Exogenous Hydrogen Sulfide (H₂S) Reduces Blood Pressure and Prevents the Progression of Diabetic Nephropathy in Spontaneously Hypertensive Rats

Abstract

The coexistence of hypertension and diabetes results in the rapid development of nephropathy. Hydrogen sulfide (H₂S) is claimed to control the vascular and renal functions. This study tested the hypothesis that exogenous H₂S lowers the blood pressure and decreases the progression of nephropathy in spontaneously hypertensive rats (SHR) that were diabetic. Eighteen SHR were divided into three groups: SHR, SHR diabetic, and SHR diabetic treated with a group of Wistar–Kyoto rats serving as normotensive nondiabetic control. Diabetes was induced with streptozotocin (STZ) in two groups and one diabetic group received sodium hydrosulfide (NaHS), a H₂S donor for 5 weeks. Blood pressure was measured in conscious and anesthetized states and renal cortical blood perfusion in acute studies. Plasma and urinary H₂S levels, creatinine concentrations, and electrolytes were measured on three different occasions throughout the 35-day period. Diabetic SHR had higher blood pressure, lower plasma and urinary H₂S levels, and renal dysfunction as evidenced by increased plasma creatinine, creatinine clearance, and decreased urinary sodium-to-potassium ratio and renal cortical blood perfusion. NaHS reduced blood pressure, increased H₂S levels in plasma and urinary excretion, and reversed the STZ-induced renal dysfunction. The findings of this study suggest that the administration of exogenous H₂S lowers the blood pressure and confers protection against the progression of STZ-induced nephropathy in SHR.

• hydrogen sulfide
• nephropathy
• spontaneously hypertensive rats
• streptozotocin

INTRODUCTION

Hypertension and diabetes are common comorbidities that often coexist. During diabetes, the chances of developing hypertension are 2.5 times greater as compared with the normal state.¹ Similarly, previous data have suggested that there is increased incidence of diabetes during hypertension and about 20% of hypertensive individuals have coexisting diabetes.² Increased activity of the renin angiotensin aldosterone system (RAAS) in hypertension and diabetes has been proposed.³ This upregulation of RAAS results in increased oxidative stress⁴ and the formation of reactive oxygen species (ROS), which in turn affect glucose utilization and blood pressure in a setting of hypertension and diabetes.⁵ Similarly aldosterone-induced increased sympathetic nervous system activity and reduced baroreceptor activity have also been observed.⁶ Nephropathy is one of the major microvascular complications of diabetes. Hypertension, whether related to diabetes or not, additionally damages the kidney resulting in complex patterns of nephropathy.⁷ It has been reported that in the presence of hypertension the induction of diabetes with streptozotocin (STZ) results in accelerated renal injury.⁸ The existence of hypertension with diabetes disturbs renal hemodynamics, increases glomerular basement membrane thickness, and promotes fibrosis and inflammation through the activation of RAAS.⁹ Renal disorders are mostly responsible for the secondary hypertension. However, it is also possible that hypertension may act as a pathogenic factor in the development of nephropathy in association with diabetes.¹⁰

Endogenous hydrogen sulfide (H₂S) has been recognized as a novel gaseous transmitter¹¹ with the concentration of ∼46 μM in rat serum.¹² Recent studies have shown that vascular tissues are also capable of generating measurable amounts of H₂S.¹² The two pyridoxal phosphate-dependent enzymes, cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE), are responsible for the majority of the endogenous production of H₂S in mammalian tissues that use l-cysteine as the main substrate.¹³ The expression of CBS and CSE has been identified in many human and other mammalian cells, including those from liver, heart, blood vessels, kidney, brain, skin fibroblasts, and blood lymphocytes.¹⁴ Since the realization that H₂S is an important biomolecule, its antihypertensive activity has been widely studied. It has been reported that it has the ability to relax vascular smooth muscle and reduce blood pressure.¹² H₂S has been linked as pathogenic factor in different settings of hypertension, including spontaneous hypertension,¹⁵ renovascular hypertension,¹⁶ and l-nitro-arginine methyl ester (L-NAME)-induced hypertension.¹⁷ Similarly it has been suggested that suppressed CSE-catalyzed endogenous H₂S production in renal tissue by persistent hyperglycemia plays a role in the pathogenesis of diabetic nephropathy.¹⁸

Previous studies have led to the common view that hypertension combined with diabetes results in profound renal injury, and H₂S is involved in the pathogenesis of hypertension and diabetic nephropathy. This study explores the antihypertensive and renoprotective potentials of exogenous H₂S in spontaneously hypertensive rats (SHR) that were diabetic.

MATERIALS AND METHODS

Experimental Animals

SHR and Wistar–Kyoto (WKY) rats weighing 235 ± 15 g were obtained from the animal house and breeding facility of Universiti Sains Malaysia. Rats were acclimatized for 1 week before any experimental procedure. All the animals were housed in the same environmental conditions with free access to food (Gold Coin, Sdn. Bhd., Penang, Malaysia) and drinking water ad libitum. The measurement of conscious blood pressure was carried out in SHR to demonstrate the hypertension using the tail-cuff method. Only animals exhibiting a systolic blood pressure of 150 mmHg or above were used. The procedures and protocols used in this study were according to the guidelines of Universiti Sains Malaysia animal ethics committee.

Experimental Protocol

SHR were randomly divided into three groups (n = 6): SHR (II), SHR diabetic (III), and SHR diabetic treated (IV). Equal numbers of WKY rats of matching body weight and age served as normotensive control (I). Diabetes was induced in groups III and IV with a single injection of STZ (Nova Laboratories, Sdn. Bhd., Selangor, Malaysia) at a dose of 40 mg/kg. STZ was dissolved in freshly prepared ice-cold sodium citrate buffer (0.1 mol/L, pH 4.5) and injected intraperitoneally.¹⁹ The SHR group of rats (II) received an equal amount of buffer intraperitoneally. After 3 days, a drop of blood was obtained by nicking the tail of overnight fasted rats and glucose concentrations were obtained using glucometer (GlucoSure plus, Apex Biotechnology Corp., Hsinchu, Taiwan) to determine the extent of hyperglycemia. Only animals exhibiting fasting blood glucose above or equal to 350 mg/dL were included for further study. After the confirmation of diabetes the rats of group IV were treated with sodium hydrosulfide (NaHS) (Sigma Aldrich, Selangor, Malaysia), a donor of exogenous H₂S.^15–17 NaHS was freshly prepared in saline and was injected at dose of 56 μmol/kg intraperitoneally at the same time daily for 5 weeks.¹⁵ Groups II and III served as SHR and SHR diabetic control, respectively, and did not receive any treatment.

Blood Pressure Measurement

The arterial blood pressure was determined indirectly by the tail-cuff method using the Model 29 pulse amplifier and Model 20NW cuff pump coupled to a computerized data acquisition system (PowerLab®, ADInstruments, Sydney, Australia) on days 0 and 21. On day 34 of this study, an invasive blood pressure measurement was obtained under ketamine and xylazine anesthesia in surgically prepared animals.

Collection of Metabolic Data and Plasma

Metabolic data were collected from all the groups of animals on days 0, 21, and 34 of 35 days of this study. Rats were placed in metabolic cages for 24 h after which water intake and urine output were measured. Similarly, blood samples were obtained on days 0, 21, and 34 and placed in a pre-cooled heparinized Eppendorf tube and plasma was obtained by centrifugation of the blood sample at 2500 rpm for 5 min. Plasma and urine samples were stored at −30°C until analysis for H₂S, creatinine, sodium, and potassium.

Surgical Procedure

All the rats were fasted overnight before the acute experiment and anesthetized with a mixture of ketamine (Ilium, Smithfield, NSW, Australia) 80 mg/kg and xylazine (Ilium) 10 mg/kg intraperitoneally,²⁰ and were supplemented intravenously with ketamine at a dose of 50 mg/kg if required. Tracheotomy was then performed to maintain a clear airway by using the endotracheal cannula (PP 240, Portex Ltd., Kent, UK). The left jugular vein was catheterized with PP 50 tubing (Portex Ltd.) to permit the infusion of supplementary anesthesia and drugs as necessary. The right carotid artery was cannulated and the cannula was advanced up to the aortic arch. Thereafter, a midline abdominal incision was made and the left kidney and iliac artery were exposed. The left iliac artery was catheterized and the cannula was pushed up to the abdominal aorta just proximal to the point where iliac bifurcation starts. Both the cannulas were connected to pressure transducers (P23 ID Gould, Statham Instruments, London, UK) linked to a data acquisition system (PowerLab®, ADInstruments) through a Quad Amp (ADInstruments) using chart Pro (V.5.5) software (ADInstruments). A laser Doppler flow probe (OxyFlow, ADInstruments was positioned on the dorsal surface of the posterior end of the exposed left kidney in order to measure renal cortical blood perfusion. The probe was connected to a laser Doppler flowmeter (ADInstruments) which was directly linked to data acquisition system (PowerLab®, ADInstruments).

The animals were allowed to stabilize for 1 h upon completion of the above surgical procedure. After the stabilization period, renal cortical blood perfusion, mean arterial pressure, systolic blood pressure, pulse pressure, and heart rate were recorded continuously for 30 min.

Measurement of Plasma and Urine H₂S Levels

H₂S levels in plasma and urine were measured spectrophotometrically according to a previously described method.¹⁵ Briefly, 100 μL aliquots of the samples were mixed with 50 μL distilled water in microcentrifuge tubes containing 300 μL zinc acetate (1% w/v) to trap H₂S. The reaction was terminated after 5 min by adding 200 μL N, N-2-dimethyl-p-phenylenediamine sulfate (20 mM in 7.2 M HCl) and immediately followed by the addition of 200 μL FeCl₃ (30 mM in 1.2 M HCl). The mixture was kept in the dark for 20 min. In order to precipitate protein from the samples, 150 μL trichloroacetic acid (10% w/v) was added. The mixture was centrifuged at 10,000 rpm for 10 min. The absorbance of the resulting supernatant was measured at 670 nm using a 96-well plate reader (BioTek Instruments, Inc., VT, USA). All samples were assayed in duplicates. Finally, H₂S concentration in the plasma or urine was calculated against the calibration curve of standard H₂S solutions (NaHS: 3.125–100 μM). All chemicals used were obtained in pure form from Sigma Aldrich.

Biochemical Analysis and Urinary Sodium and Potassium Ratio

Plasma and urinary creatinine concentrations were measured spectrophotometrically (Jaffe’s reaction). Sodium and potassium concentrations in urine were measured using a flame photometer (Jenway Ltd., Felsted, UK). Creatinine clearance was calculated from plasma and urinary creatinine values along with urine flow rate and was expressed per 100 g body weight. Urinary sodium-to-potassium ratio was calculated by dividing the urinary sodium concentration by potassium concentration.

Statistical Analysis

All the data were expressed as means ± SEM. Statistical significance was set at p < 0.05. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Bonferroni/Dunn all means post hoc test using the statistical package Superanova (Abacus Inc., Lincoln, CA, USA).

RESULTS

The single intraperitoneal (i.p.) injection of STZ effectively induced diabetes in SHR as reflected by the significant hyperglycemia (p < 0.05), decrease in body weight, polyuria, and polydipsia that occurred in the SHR diabetic groups as compared with nondiabetic controls (Table 1). There were no significant differences (all p > 0.0) in blood glucose concentration and body weight at the end of the treatment period between the SHR diabetic-treated group and the SHR diabetic control (Table 1). On the other hand, the SHR diabetic-treated group exhibited an increased urine flow rate and water intake in the later part of the treatment period as compared with the SHR diabetic control (both p < 0.05; Table 1).

Table 1. Effect of streptozotocin (STZ) and exogenous hydrogen sulfide (H₂S) on blood glucose, body weight, urine flow rate, and water intake in STZ-induced diabetic spontaneously hypertensive rats (SHR).

		Days of observation
Parameters	Groups	Day 0	Day 21	Day 34
Blood glucose (mg/dL)	WKY (I)	86.69 ± 1.75	85.04 ± 2.81	89.65 ± 3.54
	SHR (II)	88.37 ± 1.89	87.16 ± 1.87	88.42 ± 3.54
	SHR diabetic (III)	388.81 ± 8.95^,**	393.06 ± 7.50^,**	391.52 ± 5.68^,**
	SHR diabetic treated (IV)	378.81 ± 7.79	383.37 ± 9.42	380.64 ± 7.56
Body weight (g)	WKY (I)	241.83 ± 1.13	276.66 ± 2.55	299.33 ± 1.42
	SHR (II)	236.50 ± 2.07	279.50 ± 1.89	302.50 ± 2.01
	SHR diabetic (III)	248.00 ± 2.82	226.00 ± 2.54^,**	210.83 ± 2.22^,**
	SHR diabetic treated (IV)	243.83 ± 2.18	221.16 ± 1.13	200.00 ± 2.81
Urine flow rate (μL/min/100 g body weight)	WKY (I)	2.55 ± 0.05	2.38 ± 0.10	2.45 ± 0.033
	SHR (II)	2.63 ± 0.04	2.69 ± 0.08	2.70 ± 0.07
	SHR diabetic (III)	11.53 ± 0.48^,**	12.48 ± 0.86^,**	15.81 ± 0.80^,**
	SHR diabetic treated (IV)	11.97 ± 0.44	14.70 ± 0.89	21.87 ± 1.01^,***
Water intake (mL/24 h)	WKY (I)	54.00 ± 1.54	51.33 ± 2.88	55.34 ± 1.40
	SHR (II)	52.16 ± 1.40	54.50 ± 2.01	53.66 ± 2.31
	SHR diabetic (III)	72.66 ± 3.17^,**	70.00 ± 2.88^,**	69.16 ± 3.68^,**
	SHR diabetic treated (IV)	75.00 ± 4.47	71.83 ± 4.09	109.83 ± 2.54^,***

Notes: The values are mean ± SEM (n = 6). Statistical analysis was done by one-way analysis of variance (ANOVA) followed by Bonferroni/Dunn all means post hoc test for all groups in respective days. WKY, Wistar–Kyoto.

*p < 0.05 versus WKY control; ^**p < 0.05 versus SHR control; ^***p< 0.05 versus SHR diabetic control.

There was no significant difference in plasma creatinine (Table 2) between the SHR and WKY control group of rats (all p > 0.05); however, the induction of diabetes resulted in the elevation of plasma creatinine in the SHR diabetic group at all three observation time points compared with the SHR control group. The administration of NaHS significantly reduced the plasma creatinine (Table 2) in the treated group as compared with the SHR diabetic control group on days 21 and 34 of treatment (p < 0.05). Moreover, there was a higher creatinine clearance (Table 2) in the SHR diabetic group as compared with the SHR control group (p < 0.05). It was found that the SHR diabetic NaHS-treated rats exhibited increased creatinine clearance (Table 2) on days 21 and 34 compared with the SHR diabetic control group (both p < 0.05). The sodium-to-potassium ratio of the SHR group was significantly higher (p < 0.05) compared with the WKY control group (Table 2). In addition, the urinary sodium-to-potassium ratio of the diabetic SHR group was significantly lower compared with the SHR control group on days 21 and 34 of the study period (both p < 0.05). With NaHS treatment, it was found that the sodium-to-potassium ratio of the treated SHR group was significantly higher by day 34 of treatment as compared with the SHR diabetic control group (p < 0.05) (Table 2).

Table 2. Effect of exogenous hydrogen sulfide (H₂S) (56 μmol/kg intraperitoneally) on plasma creatinine, creatinine clearance, and urinary sodium-to-potassium ratio in streptozotocin (STZ)-induced diabetic spontaneously hypertensive rats (SHR).

		Days of observation
Parameters	Groups	Day 0	Day 21	Day 34
Plasma creatinine (mg/dL)	WKY (I)	0.84 ± 0.02	0.89 ± 0.03	0.96 ± 0.01
	SHR (II)	0.86 ± 0.03	0.86 ± 0.04	0.88 ± 0.04
	SHR diabetic (III)	1.14 ± 0.04^,**	1.28 ± 0.06^,**	1.37 ± 0.07^,**
	SHR diabetic treated (IV)	1.09 ± 0.03	1.05 ± 0.07^***	1.03 ± 0.04^***
Creatinine clearance (mL/min/100 g body weight)	WKY (I)	0.46 ± 0.01	0.41 ± 0.01	0.39 ± 0.01
	SHR (II)	0.30 ± 0.01	0.33 ± 0.01	0.31 ± 0.01
	SHR diabetic (III)	0.88 ± 0.06^***	0.93 ± 0.08^****	1.14 ± 0.11^***
	SHR diabetic treated (IV)	0.94 ± 0.04	1.41 ± 0.12^,***	2.13 ± 0.13^,***
Urinary sodium-to-potassium ratio	WKY (I)	3.12 ± 0.06	3.22 ± 0.09	3.01 ± 0.13
	SHR (II)	3.81 ± 0.12	4.02 ± 0.21	3.74 ± 0.12
	SHR diabetic (III)	3.74 ± 0.18	2.76 ± 0.15^**	2.56 ± 0.11^**
	SHR diabetic treated (IV)	3.80 ± 0.17	3.37 ± 0.14	3.33 ± 0.13^***

Notes: The values are mean ± SEM (n = 6). Statistical analysis was done by one-way analysis of variance (ANOVA) followed by Bonferroni/Dunn all means post hoc test for all groups in respective days. WKY, Wistar–Kyoto.

*p < 0.05 versus WKY control; ^**p < 0.05 versus SHR control; ^***p < 0.05 versus SHR diabetic control.

Figure 1. Renal cortical blood perfusion of Wistar–Kyoto (WKY), spontaneously hypertensive (SHR), SHR diabetic, and SHR diabetic-treated groups of rats. The values are mean ± SEM (n = 6). Statistical analysis was done by one-way analysis of variance (ANOVA) followed by Bonferroni/Dunn all means post hoc test for all groups. Observation was made on day 34 of this study.

Note: *p < 0.05 versus WKY control; ^**p < 0.05 versus SHR control; ^***p < 0.05 versus SHR diabetic control.

Figure 2. Mean arterial blood pressure (A) and systolic blood pressure (B) of Wistar–Kyoto (WKY), spontaneously hypertensive (SHR), SHR diabetic, and SHR diabetic-treated groups of rats. Measured in conscious state on days 0 and 21 and in anesthetized state on day 34. The values are mean ± SEM (n = 6). Statistical analysis was done by one-way analysis of variance (ANOVA) followed by Bonferroni/Dunn all means post hoc test for all groups in respective days.

Note: *p < 0.05 versus WKY control; ^***p < 0.05 versus SHR diabetic control.

Renal cortical blood perfusion in the SHR control and SHR diabetic groups (Figure 1) was significantly lower compared with the WKY control group (all p < 0.05). Moreover, the SHR diabetic group of rats exhibited a lower renal cortical perfusion compared with the SHR control group (p < 0.05). With NaHS treatment, the renal cortical blood perfusion of the treated group (Figure 1) was significantly higher compared with the SHR diabetic control group (i.e., 158 ± 3 vs. 139 ± 3 BPU; p < 0.05).

Figure 3. Plasma (A) and urinary (B) hydrogen sulfide (H₂S) of Wistar–Kyoto (WKY), spontaneously hypertensive (SHR), SHR diabetic, and SHR diabetic-treated groups of rats. The values are mean ± SEM (n = 6). Statistical analysis was done by one-way analysis of variance (ANOVA) followed by Bonferroni/Dunn all means post hoc test for all groups in respective days.

Note: *p < 0.05 versus WKY control; ^**p < 0.05 versus SHR control; ^***p < 0.05 versus SHR diabetic control.

The mean arterial blood pressure and systolic blood pressure (Figure 2) of SHR, SHR diabetic, and diabetic SHR-treated groups remained significantly higher on all three points of observation as compared with the WKY control group (all p < 0.05). There was no significant difference in mean arterial blood pressure and systolic blood pressure between the SHR and SHR diabetic groups (all p > 0.05). It was evident that with NaHS treatment (Figure 2), the mean arterial blood pressure of the treated SHR was significantly lower on days 21 and 34 compared with the SHR diabetic control, that is, 141 ± 2 versus 152 ± 3 mmHg and 132 ± 3 versus 145 ± 2 mmHg, respectively (all p < 0.05). Similarly, the systolic blood pressure was significantly lower on days 21 and 34 of treatment in the SHR diabetic-treated group compared with the SHR diabetic control, that is, 160 ± 2 versus 172 ± 2 mmHg and 150 ± 4 versus 164 ± 2 mmHg, respectively (all p < 0.05; Figure 2).

Plasma and urinary levels of H₂S (Figure 3) in the SHR and SHR diabetic groups were significantly lower on all 3 days of observation compared with the WKY control group (all p < 0.05). Following the induction and progression of diabetes, the SHR diabetic group exhibited a significantly lower level of H₂S in both plasma and urine (Figure 3) on day 34 compared with the SHR control group (both p < 0.05). With NaHS treatment, it was found that the plasma and urinary levels of H₂S in the treated diabetic SHR (Figure 3) were significantly (all p < 0.05) higher on days 21 and 34 compared with the SHR diabetic control, that is, 35.9 ± 2.3 versus 26.5 ± 1.0 μM on day 21 and 42.9 ± 1.7 versus 17.2 ± 0.7 μM on day 34 in plasma, and 32.2 ± 1.23 versus 22.6 ± 1.0 μM on day 21 and 37.1 ± 1.9 versus 15.1 ± 0.7 μM on day 34 in urine.

DISCUSSION

This study investigated the antihypertensive and renoprotective effects of H₂S in a combined state of hypertension and diabetes. This was undertaken by studying the effects of supplementation of H₂S in SHR diabetic rats. The major findings of this study were that the exogenously administered H₂S in the form of NaHS reduced blood pressure, increased H₂S levels, significantly reversed the STZ-induced increase in plasma creatinine and decrease in urinary sodium-to-potassium ratio, and reduced renal cortical blood perfusion.

In this study STZ was used to induce diabetes in the SHR and it was found that the SHR presented with typical features of diabetes mellitus, that is, hyperglycemia, polyuria, and polydipsia. The degree of hyperglycemia produced by STZ was about the same in all groups, and the action of STZ is known to produce an irreversible O-glycosylation of intracellular proteins resulting in β-cell death.²¹ Moreover, hyperglycemia together with the significant weight loss in the STZ-treated group of rats is a characteristic feature of insulin-dependent diabetes mellitus. These findings are consistent with earlier studies²² and can be explained on the basis of a lack of insulin. The NaHS-treated rats remained hyperglycemic and continued to lose weight throughout the study period, suggesting that the exogenous H₂S did not have any effect on blood glucose and body weight. On the other hand, it was apparent that with NaHS treatment the urine flow rate was elevated. This finding is in accord with an earlier study demonstrating increased urine volume in a dose-dependent manner following administration of H₂S into the renal artery.²³ It was previously reported that the abnormalities in sodium reabsorption and excretion in diabetes have been linked to the developing nephropathy.^24,25 In 2009, it was reported by Xia et al. that exogenous H₂S in the form of NaHS increased urinary sodium excretion by inhibiting the renal Na⁺/K⁺-2Cl co-transport mechanism and renal Na⁺/K⁺-ATPase activity. The observation of an increased urine flow rate with NaHS treatment can possibly be explained on the basis of a natriuresis produced by the exogenous H₂S such that the water passively followed the sodium which resulted in an increase in urine output.

It has been reported that hypertension combined with diabetes results in an accelerated progression of nephropathy in diabetic SHR and is a suitable model for the evaluation of antihypertensive therapy.¹⁰ Plasma creatinine²⁶ and creatinine clearance, a measure of glomerular filtration rate (GFR),⁸ are commonly used to evaluate the renal hemodynamic function. Khan et al.²⁷ reported that the SHR-STZ model of genetic hypertension and diabetes is one which develops the typical changes in renal injury early in the course of the disease. Interestingly, we have also demonstrated renal injury in terms of increased plasma creatinine and creatinine clearance in the diabetic SHR group. Together, these findings suggest that a single i.p. injection of STZ in SHR rats produced a consistent model of experimental diabetic nephropathy. It is widely accepted that there is a considerable increase in GFR in the early stage of diabetes,^28,29 and a comparable hyperfiltration, reflected by the increased creatinine clearance, was found in this study. Many factors, including hyperglycemia, prostanoids, nitrogen oxide, atrial natriuretic factor, growth hormone, glucagon, insulin, angiotensin II (ANG II), and others, have been considered as potential agents causing hyperperfusion and hyperfiltration.³⁰ A primary cause of hyperfiltration is likely to be an increased glomerular capillary pressure and glomerular plasma flow⁷ which could be due to a defect in the autoregulatory mechanism, as enhanced systemic and renal perfusion pressures are associated with preglomerular vasoconstriction in order to keep the GFR at a constant rate.³¹ Furthermore, it was also found that the SHR diabetic group exhibited a decreased renal cortical blood perfusion compared with the SHR control group. The results of this study can be best explained on the basis of augmented renovascular resistance probably due to the hyperglycemic-induced activation of local ANG II.³² The administration of exogenous H₂S caused plasma creatinine concentration in the treated rats to decrease, but creatinine clearance and renal cortical blood perfusion were significantly higher compared with the SHR diabetic control. An increase in creatinine clearance and renal cortical blood perfusion can be explained on the basis of a vasodilator effect of H₂S possibly causing the dilatation of afferent arterioles. The findings are consistent with a previous report indicating that exogenously administered H₂S produces a greater vasodilatation of preglomerular arterioles than postglomerular arterioles.²³ However, Sprague Dawley (SD) normotensive and nondiabetic rats were used in that study.²³ The evidence of this study and previous reports would support the view that the exogenous H₂S causes the relaxation of renal blood vessels in a setting of increased renovascular resistance. However, the above suggestion requires further exploration.

The RAAS plays a fundamental role in the development of diabetic nephropathy.^33–35 Hyperglycemia-mediated increased production of ROS is responsible for the upregulation of RAAS.³⁶ Recently, aldosterone acting independently from ANG II has been linked to the development of nephropathy.³⁷ The urinary sodium-to-potassium ratio is a marker of renal reabsorptive function and is inversely proportional to the aldosterone level acting on renal tubules.^38,39 In this study, diabetic SHR exhibited a significantly lower urinary sodium-to-potassium ratio indicative of increased aldosterone activity. By contrast, treating the rats with exogenous H₂S increased the sodium-to-potassium ratio. This would suggest that exogenous H₂S decreased aldosterone activity either indirectly via the inhibition of angiotensin-converting enzyme⁴⁰ or directly via antagonism of aldosterone. However, a possible direct antagonism of aldosterone action by H₂S needs to be further investigated.

The complex interaction between vasodilator and vasoconstrictor substances plays an important role in preserving the vascular wall integrity functionally as well as structurally.⁴¹ H₂S has been shown to be a vasodilator molecule because by using the gas in water or by using the NaHS (as a donor of H₂S) there was a vasodilatation of different blood vessels, including rat aorta, portal vein, mesenteric, and hepatic arteries.⁴² In this study, systolic blood pressure and mean arterial blood pressure of the SHR group were significantly higher compared with the WKY control which was associated with decreased plasma and urinary levels of H₂S. These findings imply that H₂S is a vasodilator molecule contributing to the maintenance of the normotensive state and is in line with a previous report suggesting that the downregulation of CSE/H₂S system is involved in the development of spontaneous hypertension.¹⁵ Endothelial dysfunction is considered to be the first step in the pathogenesis of micro- and macrovascular complications in hypertension and diabetes.⁴³ Hyperglycemia associated with diabetes modifies endothelial function through a number of complex mechanisms, including oxidative stress,⁴⁴ glycation of protein and lipids,⁴⁵ and activation of protein kinase C.⁴⁶ Recently, the vascular endothelium was reported to express 3-mercaptopyruvate sulfurtransferase and cysteine aminotransferase which produce H₂S from cysteine and α-ketoglutarate⁴⁷ suggesting an alternative source of H₂S rather than vascular smooth muscle cells. In this study, plasma and urinary levels of H₂S in the diabetic rats were significantly decreased in later part of this study without changes in blood pressure as compared with the SHR control group which concurs with an earlier study.⁴⁸ This was possibly due to the endothelial dysfunction which is a common occurrence in diabetes.^43,49 The possible mechanisms of reduced plasma and urinary H₂S in hypertensive diabetic rats include the persistent hyperglycemia, as found in this study, and increased levels of ROS due to enhanced formation of free radicals. The augmented production of ROS might have then resulted in oxidative stress and compromised endothelial function.⁴⁹ Moreover, treating the diabetic hypertensive rats with NaHS (donor of exogenous H₂S) reduced the systolic and mean arterial blood pressures and increased plasma and urinary H₂S levels. This may be explained on the basis of earlier findings that exogenous H₂S relaxes vascular smooth muscle cells through the opening of K_ATP channels while the administration of NaHS possibly upregulates CSE enzyme activity resulting in increased endogenous H₂S production.⁵⁰ However, the measurement of CSE messenger ribonucleic acid (mRNA) by quantitative reverse transcription polymerase chain reaction should be done in order to comment precisely on the downregulation and upregulation of CSE/H₂S system. Alternatively, the increased plasma and urinary H₂S levels in diabetic-treated rats suggests that exogenous H₂S might have improved endothelial function due to its reducing oxidative stress property as it has been reported to be a potent antioxidant molecule.^51,52 Nevertheless, the above hypothesis deserves further verification.

CONCLUSION

The data obtained from this study showed that decreasing the blood pressure by the use of exogenous H₂S in the form of NaHS significantly attenuated the progression of early diabetic nephropathy in SHR diabetic rats. On the basis of the above findings, it has been suggested that the pharmacological augmentation of H₂S may have the beneficial effect in the progression of nephropathy in a combined state of hypertension and diabetes.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.