The Chronic Elevated Consumption of Hibiscus sabdariffa Linnaeus Results in Kidney Damage Associated with Excess H2S

TL;DR

Chronic consumption of Hibiscus sabdariffa infusion increases hydrogen sulfide in the kidneys, leading to damage.

Contribution

This study shows that long-term Hibiscus sabdariffa intake causes kidney damage through elevated H2S levels.

Findings

Chronic HSL consumption increases H2S and reduces renal vasodilatation and OXPHOS.

Elevated CBS and CSE activity from HSL leads to kidney damage.

GSH/GSSG ratio and NrF2 expression are increased in HSL-treated rats.

Abstract

Hydrogen sulfide (H2S) is essential for renal function; however, it is toxic at high concentrations. H2S is increased during reductive stress (RS). Increased antioxidant capacity and reduced/oxidized glutathione (GSH/GSSG) characterize a rat model of RS associated with chronic consumption of 6% Hibiscus sabdariffa Linnaeus (HSL). Here, we evaluate if chronic consumption of an infusion of HSL causes kidney damage associated with an increase in H2S. Twenty-one Wistar rats were divided into three groups. Group 1: rats received plain tap water ad libitum (G1); Group 2: rats received an ad libitum infusion of 6% HSL for one month (G2); and Group 3: rats consumed a 6% HSL infusion for one month and were then given natural water for another month (G3). We evaluated renal vasodilatation, cystathionine–β–synthase (CBS), cystathionine–γ–lyase (CSE), 3–mercaptopyruvate-sulfur-transferase (3–MST), γ-glutamylcysteine synthetase (GCLC), Nrf2, total OXPHOS, H2S concentration, GSH/GSSG and oxidized/reduced thiols in the kidney. Renal vasodilatation and total OXPHOS in complex IV and I and oxidized/reduced thiols were decreased (p ≤ 0.01) but H2S, CBS, SCE, GCLC, and NrF2 expression and GSH/GSSG were increased (p ≤ 0.04). The HSL infusion provided cysteine that was metabolized by CBS and CSE, elevating chronic H2S and favoring renal damage.

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Full text

1. Introduction

Hydrogen sulfide (H_2_S) is a gaseous signaling molecule with vasodilator properties that in some ways resembles nitric oxide. Its synthesis occurs in the cytosol with the participation of two enzymes. The first one is cystathionine–β–synthase (CBS), which utilizes to L-homocysteine and serine amino acids to produce L-cystationine. Then, this molecule is used by the second enzyme, cystathionine–γ–lyase (CSE), to form L-cysteine. H_2_S is produced by transsulfuration and direct desulfuration of the metabolites of these two enzymes. In contrast to the synthesis of H_2_S in the cytosol, its synthesis in mitochondria depends on 3–mercaptopyruvate, which derives from L-cysteine and is used as a substrate of the 3–mercaptopyruvate-sulfur-transferase (3–MST). This enzyme has to be imported into the mitochondria and the import process is a limiting and critical step for this pathway [1]. Once formed, H_2_S can react with the cysteine residues of proteins to form persulfide bonds (sulfhydration process). H_2_S may also increase the activity of the key enzyme γ-glutamylcysteine synthetase (GCLC) that participates in the synthesis of glutathione (GSH) starting from cysteine, and it may directly act by reducing glutathione disulfide (GSSG). This allows, in part, for the maintenance of constant levels of GSH [2].

H_2_S plays an important role in the renal system which is in charge of removing toxic substances produced by cellular metabolism through glomerular filtration [3]. In this context, the three enzymes responsible for the synthesis of H_2_S are present in the renal proximal tubules, glomeruli, interstitium and interlobular arteries [4], and their physiological levels are crucial for renal physiology through a hormesis mechanism. For example, at nanomolar concentrations, H_2_S participates as an oxygen sensor; it releases renin and inhibits the Na^+^–K^+^ ATPase enzyme and the Na^1+^–K^1+^–Cl^2−^ cotransporter in the ascending limb of the loop of Henle, increasing the Na^+^ and K^+^ excretion and the reabsorption of approximately 20–25% of Na^+^ Cl^−^ [5]. H_2_S is also a hyperpolarizing vasodilator in endothelial cells through activation of the ATP-sensitive channels and the S–sulfhydration channel which lowers blood pressure [6]. In several pathologies such as glomerular sclerosis and glomerulonephritis, it is decreased. However, in diabetic nephropathy associated with type I diabetes, there is an increase in the H_2_S concentrations [6,7], which is associated with overexpression of CSE that leads to endoplasmic reticulum stress (ERS) [7].

However, at high concentrations, this gas has toxic effects. For example, at 1 µM concentration it may inhibit the cytochrome c oxidase (complex IV) in the mitochondrial transport chain (MTC) [8]. This causes the electrons to accumulate in complex I (indirect inhibition), and then NADH cannot be oxidized to NAD^+^, leading to an increased ratio of NADH/NAD^+^. Therefore, a state of reductive stress (RS) is favored [9].

Furthermore, H_2_S may act as a mutagen, damaging DNA and generating cleavage that promotes single-strand DNA. This involves H_2_S autoxidation which generates reactive sulfur radicals (RSRs) such as sulfanyl, persulfide, sulfur dioxide and sulfite through the trace metal-mediated Fenton–Weiss reaction [10]. In turn, the cleavage of the DNA strand is associated with the increase in H_2_S and favors the formation of elemental sulfur that reacts with HS– to generate polysulfides that give rise to more RSS [11]. In other words, H_2_S produces two different responses when at low and high concentrations, being cytoprotective or cytotoxic respectively (hormesis process) [12]. Ergo, while physiological levels are essential for homeostasis, its excessive accumulation can induce reductive stress (RS) [13].

It is also important to recall that RS is characterized by the excess of the reducing equivalent couples, such as reduced/oxidized nicotinamide adenine dinucleotide (NADH/NAD^+^), reduced/oxidized nicotinamide adenine dinucleotide phosphate (NADPH^+^/NADP^+^), and reduced/oxidized glutathione (GSH/GSSG), or the overexpression of the enzymatic and non-enzymatic antioxidant systems, which can decrease reactive oxygen species (ROS).

Hibiscus sabdariffa Linnaeus (HSL) is native to tropical Africa and is known as Jamaica flower in Mexico [14]. In traditional medicine, the calyxes of this plant are used to treat pyrexia, hypertension, liver damage, and kidney diseases, and they also contribute to reducing cholesterol and blood pressure. It is used to prepare various drinks and jams [14]. However, there is no well-founded consensus on the percentage of HSL used in the formulations of many beverages, which may range from 0.5 to 1%. In addition, several studies describe the beneficial antioxidant properties of HSL in pathological conditions such as in metabolic syndrome. But in these pathologies, there is damage due to oxidative stress (OS), where the antioxidants provided by HSL reduce OS, resulting in significant improvement [15]. Its beneficial effects are attributed to a high content of several antioxidant molecules such as polyphenols, flavonoids, minerals, and essential and nonessential amino acids, including cysteine, serine, methionine, glycine and glutamate [16]. In this sense, the anthocyanins and polyphenols present in the HSL can oxidize H_2_S to more stable forms, such as polysulfides and thiosulfates, and this results in H_2_S remaining available for the vasodilation process and mitochondrial protection [17]. However, chronic and excessive consumption of this plant in clinically healthy humans and animals may have adverse effects, since an excess of antioxidants can decrease ROS that under basal conditions [18] participate in various cellular pathways. Therefore, evaluating the possible toxic effect of an infusion of HSL at high concentrations is justified. In this sense, three studies by our investigation group have demonstrated that RS can be induced in rats by chronic excess consumption of HSL at 6%. This model is characterized by an increase in the total antioxidant capacity that favors the increase in the redox couples GSH/GSSG and NADPH^+^/NADP^+^ and overactivity of the antioxidant enzymes, thus contributing to decreased ROS [19]. This results in hypertension associated with an increase in the vasoconstriction response and anatomic changes both in the thoracic aorta and in the efferent renal artery, with elevated inflammation markers in plasma. These changes are related to deterioration of renal function [18,19].

Despite the aforementioned factors, there are few studies in the literature that provide information on whether the intake of HSL favors the synthesis of H_2_S and on whether it may cause a permanent chronic increase in the synthesis of H_2_S, resulting in kidney damage. Therefore, the objective of this study was to evaluate if chronic excess consumption of a 6% HSL infusion results in kidney damage associated with an increase in the H_2_S concentration.

2. Results

2.1. Renal Function Markers and General Characteristics of Experimental Rats

Table 1 shows the markers of renal function and general characteristics of the rats that formed the experimental groups. The rats of G2 that consumed the 6% HSL infusion did not show significant changes in body weight but there was a decrease in urine volume, creatinine clearance (CCr), and water consumption (p ≤ 0.01) and an increase in the systolic blood pressure (SBP) and albuminuria (p = 0.001) when compared with the rats of the G1 and G3 groups respectively. The same table shows the urine cysteine concentration and adenochrome and glucose-6-phosphate dehydrogenase (G6PDH) in the kidney homogenate. The rats of the G2 group presented a significant increase (p = 0.01) in comparison with the rats of the G1 and G3 groups, respectively.

2.2. Effect of the H2S on Δ-Perfusion Pressure (Δ-PP) and H2S Concentration in the Kidney Homogenate

Figure 1 shows the effect of the H_2_S on the perfusion and on the Δ-PP in the left kidney. A significant decrease in perfusion was observed when H_2_S (80 μM) was perfused into the left kidney of the rats of the G2 group in comparison with G1 and G2 respectively (p = 0.003 and p = 0.01), as shown in Figure 1a. In the same figure, Figure 1b shows the total H_2_S concentration in kidney homogenate, illustrating that there was a statistically significant increase in the G2 group in comparison with rats in the G1 and G3 groups (p = 0.03 and p = 0.02) respectively.

2.3. Anatomical Changes in the Kidney Evidenced by Histology

Figure 2a (G1), Figure 2b (G2) and Figure 2c (G3) show the representative histological sections of two glomeruli, and Figure 2d (G1), Figure 2e (G2) and Figure 2f (G3) present the representative histological sections of tubules in the renal cortex of the experimental groups. The rats of the G2 group presented retraction of the glomerular tuft, fibrosis, increase in urinary space and thickening of the tubular membrane in the tubular interstitium in comparison with the kidneys of the rats of the G1 and G3 groups. The density analysis area of the glomeruli in the rats of G2 was decreased (p < 0.001) in comparison with the G1, but without significant difference vs. the G3 group.

2.4. Immunolabeling the Analyzed Area of Enzymes Involved in the H2S Pathway

Figure 3 shows representative photomicrographs of the immunolabeling area in the kidney cortex when antibodies against CBS (Figure 3a–c), CSE (Figure 3d–f), 3–MST (Figure 3g–i) and GCLC (Figure 3j–l) were used. The brown mark shows the location of the enzymes of interest. In the G2 group, the signal was more intense in CBS, CSE and GCLC with respect to the G1 and G3 groups but without changes in the 3–MST. The intensity of the mark was located in the basal membrane of the cortical tubules, which resulted in a significant increase when performing densitophotometry. In this sense, Figure 4 shows the density analysis of the area in the kidney cortex of the enzymes involved in the H_2_S pathway. The rats of G2 present significant increases in CBS, CSE and GCLC (p ≤ 0.004 and p ≤ 0.01) in comparison with G1 and G3 respectively, but without significant differences in the 3–MST.

2.5. Expression of the Enzymes Involved in the H2S Pathway

Figure 5 shows the expressions of the enzymes that participate in the H_2_S synthesis pathway in the kidney homogenate. A similar tendency as in the immunohistochemistry is shown. The expressions of CBS, CSE and GCLC show significant increases in G2 (p ≤ 0.04, p ≤ 0.04 and p ≤ 0.03 respectively) in comparison with G1 and G3.

2.6. Ratios of the Reduced and Oxidized Glutathiones and Thiols

Figure 6 shows the GSH/GSSG ratio (Figure 6a) and oxidized thiol/reduced thiol ratio (Figure 6b) in the kidney homogenate of the experimental groups, where an increase (p = 0.03) and decrease (p = 0.01) respectively are present in the G2 group in comparison with G1. In G3, there is also an increase (p = 0.04) in comparison with G1.

2.7. Expression of Nrf2 and Total OXPHOS

Figure 7 shows the expression of Nrf2 (Figure 7a) where there is an increase (p = 0.03) in the G2 group vs. G1. However, the total OXPHOS mitochondrial complexes (Figure 7b) present a significant decrease (p = 0.03 and p = 0.01) in complexes (C) I and CIV respectively in G2 vs. G1.

2.8. Linear Regression and Sperman Correlation

To demonstrate if there is an interdependence between H_2_S concentration and the modulation of the activity of the first enzymes in providing the NADPH, we perform a linear regression. The activity of G6PH is necessary to maintain the NADH/NADPH couple. This result is shown in Figure 8, where a positive correlation with a significant difference (p = 0.01 and r^2^ = 0.61) is shown.

3. Discussion

Low doses of HSL have antioxidant properties that decrease the pathological consequences of metabolic syndrome and kidney damage, among other diseases. However, damaging effects may be expected at high doses [20]. The elevated ingestion of HSL induces RS characterized by an increase in the GSH/GSSG and NADP^+^/NADPH^+^ couples and the total antioxidant capacity, and can favor renal damage, arterial hypertension and inflammation [16,17,18]. H_2_S penetrates cell membranes without using transporters since it has lipophilic characteristics and it participates as a signaling messenger regulating important metabolic pathways in the organism, such as the expression of antioxidant response elements through the Kelch-like ECH-associated protein 1 (Keap1)/nuclear transcription factor erythroid-related factor (Nrf2) pathway [1]. However, few studies have shown if the elevated intake of HSL favors the synthesis of H_2_S and if this permanent chronic increase may be associated with renal damage [21]. Therefore, the aim of this study was to evaluate if chronic consumption of a 6% HSL infusion results in renal damage related to the chronic increase in the H_2_S.concentration.

HSL is a source of cysteine, serine, glycine, glutamate and methionine, among other molecules [14]. These amino acids are essential substrates for the enzymes involved in the H_2_S synthesis pathways. Under physiological conditions, the CSE level in the renal cortex is 20-fold higher than CBS, which suggests that it is the first enzyme that participates in the H_2_S synthesis in the kidney. Our results show a three to one increase in this enzyme with respect to the CBS [22]. Our results achieved by using immunohistochemistry and Western blot also show that the expressions of CBS and CSE are increased in the renal cortex. This suggests that the chronic consumption of the 6% infusion of HSL provides the methionine, serine and cysteine that favor the overexpression of CBS and CSE. CBS requires serine to produce homocysteine which then leads to the formation of cysteine through transsulfuration. Cysteine is then reused by CBS and CSE for the production of H_2_S [2]. Therefore, a chronic elevated supply of these amino acids directly contributes to H_2_S synthesis. In this sense, our results show an increase in the cysteine in the urine of rats in G2. The methionine amino acid supplied by the HSL infusion results in cysteine by a transmethylation process and this molecule is then used for the synthesis of H_2_S [23]. Therefore, the excess H_2_S concentration supplied by these metabolic pathways may result in toxic effects because it favors an increase in the cysteine persulfide residue (–CySSH, sulfhydration process) and oxidation of GSH that leads to GSSG. This may decrease ROS which, at normal physiological concentrations, are necessary for the regulation of different cellular pathways but are depleted in RS. Furthermore, in an RS state, the sulfhydrylation process may increase as a compensatory response due to the imbalance in RSR production [24]. Moreover, the increase in cysteine may also be toxic because it induces protein misfolding and ERS [25].

Our results regarding 3–MST showed no significant differences, and this suggests that chronic intake of a 6% HSL infusion does not modulate the overexpression of this enzyme. In this regard, the expression/activity of this enzyme depends on D-cysteine rather than on L-cysteine, and D-cysteine is present in the kidneys in rats [26]. This pathway produces 60 times more H_2_S from D-cysteine than from L-cysteine [27]. Nevertheless HSL does not contribute to the amount of this enantiomer of cysteine, and this could be a possible explanation for the lack of significant differences in this enzyme.

However, our results show that the cysteine excess, directly provided by the chronic consumption of 6% HSL in rats or metabolized by different pathways after its ingestion, results in elevated pathological concentrations of H_2_S in the kidney which translate into systemic physiological conditions such as decreased Δ-PP and an elevated GSH/GSSG ratio in the kidney homogenate.

The excess H_2_S favors vasodilatation in the afferent arteriole that contributes to the decrease in Δ-PP. However, this condition can be reversible if the insult is removed. This is evidenced by the Δ-PP restoration in the kidneys of the G3 group. In this sense, H_2_S is considered an endothelium-derived hyperpolarizing factor that acts through the activation of the ATP-sensitive intermediate and small-conductance K^+^ channels. For the activation of these channels, there is an essential S–sulfhydration step [6]. Vasodilation increases renal blood flow [6,28]. H_2_S can also directly inhibit the renin–angiotensin–aldosterone system, interfering with zinc at the active center of angiotensin-converting enzyme. This results in a decrease in the SBP and an increase in the glomerular filtration rate [28].

RS is associated with chronic HSL consumption, and there exists an increase in the vasoconstrictor response to norepinephrine which favors Δ-PP in the perfused isolated kidney. This condition leads to an increase in the SBP and a decreased glomerular filtration rate [15]. Hence, our results seem contradictory. However, chronic vasodilation of the renal afferent arteriole and vasoconstriction of the efferent arterioles originate a scenario of double injury on the glomerulus and renal tubules [29,30]. The increased blood flow associated with vasodilation of the afferent arteriole causes increases in pressure in the glomerular tuft, which is kept constant by vasoconstriction in the efferent arteriole [20], maximizing glomerular pressure, destroying the filtration barrier and causing scarring and a dynamic imbalance at different points of the renal microcircuit [31]. This condition perpetuates renal failure and this altered physiological mechanism may lead to glomerular sclerosis that impairs perfusion to the peritubular capillaries [32]. This causes chronic ischemia in the interstitial tissue and thickening of the tubular membranes associated with the lack of oxygen [33]. This dynamic imbalance at different points of the renal microcircuit perpetuates renal failure which is characterized by an increase in proteinuria and low CCr, which are shown in our results [34]. G6PD is the rate-limiting enzyme of the pentose phosphate pathway whose main function is to generate NADPH [35]. Under reducing-stress conditions, there is an excess of reducing equivalents, and H_2_S, through S-sulfidation, promotes G6PD activity either by direct modification of the enzyme or by activation of Nrf2, resulting in an increase in NADPH [36]. This, in turn, favors increased activity of the enzymes in the enzymatic antioxidant system and the reduction in ROS. In this regard, our results show that in the G2 kidney homogenate, there is a decrease in O_2_^−^ evidenced by reduced oxidation of adrenaline to enzyme-mediated adrenochrome [15].

On the other hand, H_2_S induces Nrf2 dissociation from Keap1 through S–sulfhydration which favors the translocation of Nrf2 to the nucleus, inducing downstream inhibition of the antioxidant enzymes involved in the decrease in ROS [37,38]. Our results show an increase in the expression of the phosphorylated NrF2. This suggests that the increase in H_2_S contributes to the expression of phosphorylated NrF2 and to a state of RS caused by overstimulation of Nrf2, which leads to a positive feedback process [39,40,41]. This, in turn, favors the transcription of various antioxidant enzymes [39], such as CBS, CSE, and expression of the two subunits of the GCLC enzyme that catalyze the first step in GSH synthesis. Therefore, the GSH/GSSG ratio remains constant [41]. The S–sulfhydration process also induces sulfhydration of tyrosine phosphatase–1β located on the cytoplasmic face of the endoplasmic reticulum (ER) and this contributes to perpetuating the ERS [42]. In addition, the 6% infusion of HSL can, by itself, contribute to increasing GSH since it provides the essential amino acids for its synthesis, namely cysteine, glycine, and glutamic acid, and this may contribute to maintaining the increase in the GSH/GSSG ratio.

Furthermore, our results show a decrease in the oxidized/reduced thiol ratios in the G2 group. In this regard, RS decreases the content of thiol groups, resulting in misfolding of proteins in the ESR which leads to their accumulation with a loss of disulfide bridges [39]. In this sense, the equilibrium between the oxidized/reduced thiols is very important since it regulates the functionality and activity of enzymes and proteins, and low concentrations of H_2_S favor a reductive state on thiols. However, in high concentrations, it promotes an oxidized state [43]. Therefore, our results suggest that the increase in H_2_S is related to the increase in the oxidized/reduced thiol ratio, adding another redox couple to the RS state [44].

On the other hand, one of the main effects of the increase in the concentration of H_2_S is the inhibition of complex IV in the mitochondrial ETC, since it binds to the heme–copper active site, blocking oxygen reduction and decreasing proton translocation [45]. It consequently decreases ATP production by complex V (ATPase), which leads to a non-canonical operation of the ETC, which triggers a secondary and retrograde inhibition of complex I. In this condition, the decrease and increase in the coupling redox NAD^+^/NADH are drastically altered [46]. This contributes to the positive feedback loop that leads to an RS state. Our results show that the expression of CIV and CI complexes in total OXPHOS is decreased in the kidney homogenate in the G2 group. In this sense, the organ with the second highest number of mitochondria in the body is the kidney, and these organelles are most concentrated in the renal tubules [47]. The fibrosis present in the tubular interstitium has a strong relation with the ETC decoupling and the decrease in ATP production [48,49]. This causes an inflammatory process that perpetuates tubulointerstitial fibrosis, because in this state, macrophages are activated and they secrete pro-inflammatory interleukins such as TNF-α and IL-1β [15,20]. Figure 9 shows the possible mechanism by which the chronic consumption of the HSL infusion at 6% may over-regulate the H_2_S pathway and contribute to an SR state which favors renal damage.

4. Materials and Methods

4.1. Rat Groups

Twenty-one male Wistar rats were used to form 3 groups with 7 animals each. Group 1: Rats that received plain tap water ad libitum for one month (G1). Group 2: Rats that received ad libitum a 6% infusion of HSL to for one month (G2). Group 3: Rats that received ad libitum a 6% infusion of HSL for one month, after which they were given tap water for another month (G3). The animals were housed for 4 weeks under the following conditions: a 12 h light/12 h dark cycle, room temperature from 18 to 26 °C, and relative humidity ranging from 40 to 70%. The commercial food the rodents consumed was solid rodent kibble supplemented with 23% crude protein, 4.5% crude fat, 6% crude fiber, 8% ash, and 2.5% minerals (Labdiet 5008; PMI Nutrition International, Richmond, IN Indiana EE. UU.) ad libitum. This study was designed and carried out in compliance with the Laboratory Animal Care Committee of the National Institute of Cardiology Ignacio Chávez (INC/CICUAL/009/2023).

4.2. Preparation of the HSL Infusion

A total of 60 g of HSL calyces were added to one liter of boiling water. This was kept boiling for 10 min, allowed to cool and then filtered. This solution was provided ad libitum to the rats. The 6% HSL infusion contained cyanidin-3-glucoside (549.67 ± 26.75 mg/L), quercetin (198.48 ± 31.56 mg/L), and polyphenols (52.82 ± 0.38 mmol/L).

4.3. Systolic Blood Pressure Measurement and Urine Collection

At the end of treatment and before euthanizing the animals, SBP was measured using a plethysmograph (Narco Bio-system Houston, Texas, EE. UU). Before euthanasia, the rats were weighed and placed in metabolic cages after fasting for 24 h. Urine was collected and stored at −30 °C. The albuminuria was subsequently determined using the bromocresol green method, and urinary creatinine (UCr) and serum creatinine (SCr) concentrations were determined to calculate creatinine clearance (CCr) using the formula: CCr = (SCr/UCr (Urine Volume)/1440 min [15].

4.4. Calculation for the Sample Size

The mean systolic blood pressure (SBP) in healthy rats was reported to be between 116 mmHg and 112.8 mmHg, with a variance of 19, in rats from the National Institute of Cardiology Ignacio Chavez. Based on this, the sample size per group was estimated by μ (SBP of Wistar rats), with 95% confidence and a maximum error (ME) of 3.2 mmHg, according to the following formula:

$$[eqn]$$ $$[eqn]$$ $$[eqn]$$

where ME = maximum error, σ̅_x_ = # of standard deviations of the mean estimator, σ^2^x = variance in the SBP of the rats in our institute, and SS = sample size [15].

4.5. Isolated and Perfused Kidney

Rats were anesthetized by an intraperitoneal injection of sodium pentobarbital (63 mg/kg of body weight). The right kidney was exposed via a midline laparotomy, and the mesenteric and renal arteries and surrounding tissue were cleared. The right renal artery was cannulated via the mesenteric artery to avoid interruption of blood flow, and the kidney was removed, suspended, and perfused at a constant flow rate using a peristaltic pump (MasterFlex Easy-load II, no. 77200-50; Cole-Parmer Instrument Co., Vermon Hills, IL, USA) at 37 °C and oxygenated with 95% O_2_ and 5% CO_2_ with Krebs solution at pH 7.4. The basal perfusion pressure (PP) was adjusted to 80–90 mmHg. The average flow rate of the perfusion solution was 8–9 mL/min. PP was measured with a transducer (Grass Telefactor, Grass Technologies, Astro Med, West Warwick, RI, USA) coupled with a Grass model 79D polygraph and online software (Grass PolyView, West Warwick, RI, USA). Data are expressed as changes (Δ) in PP in millimeters of mercury (mmHg). After at least 15 min of perfusion and once a stable Δ-PP was obtained, vasodilator responses to H_2_S were determined. The bolus concentration was 80 μM H_2_S. Changes in Δ-PP due to H_2_S were calculated by taking the mean of the pulsatile tracings before administration and the mean of the tracings at the peak Δ-PP value after administration. Data are expressed as changes in Δ-PP in mmHg. The concentrations of H_2_S were selected from published data, as they seemed the most suitable after obtaining changes in Δ-PP [15].

4.6. Anatomical Changes in the Kidney by a Histological Process

To demonstrate the anatomical and structural changes in the renal tissue, the left kidney was dissected and washed with 0.9% saline solution for 30 s. The capsule was removed and cut in half. One half was processed for light microscopy according to standard techniques, which are briefly described below. One half of the kidney was fixed in 10% formalin solution for 24 h, gradually dehydrated in ethanol, cleared in xylene, embedded in paraffin, and cut into 5 µM thick slices using a microtome (Leica RM212RT, Wetzlar, Germany). The paraffin sections were stained with Masson’s trichrome and Jones’s methenamine technique. For immunohistochemistry the antibodies were CBS antibody mouse IgG_1_κ (sc-133154 Santa Cruz Biotechnology Dallas, Dallas, TX, USA), CSE antibody mouse (F-1) (sc-374249 Santa Cruz Biotechnology), MST-3 antibody mouse (48) (sc-135993 Santa Cruz Biotechnology), and GCLC antibody [EP13475] ab190685 (Abcam, Cambridge, UK). The primary antibodies were used at a 1:50 dilution and the secondary antibody at a 1:100 dilution. Histological and immunohistochemistry sections were analyzed at 25× and 12.5× magnification using a model 63,300 optical microscope (Carl Zeiss, Oberkochen, Germany) equipped with a Tucsen digital camera (18 megapixels) coupled with TSview 7.3.1 software. Blinded evaluation was used to analyze histological and immunohistochemistry sections and it was validated with the scoring system Allred Score. The intensity of light in the microscope was adjusted and remained constant. The glomerular area was analyzed by densitometry using Sigma Scan Pro 5 Image Analysis software (Systat Software Inc., San Jose, CA, USA). For the immunohistochemistry density area, the parameters of analysis in the software were adjusted and remained constant for each of the antibodies. The density values are expressed in arbitrary pixel units. The other half of the kidney was homogenized in a cold sucrose buffer consisting of 25 mM sucrose, 10 mM Tris, and 1 mM EDTA at pH 7.35 with protease inhibitors (1 mM PMSF, 2 μM pepstatin, 2 μM leupeptin, and 0.1% aprotinin). The homogenate was then centrifuged at 900× g for 10 min at 4 °C, and the supernatant was separated and stored at −30 °C until used. Total protein was determined using the Bradford method.

4.7. Determination of Polyphenols, Total Flavonoids and Anthocyanins in HSL Infusion

A total of 100 μL of the HSL infusion was diluted 1:10 in distilled water. Then, 500 μL of Folin–Ciocalteu reagent (2 N) was added and the mixture was homogenized and incubated for 3 min at room temperature. Then, 3 mL of 2% sodium bicarbonate was added, the mixture was incubated for 15 min at room temperature, and the absorbance was read at 750 nm. The total flavonoids present in the HSL infusion were determined using the Jia method and the absorbance was determined at 510 nm [50]. The anthocyanins were determined according to the method described by Lee, and the absorbance was measured at 520 nm and 700 nm [51].

4.8. GSH/GSSG and Oxidized Thiol/Reduced Thiol Ratios

To 100 μg of protein from the kidney homogenate, 100 μL of 0.05 M KH_2_PO_4_ pH 7.35 (plus 100 μL of 5% Na_2_WO_4_) was added and then 100 μL of H_2_SO_4_ was added. The mixture was homogenized and centrifuged for 5 min at 5000 rpm. The supernatant was recovered and 700 μL of KH_2_PO_4_ plus 100 μL of 10 M Ellman’s reagent was added. The mixture was incubated at room temperature for 5 min, and the absorbance was read at 412 nm [13]. To quantify GSSG, the procedure was the same as that for quantifying GSH, but after deproteinizing the kidney homogenate, 4 µL of 8-vinylpyridine was added to oxidize the present GSH. For the reduced thiol ratio, 100 µg of protein from the kidney homogenate was used, to which 100 µL of KBH_4_ dissolved in a 10 mM methanol/water (1:1 v/v) mixture was added. The mixture was homogenized and incubated for 3 min. Then, 750 μL of buffer containing 6.7 mM formaldehyde, 10 mM EDTA, and 100 mM TRIS at pH 8.2 was incorporated and the mixture was incubated for 3 min. Then, 100 μL of 10 mM Ellman’s reagent was added. The samples were incubated at room temperature for 4 min, and the absorbance was read at 415 nm [15,52]. To quantify oxidized thiols, the procedure was the same as that for quantifying reduced thiols but without KBH_4_.

4.9. Nuclear Factor Erythroid 2 and Total OXPHOS

A total of 50 μg of protein from the kidney homogenate was separated on an 8% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes. The blot was blocked for 1 h at room temperature using Tris-buffered saline (TBS)-0.01% Tween plus 5% skim milk. The membranes were incubated overnight at 4 °C with the primary anti-phospho-NrF2-S40 monoclonal antibody produced in rabbit SAB5701902-100UL (Sigma-Aldrich EE. UU. Massachusetts) and total OXPHOS rodent WB Antibody Cocktail ab110413 (Abcam). The blot was then incubated with β-actin antibody (sc-81178) and ANT1 antibody (E-7) as a loading control. For Western blot, the antibodies were CBS antibody mouse IgG_1_κ: sc-133154, CSE antibody mouse (F-1): sc-374249, MST-3 antibody mouse (46): sc-135993, and GCLC antibody [EP13475], which were provided by Santa Cruz Biotechnology.

Images of the films were digitally acquired using a GS-800 densitometer with Quantity One software version 4 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and were reported as arbitrary units (AU).

4.10. Hydrogen Sulfide Concentration

The H_2_S concentration was measured according to the method described by Padiya et al. [53]. A total of 100 µg of protein from the kidney homogenate was incubated with 188 µL of 1% zinc acetate, 100 µL distilled water, and 188 µL 20 mM of N, N-dimethyl-phenylene diamine dihydro chloride in 7.2 M of HCL and 150 µL 30 mM of FeCl_3_ in 1.2 M of HCL. The mixture was incubated for 20 min and 376 µL of 10% C_2_HCL_3_O_2_ was added. The samples were centrifuged at 5000 rpm, and the supernatant was measured at a wavelength of 670 nm.

4.11. Cysteine Concentration

The cysteine concentration was measured according to the method described by Wu et al. [54]. A total of 250 µL of urine was deproteinized with 250 µL of 10% trichloroacetic acid and centrifuged to 5000 rpm for 5 min. Then, the supernatant was recovered and 100 mL of 5% sodium cyanide was added. It was incubated for 20 min and then 100 mL of 5% sodium nitroprusside plus 100 mL of 10% sodium hydroxide was added and the reading was taken at 530 nm.

4.12. Superoxide Anion Detection

The O_2_^−^ anion in kidney homogenates was determined by the irreversible oxidation of adrenaline to adenochrome as follows: 50 μg of protein was added to 2 mL of glycine buffer (50 mM) at pH 10.2, plus 50 μL of drenaline (60 mM), and incubated and monitored at 30 °C for 6 min at 480 nm with an extinction coefficient of 4.0 mM^−1^ cm^−1^ [55].

4.13. Glucose-6-Phosphate Dehydrogenase Activity (G6PD)

G6PD activity was quantified using the kit provided by Sigma-Aldrich (MAR015-1KT) and, according to the manufacturer’s specifications, 50 μg of protein from the kidney homogenate was used and monitored at a wavelength of 450–490 nm using a visible-light microplate reader (Stat Fax 3200 Awareness Technology, Palm City, FL, USA).

4.14. Statistical Analysis

Statistical analysis and graphs were performed using Sigma Plot software (SigmaPlot^®^ version 15.0, Jamdel Corporation Palo Alto, Palo Alto, CA, USA). Data are presented as mean ± standard error. Statistical significance was determined using one-way ANOVA and Tukey’s post hoc test. We created graphs and conducted a Spearman correlation and linear regression. The GraphPad-Prism 8 Software. Inc. (San Diego, CA, USA), 1995–2023, was used to generate the graphs of Figure 8. p ≤ 0.05 was considered significant.

5. Conclusions

Our findings suggest that the chronic consumption of a 6% infusion of HSL provides a high cysteine concentration by the increased availability of cysteine inducing the overexpression of CBS and CSE, a process further amplified by the upregulation of the Nrf2 transcription factor. This “feed-forward” mechanism raises H_2_S to supra-physiological levels which contribute to a shift in the cellular redox environment toward an RS state.

Study Limitations

While this study attempts to elucidate the role of H_2_S in renal syndrome (RS) associated with chronic consumption of 6% HSL, resulting in renal failure, it is limited by the use of specific inhibitors of the enzymes involved in the H_2_S pathway. This prevents the definition of a precise dose–response threshold for the transition from hormesis to toxicity. Furthermore, although the Nrf2/H_2_S/RS axis is clear, the specific protein targets downstream of this reducing environment have not yet been identified. Future research should focus on proteomic analysis to identify the thiol-modified proteins responsible for the loss of renal function. Another limitation is that this study did not assess ETC activity and ATP depletion, which could have contributed to a greater understanding of the decline in renal function.