxolone methyl group showed an increase in inulin measured GFR of 5.95 ml/min/1.73 m² vs. −0.69 ml/min/1.73 m² for the placebo group. However, more patients in the bardoxolone group experienced peripheral edema and discontinued treatment compared to the placebo group⁸³, again raising safety concerns. Animal studies suggest that bardoxolone has a narrow therapeutic window, since its metabolites could also be toxic⁸⁴.

Sulforaphane may be a safer alternative than bardoxolone in the activation of the Nrf2 pathway. In vitro studies suggest differential action of bardoxolone and sulforaphane. In human dermal microvascular endothelial cells, both bardoxolone and sulforaphane augmented Nrf2 expression, but bardoxolone was 2–5 times more potent in Nrf2 activation than sulforaphane at equal concentration⁸⁵. Both agents decreased oxidative stress; however, at similar concentrations, bardoxolone was found to confer more cytotoxicity, characterized by an increase in both apoptosis and necrosis⁸⁵. Bardoxolone, but not sulforaphane, also adversely affected mitochondrial function, causing a significant increase in proton leakage, and decrease in spare respiratory capacity and mitochondrial membrane potential⁸⁵. At low dose ex vivo, sulforaphane can induce the expression of genes in human peripheral blood mononuclear cellsthat encode the enzymes Aldo-Keto Reductase Family 1 Member C1 (AKR1C1) and NAD(P)H dehydrogenase, quinone 1 (NQO1) that play a role in reducing radical species^{86, 87}. At higher doses, sulforaphane can induce not only higher expression of AKR1C1 and NQO1, but also expression of heme oxygenase (HO-1)⁸⁷.

The observation that only Gstm1 knockout mice and humans carrying the GSTM1(0) null allele derive protection from kidney injury and progression from consumption of cruciferous vegetables brings to focus the interaction between gene and environment. Based on existing evidence that the GSTM1 enzyme has activity against multiple substrates, this observation supports the notion that, in addition to its known role in metabolizing damaging reactive oxygen species and aldehydes such as 4-HNE^{30, 88}, GSTM1 enzyme, along with other GSTs, may be involved in the breakdown of antioxidant-promoting compounds. Thus, deficiency in the GSTM1 enzyme may increase the bioavailability of protective compounds relevant in CKD. In this regard, the bioavailability of sulforaphane has been shown to be influenced by GSTM1, as free sulforaphane and sulforaphane metabolites are increased in GSTM1 null subjects compared to those with the active allele⁸⁹. Moreover, since GSTs are heterogeneous enzymes with significant overlapping substrate specificities, the findings suggest that endogenous levels of other phase II enzymes cannot compensate adequately for the loss of GSTM1, and thus their induction by a pharmacologic/dietary factor such as sulforaphane may be required to lower oxidative stress in a disease state such as hypertensive kidney disease (Figure 1).

Figure 1: Sulforaphane-Nrf2-GSTM1 pathway in the prevention of kidney disease progression: Gene - diet interaction.

Glucoraphanine in cruciferous vegetables is hydrolyzed to sulforaphane by myrosinase or by gut microbiome. Sulforaphane inhibits the KEAP-1 mediated degradation of Nrf2, allowing Nrf2 to migrate from the cell cytoplasm into the nucleus where it binds to the antioxidant responsive element in the promoter regions of Nrf2 target genes, including GSTM1, thereby increasing their transcription, and hence their activities. The result is a decrease in reactive oxygen species (ROS), reactive aldehydes (RAs), and factors involved in inflammation and kidney injury. In a disease state such as hypertensive kidney disease, those homozygous for the GSTM1 active allele (GSTM1+) have decreased risk of kidney disease progression through the activation of GSTM1 enzyme. While those carrying the GSTM1 null allele (GSTM1(0)) may be at higher risk of disease progression, they could be protected due to increased bioavailability of sulforaphane to enhance Nrf2-mediated transcription of other antioxidant genes.

Figure was modified from Reference # 74.

Image of cruciferous vegetables taken from https://www.eatright.org/food/vitamins-and-supplements/nutrient-rich-foods/the-beginners-guide-to-cruciferous-vegetables

Image of the kidney taken from https://www.nephrologyspecialistsoftulsa.com/chronic-kidney-disease.php

Conclusion

In summary, GSTM1 has emerged as a factor in kidney disease. While deficiency in GSTM1 increases susceptibility to kidney disease progression due to enhanced oxidative stress, it may also increase the bioavailability of key antioxidants such as sulforaphane with therapeutic potential in kidney disease. Studies are needed to determine the effect of sulforaphane on long term outcomes such as disease progression and mortality in patients with CKD, and whether its effect is modified by GSTM1 genotype in a precision medicine approach.