• Users Online: 61
  • Home
  • Print this page
  • Email this page
Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 


 
 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 34  |  Issue : 1  |  Page : 17-27

Renoprotective effect of saxagliptin and Hibiscus sabdariffa Linn extract in Nω-nitro-L-arginine methyl ester-induced hypertensive nephropathy in male albino rats: the role of proinflammatory and fibrogenic cytokines


1 Department of Pharmacology, Faculty of Medicine, Benha University, Benha, Egypt
2 Department of Physiology, Faculty of Medicine, Benha University, Benha, Egypt

Date of Submission14-Nov-2016
Date of Acceptance20-Dec-2016
Date of Web Publication24-May-2017

Correspondence Address:
Omaima M Abd Allah
Department of Pharmacology, Faculty of Medicine, Benha University, Al-Sahaa Street, Diverted from Farid Nada Street, Benha, Qalyubia Governorate, 13518
Egypt
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-208X.206902

Rights and Permissions
  Abstract 


Objective
The purpose of this paper is to evaluate the possible prophylactic effects of saxagliptin (SAX), a dipeptidyl peptidase 4 inhibitor, and Hibiscus sabdariffa Linn extract (HSE) and their combination against Nω-nitro-l-arginine methyl ester (l-NAME)-induced hypertensive nephropathy in rats; in addition, their effects on proinflammatory and fibrogenic cytokines involved in renal injury induced by hypertension were investigated.
Materials and methods
Rats were randomly divided into five groups (eight each) as follows: control group; l-NAME-treated group (50 mg/kg/day in drinking water); SAX+l-NAME-treated group (10 mg/kg/day postoperatively); HSE+l-NAME-treated group (100 mg/kg/day post operatively); SAX+HSE+l-NAME-treated group that received the same doses. Systolic blood pressure was measured. Kidney function tests (blood urea nitrogen, serum creatinine, total protein contents in 24-h urine, and creatinine clearance rate) and renal tissue oxidative biomarkers (malondialdehyde, glutathione, and glutathione peroxidase activity) were assessed. Proinflammatory cytokines (tumor necrosis factor-α and interleukin-6) levels were assessed in renal tissue homogenate; in addition, renal tissue transforming growth factor-β1 mRNA expression level was assayed. Histopathological analysis of the kidney and scoring were also performed.
Results
Administration of either SAX or HSE for 8 weeks attenuated l-NAME-induced increased oxidative stress, inflammation, and fibrosis in the kidney of rats, associated with improvement of the impaired renal function and histopathological changes, but their combination was found to be more effective in the protection against l-NAME-induced hypertensive renal damage than each drug alone.
Conclusion
A combination of SAX and HSE protected the kidney tissue against l-NAME-induced hypertensive nephropathy through their antioxidant, anti-inflammatory, and antifibrotic activities.

Keywords: hibiscus, hypertensive nephropathy, rat, saxagliptin, transforming growth factor-β1


How to cite this article:
Abd Allah OM, Shoman AA. Renoprotective effect of saxagliptin and Hibiscus sabdariffa Linn extract in Nω-nitro-L-arginine methyl ester-induced hypertensive nephropathy in male albino rats: the role of proinflammatory and fibrogenic cytokines. Benha Med J 2017;34:17-27

How to cite this URL:
Abd Allah OM, Shoman AA. Renoprotective effect of saxagliptin and Hibiscus sabdariffa Linn extract in Nω-nitro-L-arginine methyl ester-induced hypertensive nephropathy in male albino rats: the role of proinflammatory and fibrogenic cytokines. Benha Med J [serial online] 2017 [cited 2017 Jun 22];34:17-27. Available from: http://www.bmfj.eg.net/text.asp?2017/34/1/17/206902




  Introduction Top


Systemic hypertension is the second major cause of end-stage renal disease, with diabetes mellitus being the first. Early detection and prevention of renal dysfunction could improve both renal and cardiovascular morbidity and mortality [1]. Glomerular sclerosis (GS), tubular interstitial fibrosis, and vascular sclerosis have been found to be important factors in the progression of chronic kidney injury [2]. Accordingly, drugs that aim to protect against glomerular injury can be of great value.

Multiple mechanisms have been validated to be involved in hypertensive nephropathy. Besides elevated blood pressure, numerous local factors including angiotensin II are known to be involved in the development of renal fibrosis. Angiotensin II stimulates transforming growth factor-β1 (TGF-β1) gene expression and protein release [3]. TGF-β1 induces renal fibrosis by activating interstitial fibroblasts to become myofibroblasts, which produce large amounts of matrix components [4]. Experimental and clinical studies have verified the pathogenic role of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the development of renal injury and the potential benefit of modulating these cytokines activity as a therapeutic target in chronic renal diseases [5],[6].

Recent data have shown that even with excellent blood pressure control and the use of a renin–angiotensin system inhibitor, progression of renal dysfunction is relentless [7]. Therefore, combination therapy may be theoretically favored by the fact that multiple factors contribute to hypertensive nephropathy, and achieving control of nephrosclerosis with a single agent acting through one particular mechanism may not be possible. A combination of conventional drugs with herbal medicinal products is possibly of great interest for the improvement of therapy with much less adverse effects.

Dipeptidyl peptidase 4 (DPP4) inhibitors are members of a novel class of oral antihyperglycemic agents used to treat type 2 diabetes [8]. DPP4 inhibitors have pleiotropic actions in diabetic patients, resulting in favorable effects on postprandial glycemia and lipemia, endothelial dysfunction, silent inflammation, oxidative stress, and blood pressure [9]. In addition to lowering blood glucose, DPP4 inhibitors have been shown to attenuate hepatic fibrosis in rats [10]. Saxagliptin (SAX) is a DPP4 inhibitor that prevents the degradation of endogenous glucagon-like peptide 1 and prolongs its actions on insulin and glucagon secretion [8].

Hibiscus sabdariffa Linn extract (HSE) has been reported to possess antioxidative and antihypertensive activities in animal and human studies [11]. Several mechanisms have contributed to the hypotensive action of HSE including its diuretic activity which is mainly referred to modulation of the aldosterone action, vasodilatory activities, and inhibition of Ca2+ influx [12],[13]. Inhibition of angiotensin-converting enzyme (ACE) by HSE was also reported [14] and this could be another important antihypertensive mechanism of action. Several clinical trials confirmed the antihypertensive effectiveness and safety of HSE [15],[16].

Since SAX and HSE probably have different mechanisms of action with complementary effects, we hypothesize that simultaneous use of both therapies could be beneficial for prophylaxis against hypertensive nephropathy. Thus the aim of this study was to evaluate the prophylactic effects of SAX and HSE, separately and as combined therapy, against Nω-nitro-l-arginine methyl ester (l-NAME)-induced hypertensive nephropathy in rats. Furthermore, this study focused on the effect of SAX and HSE on proinflammatory and fibrogenic cytokines involved in renal injury induced by hypertension.


  Material and methods Top


Animals

Forty adult male albino rats (at 10–12 weeks old) weighing 180–200 g were used in this study after 7 days of acclimatization. The animals were purchased from Helwan farm (Holding Company for Biological Products and Vaccines; VACSERA, Giza, Egypt). The animals were housed in the animal breeding facility of the Faculty of Medicine, Benha University, Egypt, and allowed for normal rodent diet which was supplied under hygienic conditions. Animals were allocated four per cage at room temperature (22–25°C) with natural light/dark cycle, regular diet, and water access. Animal studies were performed in accordance with the institutional guidelines of the Faculty of Medicine, Benha University, Egypt, for the Care and Use of Laboratory Animals. Experimental procedures were reviewed and approved by the Ethics Committee for Animal Experimentation.

Drugs

l-NAME and HSE were purchased from Sigma-Aldrich Chemical Co. (St Louis, Missouri, USA). SAX was obtained from AstraZeneca (UK). All drugs were dissolved in distilled water and were freshly prepared before use. All the drugs and other chemicals used were of the high analytical grade.

Experimental groups

Animals were randomly allocated into five equal groups of eight rats each:
  1. Group 1: served as control group in which animals received distilled water orally for 8 weeks without any drugs.
  2. Group 2 [l-NAME-treated group (hypertensive group)]: l-NAME was received in doses of ∼0.5 mg/ml drinking water, changed daily, for 8 weeks. The normal rat drink is about 100 ml water/kg/day and so the daily intake of l-NAME was 50 mg/kg/day [17].
  3. Group 3 (SAX+l-NAME-treated group): SAX was received orally at a dose of 10 mg/kg/day [18] in concomitant with l-NAME treatment for 8 weeks.
  4. Group 4 (HSE+l-NAME-treated group): HSE was given orally at a dose of 100 mg/kg/day [19] concomitantly with l-NAME treatment for 8 weeks.
  5. Group 5 (SAX+HSE+l-NAME-treated group): rats in this group were given a combined therapy of SAX+HSE+l-NAME for 8 weeks.


Procedure of the experiment

Measurement of blood pressure

Systolic blood pressure (SBP) was recorded, on day 0 and weeks 2, 4, and 8, noninvasively in restrained conscious rats by tail-cuff plethysmography (Harvard, UK) at the same time of day. The mean of three successive measurements of each rat was recorded.

Sample collection

Rats were individually housed in metabolic cages and 24-h urine samples were collected on day 0 and weeks 2, 4, and 8. They were fasted and allowed free access to water only, for 24 h urine collection. At the end of 8 weeks, rats were killed under ether anesthesia; a midline abdominal incision was performed and blood samples were collected from the abdominal aorta. Samples were collected in clean and dry centrifuge tubes, which were left for 15 min to clot and then centrifuged at 3000 rpm for 15 min to separate the serum for biochemical analysis. At the same time, kidneys were removed, washed with physiological saline. A part of the kidney tissue was homogenized in ice cold 100 mmol/l phosphate buffer (pH 7.4). Homogenates were centrifuged and the resulting supernatant was used for biochemical analysis. The other part of the kidney tissue was preserved in 10% phosphate buffered formaldehyde for histopathological examination. Third part was stored at −80°C for DNA extraction.

Biochemical analysis

Blood urea nitrogen, serum creatinine, and urinary creatinine were estimated colorimetrically as previously described [20],[21]. In addition, total protein contents in 24-h urine samples were determined [22] using commercially available diagnostic kits from Biodiagnostic Company (Giza, Egypt). Creatinine clearance rate (Ccr) was calculated using a standard formula and expressed as milliliters per minute.

Malondialdehyde (MDA), renal lipid peroxidation product, was measured in renal tissue homogenates in nmol/mg wet weight by the method described before [23]; glutathione (GSH) was determined in μmol/g protein in renal tissues [24]; and glutathione peroxidase (GPx) activity was estimated in IU/G protein [25], using commercially available diagnostic kits from Biodiagnostic Company.

Levels of TNF-α and IL-6 in the supernatants from kidney tissue homogenates were quantitated by enzyme-linked immunosorbent assay and expressed as pg/mg of protein using Quantikine Rat Specific Kits (R&D Systems Inc., Minneapolis, Minnesota, USA), which were obtained from Clinilab Company (Cairo, Egypt), according to manufacturer’s directions.

Histopathological evaluation of the kidney

Kidneys were fixed in 10% phosphate buffered formaldehyde and embedded in paraffin. Tissue sections (4 μm) were placed on slides, stained with hematoxylin and eosin and the morphology was analyzed under light microscopy. Approximately 50 glomeruli for each rat were graded.

The extent of GS was graded from 0 to 4 by a semiquantitative score [26]: 0, normal; 1, mesangial expansion/sclerosis involving less than 25% of the tuft; 2, moderate GS (25–50%); 3, severe GS (50–75%); and 4, diffuse GS involving greater than 75% of the glomerular tuft. GS index for each rat was calculated as a mean value of all glomerular scores obtained. Tubulointerstitial lesion indexes were determined using a semiquantitative scoring system [26]. Ten fields per kidney were examined, and lesions were graded from 0 to 3 (0, no changes; 1, changes affecting <25% of the section; 2, changes affecting 25–50% of the section; and 3, changes affecting 50–100% of the section), according to the area with tubulointerstitial lesions (tubular atrophy, casts, interstitial inflammation, and fibrosis). The score index in each rat was expressed as a mean value of all scores obtained.

Extraction of RNA and quantitative real-time polymerase chain reaction

Total RNA extraction from the frozen kidney tissues was carried out using total RNA Purification Kit (Jena Bioscience GmbH, Germany) according to the manufacturer’s instructions; about 25 mg kidney tissue put in a microcentrifuge tube with 300 μl of lysis buffer containing 2-mercaptoethanol was homogenized using rotor Tissue Ruptor (Qiagen GmbH). The purity and concentration of the RNA extracted was measured using Nanodrop spectrophotometer (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA). The absorbance of Nanodrop spectrophotometer was measured at A260 and A280. The ratio of the reading at A260/A280 provides an estimate of the purity of RNA. Pure RNA has an A260/A280 ratio of 1.9–1.3. Template RNA (5 μl) and distilled water (15 μl) were added to Maxine RT premix tube. cDNA synthesis (reverse transcription) reaction using G-storm thermal cycler (G-storm Biotechnology Company, Somerset, Hamilton, Scotland, UK) was performed at a temperature of 45°C for 60 min followed by RTase inactivation step at 95°C for 5 min. This reactant was diluted by adding 30 ml nuclease-free water. Real-time-PCR was carried out using ABI 7900HT fast real-time PCR (Applied Biosystem, USA), and the prepared reaction components were carried out in 96-well PCR plate (microAmp R 90-well optical reaction plate with Barcode, code 128). The reaction was performed using qPCR Green Master from Jena Bioscience GmbH, using real-time cycler conditions of 95°C and 5 min (initial denaturation), followed by 35 cycles of 95°C, 30 s, 52°C, 1 min and 72°C, 30 s for denaturation, annealing, and extension steps, respectively. β-actin served as an internal control. The sequences of primers used in quantitative real-time-PCR are depicted in [Table 1]. All data are expressed as fold change in expression, compared with the expression in other animal groups, using the 2ΔΔCt method [27] in which Ct indicates cycle threshold, the fractional cycle number where the fluorescent signal reaches detection threshold. The normalized ΔCt value of each sample is calculated using up to an endogenous control gene (β-actin). Fold change values are presented as average fold change =2−(average ΔΔCt) for genes in treated relative to control samples using relative quantity manager program, 1.2 ABI SDS software (ABI 7900 HT; Applied Biosystems, California, USA).
Table 1 A list of primer sequences used for real-time-polymerase chain reaction

Click here to view


Statistical analysis

All data were expressed as mean±SE and analyzed with statistical package SPSS for Windows (version 22; SPSS Inc., Chicago, Illinois, USA). The data were analyzed by one-way analysis of variance followed by Tukey’s post-hoc test to determine which pairs in the group comparison were significantly different. P-value less than 0.05 was considered significant.


  Results Top


Blood pressure changes

The baseline values of SBP were similar among the groups. At the end of the second week, l-NAME-treated group had significant (P<0.05) elevated SBP compared with the control group. At 8 weeks after induction of hypertension, SBP of l-NAME-treated rats (group 2) was significantly (P<0.05) higher than that of control rats (group 1). SBP remained high throughout the study in l-NAME-treated animals. SAX treatment of group 3 produced a significant (P<0.05) decrease in SBP which did not reach the normal value; HSE treatment of group 4 alone or in combination with SAX (group 5) induced significant (P<0.05) reduction in SBP compared with group 2. Nonsignificant difference was found between group 3 and group 4. There was a significant (P<0.05) difference between group 4, treated with HSE alone, and group 5 which received combination treatment in favor of group 5 which almost reach the normal value ([Figure 1]).
Figure 1 Effect of saxagliptin (SAX), Hibiscus sabdariffa Linn extract (HSE) and their combination on Nω-nitro-l-arginine methyl ester (l-NAME)-induced changes of systolic blood pressure in rats. Data are represented as mean±SE (n=8).

Click here to view


Biochemical parameters of renal function evaluation

In l-NAME-treated rats, blood urea and serum creatinine levels were significantly (P<0.05) increased compared with the control group. Administration of either SAX (group 3) or HSE (group 4) induced significant (P<0.05) decrease in blood urea and serum creatinine levels with insignificant difference between the two groups, while coadministration of both agents (group 5) produced significant (P<0.05) reduction in both parameters compared with either group 3 or group 4 ([Table 2]).
Table 2 Effect of saxagliptin, Hibiscus sabdariffa Linn extract, and their combination on Nω-nitro-l-arginine methyl ester-induced changes of blood urea, serum creatinine, and creatinine clearance in rats

Click here to view


Regarding Ccr, it significantly (P<0.05) decreased in l-NAME-treated group (group 2) compared with control rats, but significantly (P<0.05) increased in both SAX-treated (group 3) and HSE-treated (group 4) groups compared with group 2. There was nonsignificant difference between group 3 and group 4. In the combination group (group 5), Ccr was significantly (P<0.05) increased compared with either group 3 or group 4 ([Table 2]).

There was no significant difference in initial 24-h urinary protein excretion among the studied groups. l-NAME-treated rats (group 2) showed significantly (P<0.05) higher 24-h urinary protein excretion throughout the study period compared with control rats (group 1). Rats received either SAX (group 3) or HSE (group 4) showed significantly (P<0.05) lower urinary protein levels compared with group 2. Administration of SAX alone reduced urinary protein excretion significantly (P<0.05) versus HSE alone. However, in the combination group (group 5) the reduction in urinary protein excretion was significant (P<0.05) compared with SAX alone ([Figure 2]).
Figure 2 Effect of saxagliptin (SAX), Hibiscus sabdariffa Linn extract (HSE), and their combination on Nω-nitro-l-arginine methyl ester (l-NAME)-induced changes of 24-h urinary protein in rats. Data are represented as mean±SE (n=8).

Click here to view


Biochemical parameters of oxidative stress

Administration of l-NAME to rats of group 2 resulted in significant (P<0.05) increase in renal tissue MDA with significant (P<0.05) decrease in renal tissue GSH content and GPx activity compared with the control group. Treatment of rats with either SAX or HSE for 8 weeks resulted in significant decrease (P<0.05) in renal tissue MDA with significant (P<0.05) increase in tissue GSH content and GPx activity compared with the l-NAME-treated group (group 2). However, there was a significant (P<0.05) difference between group 3 and group 4 in favor of group 4. The decrease in tissue MDA and the increase in renal tissue GSH content and GPx activity were significant (P<0.05) in SAX and HSE combination group compared with the use of each drug individually ([Table 3]).
Table 3 Effect of saxagliptin, Hibiscus sabdariffa Linn extract, and their combination on malondialdehyde, glutathione level, and glutathione peroxidase activity in renal tissues homogenate of Nω-nitro-l-arginine methyl ester-treated rats

Click here to view


Measurements of proinflammatory cytokines

l-NAME induced significant (P<0.05) increase in the levels of TNF-α and IL-6 in renal tissue homogenates of group 2 compared with control rats. Coadministration of either SAX (group 3) or HSE (group 4) with l-NAME resulted in significant (P<0.05) decrease in renal tissue levels of TNF-α and IL-6 compared with l-NAME-treated group with significant (P<0.05) difference between group 3 and group 4 in favor of SAX. Combined administration of SAX and HSE plus l-NAME (group 5) resulted in significant (P<0.05) decrease in renal tissue levels of TNF-α and IL-6 compared with group 3 and group 4 ([Figure 3] and [Figure 4]).
Figure 3 Effect of saxagliptin (SAX), Hibiscus sabdariffa Linn extract (HSE), and their combination on renal tissue level of tumor necrosis factor-α (TNF-α) in Nω-nitro-l-arginine methyl ester (l-NAME)-induced hypertensive nephropathy rat model. Data are presented as mean±SE (n=8). *Significant difference compared with the control group. Significant difference compared with the l-NAME-treated group. Significant difference compared with the SAX+l-NAME-treated group. Significant difference compared with the HSE+l-NAME-treated group.

Click here to view
Figure 4 Effect of saxagliptin (SAX), Hibiscus sabdariffa Linn extract (HSE), and their combination on renal tissue level of interleukin-6 (IL-6) in Nω-nitro-l-arginine methyl ester (l-NAME)-induced hypertensive nephropathy rat model. Data are presented as mean±SE (n=8). *Significant difference compared with the control group. Significant difference compared with the l-NAME-treated group. Significant difference compared with the SAX+l-NAME-treated group. Significant difference compared with the HSE+l-NAME-treated group.

Click here to view


Histopathological results

The kidneys of control rats had normal morphology with normal appearance of the glomeruli, tubules, and interstitium ([Figure 5]). l-NAME-treated rats showed glomerular damage evidenced by proliferative glomerulonephritis and congested glomerular capillaries ([Figure 6]). The convoluted tubules showed cloudy swelling with intracellular and extracellular hyalinosis leading to obliterated lumen. Cellular infiltration was also observed. In addition, some proximal convoluted tubules were lined by vacuolated cubical epithelium with wide lumen. This picture was significantly improved in rats given SAX or HSE alone or in combination, as evidenced by mild degenerative changes with attenuation of fibrotic changes ([Figure 7],[Figure 8],[Figure 9]).
Figure 5 Representative photomicrographs of renal tissue of control rat (group 1) showing normal glomeruli, distal, and proximal convoluted tubules. Hematoxylin and eosin, ×200.

Click here to view
Figure 6 Representative photomicrographs of renal tissue of Nω-nitro-l-arginine methyl ester-treated rat (group 2) showing thickening of the glomerular capillary basement membrane (glomerulosclerosis) and the surrounding tubules showing both intracellular and extracellular hyalinosis leading to obliterated lumen. Hematoxylin and eosin, ×400.

Click here to view
Figure 7 Representative photomicrographs of renal tissue of saxagliptin+Nω-nitro-l-arginine methyl ester-treated group (group 3) showing mild degenerative changes affecting renal tubules and glomerular capillary tuft with increased lumen space of the tubules. Hematoxylin and eosin, ×200.

Click here to view
Figure 8 Representative photomicrographs of renal tissue Hibiscus sabdariffa Linn. extract+Nω-nitro-l-arginine methyl ester-treated group (group 1) 5 showing mild degenerative changes affecting renal tubules and glomerular capillary tuft. Some tubules are dilated. Hematoxylin and eosin, ×200.

Click here to view
Figure 9 Representative photomicrographs of renal tissue of saxagliptin+Hibiscus sabdariffa Linn extract+Nω-nitro-l-arginine methyl ester-treated group showing mild degenerative changes affecting renal tubules with marked reduction of glomerular sclerosis. Hematoxylin and eosin, ×400.

Click here to view


Semiquantitative analysis showed significant (P<0.05) glomerulosclerosis in the l-NAME group compared with control rats, and this was significantly (P<0.05) improved in treated groups ([Figure 10] and [Figure 11]).
Figure 10 Effect of saxagliptin (SAX), Hibiscus sabdariffa Linn extract (HSE) and their combination on histopathological glomerular damage score in Nω-nitro-l-arginine methyl ester (l-NAME)-induced hypertensive nephropathy rat model. Data are presented as mean±SE (n=8). *Significant difference compared with the control group. Significant difference compared with the l-NAME-treated group. Significant difference compared with the SAX+l-NAME-treated group. Significant difference compared with the HSE+l-NAME-treated group.

Click here to view
Figure 11 Effect of saxagliptin (SAX), Hibiscus sabdariffa Linn extract (HSE), and their combination on histopathological tubulointerstitial damage score in Nω-nitro-l-arginine methyl ester (l-NAME)-induced hypertensive nephropathy rat model. Data are presented as mean±SE (n=8). *Significant difference compared with the control group. Significant difference compared with the l-NAME-treated group. Significant difference compared with the SAX+l-NAME-treated group. Significant difference compared with the HSE+l-NAME-treated group.

Click here to view


Quantitative determination of cortical transforming growth factor-β1 mRNA expression

The expression of gene for TGF-β1 as a marker for the occurrence of renal fibrosis is shown in [Figure 12]. Quantitative PCR estimation of renal tissue TGF-β1 mRNA expression level in l-NAME-treated rats of group 2 was significantly (P<0.05) higher compared with the control group. Administration of either SAX or HSE for 8 weeks resulted in significant decrease (P<0.05) in TGF-β1 mRNA expression in renal tissue compared with l-NAME-treated group (group 2). However, the reduction of TGF-β1 mRNA expression in the renal tissue was more significant (P<0.05) in group 3 compared with group 4. On the other hand, combined SAX and HSE therapy significantly (P<0.05) reduced renal TGF-β1 mRNA expression level compared with HSE-treated group alone.
Figure 12 Effect of saxagliptin (SAX), Hibiscus sabdariffa Linn extract (HSE), and their combination on transforming growth factor-β1 (TGF-β1) mRNA expression level in renal tissue in Nω-nitro-l-arginine methyl ester (l-NAME)-induced hypertensive nephropathy rat model. Data are presented as mean±SE (n=8). *Significant difference compared with the control group. Significant difference compared with the l-NAME-treated group. Significant difference compared with the SAX+l-NAME-treated group. Significant difference compared with the HSE+l-NAME-treated group.

Click here to view



  Discussion Top


Hypertensive nephropathy or hypertensive nephrosclerosis is a medical condition referring to damage to the kidney due to chronic high blood pressure. The current study was undertaken to assess the possible renoprotective effect of SAX, a DPP4 inhibitor, alone and in combination with HSE in a rat model of hypertensive nephropathy induced by l-NAME. Previous studies demonstrated that l-NAME decreases renal blood flow without changing glomerular filtration. These observations imply that nitric oxide (NO) inhibition may selectively increase postglomerular renal vascular resistance [28]. Another study has shown that the inhibition of NO synthesis may influence tubular reabsorption by a direct effect on tubular sodium transport or by hemodynamically mediated mechanisms involving a reduction in medullary blood flow or a rise in renal interstitial hydrostatic pressure [29]. Xavier et al. [30] suggested that inhibition of NO synthesis can directly and transiently enhance sodium reabsorption in postproximal nephron segments. However, larger doses of l-NAME produced increases in arterial pressure that overrode the initial antinatriuretic effect. So the glomerular filtration rate decreased following l-NAME administration, suggesting afferent arteriole vasoconstriction; they also explain the mechanism as they showed that bilateral renal denervation delayed and attenuated the l-NAME-induced hypertension by promoting an additional decrease in tubule sodium reabsorption in the post-proximal segments of nephrons. In this work, we used a relatively large dose of l-NAME as Bernátová et al. [31] found that chronic low-dose l-NAME treatment can increase NO production and vasorelaxation in normotensive rats. At low doses of l-NAME the blood pressure was significantly elevated on the 3rd and 6th week of treatment versus controls, after this period, blood pressure of l-NAME-treated rats returned to the control values.

Our results have shown that l-NAME administration induced significant increase in systolic arterial blood pressure which remained high throughout the study period compared with control rats; in addition, proteinuria was developed with renal inflammation marked by cellular infiltration and increase in renal tissue proinflammatory cytokines, TNF-α, and IL-6. In agreement with our findings, Ikeda et al. [17] reported that chronic inhibition of NO synthase by l-NAME in rats caused hypertension, glomerulosclerosis, interstitial fibrosis, and macrophage infiltration. In this study, l-NAME induced significant increase in renal tissue MDA with a significant decrease in renal tissue GSH content and GPx activity compared with control rats, supporting the involvement of oxidative stress in the rise in blood pressure and renal damage effects of l-NAME. In consistent with our results, Toba et al. [32] demonstrated that l-NAME administration increased oxidative stress, vascular inflammation, and ACE activity. Kobayashi et al. [33] declared that increases in oxidative stress reduced the bioavailability of NO and this reduction in the potent vasodilator contributes to the development of hypertension.

The results of our study proved that either SAX or HSE treatment had significantly lower the SBP when used in l-NAME-treated rats but not reached the normal value. However, a combined therapy using SAX and HSE significantly lowered the blood pressure to nearly the normal value compared with the control group, suggesting possible additive effects associated with combined therapy. In accordance with our results Mason et al. [18] reported that SAX reduces blood pressure and inflammation in hypertensive rats and increases NO bioavailability. These results also go along with Mistry et al. [34] who stated that sitagliptin, a DPP4 inhibitor, produced small but statistically significant reduction in blood pressure, and was generally well tolerated in nondiabetic patients with mild to moderate hypertension. The mechanism can be evidenced as Pacheco et al. [35] reported that chronic administration of sitagliptin attenuated blood pressure rising in young prehypertensive adult spontaneously hypertensive rats, partially by inhibiting Na+/H+ exchanger isoform 3 activities in renal proximal tubule; sitagliptin also decreased blood pressure through its diuretic effect that increases the urinary sodium excretion. In contrast, Jackson et al. [36] have indicated that acute administration of a DPP4 inhibitor increases blood pressure in spontaneously hypertensive rats through the vasoconstrictive effect of the neuropeptide Y1 receptors, this can be attributed to the method of administration and to the different types of dosage.

The hypotensive effect of HSE was evident, a study suggested that a variety of bioactivities has been attributed to this compound including, antihypertensive effect which is mediated through acetylcholine-like and histamine-like mechanisms as well as through direct vasorelaxant effects [12]. In addition, diuretic activity due to the modulation of the aldosterone action was found to be involved in the hypotensive action of HSE [13]. Besides, ACE inhibition was proved to be induced by HSE leading to reduction in blood pressure [14]. Another mechanism of the antihypertensive effect of HSE has been discussed by Aliyu et al. [37] as they stated that the increased blood pressure induced by sympathetic stimulation was significantly dampened by HSE suggesting that its hypotensive effect may be due to an attenuation of the discharge of the sympathetic nervous system.

The current study has shown that treatment of rats with either SAX or HSE for 8 weeks resulted in significant decrease in renal tissue MDA with significant increase in tissue GSH content and GPx activity compared with the l-NAME treated group. Nassar et al. [38] found that SAX induced reduction in the brain thiobarbituric acid reactive substances in rats, denoting antioxidant efficacy. Recently, the antioxidant activity of SAX was demonstrated in a study by Solini et al. [39], who verified that the intravascular superoxide production is attenuated by SAX. On the other hand, the antioxidant activity of HSE was more prominent compared with the SAX-treated group. Several studies documented the antioxidant efficacy of HSE [40],[41]. The beneficial effect of combined SAX and HSE was evidenced by the enhanced antioxidant effect as the decrease in tissue MDA and the increase in renal tissue GSH content and GPx activity was significant in the combined group compared with the use of each drug individually.

Besides the antioxidant properties, anti-inflammatory and antifibrotic effects of SAX and HSE were incorporated into the renoprotective effects of these compounds. Either SAX or HSE alone or in combination was able to ameliorate the l-NAME-induced deterioration in renal function and the histopathological scoring of hypertensive rats. SAX, HSE, and their combination also resulted in a significant decrease in renal tissue levels of proinflammatory cytokines, TNF-α, and IL-6 compared with the l-NAME-treated group.

Impaired renal function in l-NAME-treated rats includes significant increase in serum urea and creatinine, significant higher urinary protein excretion, and significant decrease in plasma creatinine clearance compared with the control group. Pathological evidence of kidney damage was observed in all rats treated with l-NAME. In consistent with our findings, Polichnowski et al. [42] have shown that in l-NAME-induced hypertensive rat kidney, there were outer medullary interstitial fibrosis and macrophage density. Overall, the outer medullary injury and infiltration of macrophages were mainly due to the direct effects of elevated angiotensin II and decreased NO, in addition to outer medullary tubular necrosis.

Our results have demonstrated that the renal histological picture was significantly improved in rats given SAX or HSE alone as evidenced by mild degenerative changes with more improvement in rats received combined therapy. These results agreed with the finding of Sakai et al. [43] who found that treatment with SAX for 4 weeks significantly suppressed the increase in urinary albumin excretion and tended to ameliorate glomerular injury without altering the blood glucose levels. The inhibition of the renal DPP4 activity induced by SAX may contribute to ameliorating renal injury in hypertension-related renal injury. The anti-inflammatory effect of SAX was recently evaluated by Birnbaum et al. [44] who demonstrated that SAX attenuated the increase in serum levels of inflammatory cytokines as c-reactive protein, TNF-α, IL-1β, IL-18, and IL-6 in type 1 and type 2 diabetic mice.

The renoprotective effect of HSE agreed with Lee et al. [45] research that showed the beneficial effects of HSE on streptozotocin-induced diabetic nephropathy including pathology, serum lipid profile, and oxidative marker in kidneys. Other study showed that HSE improves hyperglycemia-caused osmotic diuresis in renal proximal convoluted tubules (defined as hydropic change) in diabetic rats and shows that upregulation of nuclear factor-κ B-mediated transcription might be involved. So hibiscus possesses the potential effects to ameliorate diabetic nephropathy through improving oxidative status [46]. Our results also go along with Yang et al. [47] who found that HSE inhibits albuminuria and glomerular hyperfiltration in early diabetic nephropathy. HSE reduces tubular connective tissue growth factors and glomerular cluster of differentiation 31. HSE reversed collagen accumulation. HSE decreased angiotensin II type 1 receptor elevation, while improving the oxidative stress. Furthermore, Yang et al. [47] stated that treatment of HSE recovered morphological changes of cell junction and basement membrane. Furthermore, Kao et al. [48] reported that HSE has the ability to prevent inflammation through impairing COX-2 induction. These findings suggest that HSE has the potential to be an adjuvant therapy for hypertensive nephropathy.

In this study, the expression of gene for TGF-β1, a marker for the occurrence of renal fibrosis, of renal tissue (TGF-β1 mRNA expression level) in l-NAME-treated rats was significantly higher compared with the control group. Administration of either SAX or HSE for 8 weeks resulted in significant decrease in TGF-β1 mRNA expression in renal tissue compared with the l-NAME-treated group. However, the reduction of TGF-β1 mRNA expression in renal tissue was significant in SAX-treated group compared with HSE-treated group. On the other hand, combined SAX and HSE therapy significantly reduced renal TGF-β1 mRNA expression level compared with each used alone. The antifibrotic effect of SAX has not been fully elucidated however; our results can be supported by the finding of Kaji et al. [10] who reported significant attenuation of liver fibrosis development in rats with sitagliptin treatment along with the suppression of hepatic TGF-β1, total collagen, and tissue inhibitor of metalloproteinases-1. In addition, Li et al. [49] found that sitagliptin exhibited renoprotective effect against dyslipidemia-related kidney injury in mice; this may be due to the downregulation of phosphorylated AMP-activated protein kinase, and inhibition of TGF-β1, fibronectin, and p38/ERK mitogen-activated protein kinase signaling pathways. Regarding the effect of HSE on renal TGF-β1 mRNA expression, Yang et al. [47] concluded that HSE treatment reduced renal TGF-β1 evoked by high glucose in diabetic nephropathy.


  Conclusion Top


Our data have proved that either SAX or HSE has a significant antihypertensive and renoprotective effect when used alone. However, combination therapy was more effective in attenuating renal injury induced by l-NAME in rats. This combination has the advantage of additive renoprotective mechanisms attributed to the anti-inflammatory, antioxidant, and antifibrotic effects of both SAX and HSE in renal tissue.

Acknowledgements

The authors acknowlege all memberships of the Biochemistry and Histology Departments, Faculty of Medicine, Benha University for their valuable scientific support and kind cooperation during this research.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Lea JP, Nicholas SB. Diabetes mellitus and hypertension: key risk factors for kidney disease. J Natl Med Assoc 2002; 94:7S–15S.  Back to cited text no. 1
[PUBMED]    
2.
Bidani AK, Griffin KA. Long-term renal consequences of hypertension for normal and diseased kidneys. Curr Opin Nephrol Hypertens 2002; 11:73–80.  Back to cited text no. 2
[PUBMED]    
3.
Wolf G. Renal injury due to renin-angiotensin-aldosterone system activation of the transforming growth factor-beta pathway. Kidney Int 2006; 70:1914–1919.  Back to cited text no. 3
[PUBMED]    
4.
Border WA, Noble MA. Interaction of transforming growth factor-β and angiotensin II in renal fibrosis. Hypertension 1998; 31:181–188.  Back to cited text no. 4
    
5.
Bourraindeloup M, Christophe A, Candiani G, Calleret M, Bourin MC, Badoual T et al. N-acetylcysteine treatment normalizes serum tumor necrosis factor-α level and hinders the progression of cardiac injury in hypertensive rats. Circulation 2004; 110:2003–2009.  Back to cited text no. 5
    
6.
Dalla Vestra M, Mussap M, Gallina P, Bruseghin M, Cernigoi A, Saller A et al. Acute-phase markers of inflammation and glomerular structure in patients with type 2 diabetes. J Am Soc Nephrol 2005; 16:S78–S82.  Back to cited text no. 6
    
7.
Appel LJ, Wright JT Jr, Greene T, Agodoa LY, Astor BC, Bakris GL et al. Intensive blood-pressure control in hypertensive chronic kidney disease. N Engl J Med 2010; 363:918–929.  Back to cited text no. 7
    
8.
Scheen AJ. A review of gliptins for2014. Expert Opin Pharmacother 2015; 16:43–62.  Back to cited text no. 8
    
9.
Ussher JR, Drucker DJ. Cardiovascular biology of the incretin system. Endocr Rev 2012; 33:187–215.  Back to cited text no. 9
    
10.
Kaji K, Yoshiji H, Ikenaka Y, Noguchi R, Aihara Y, Douhara A et al. Dipeptidyl peptidase-4 inhibitor attenuates hepatic fibrosis via suppression of activated hepatic stellate cell in rats. J Gastroenterol 2014; 49:481–491.  Back to cited text no. 10
    
11.
Joven J, March I, Espinel E, Fernández-Arroyo S, Rodríguez-Gallego E, Aragonès G et al. Hibiscus sabdariffa extract lowers blood pressure and improves endothelial function. Mol Nutr Food Res 2014; 58:1374–8.  Back to cited text no. 11
    
12.
Ajay M, Chai HJ, Mustafa AM, Gilani AH, Mustafa MR. Mechanisms of the anti-hypertensive effect of Hibiscus sabdariffa L. calyces. J Ethnopharmacol 2007; 109:388–393.  Back to cited text no. 12
    
13.
Jiménez-Ferrer E, Alarcón-Alonso J, Aguilar-Rojas A, Zamilpa A, Jiménez-Ferrer CI, Tortoriello J, Herrera-Ruiz M. l. Diuretic effect of compounds from Hibiscus sabdariffa by modulation of the aldosterone activity. Planta Med 2012; 78:1893–1898.  Back to cited text no. 13
    
14.
Ojeda D, Jiménez-Ferrer E, Zamilpa A, Herrera-Arellano A, Ortoriello J, Alvarez L. Inhibition of angiotensin convertin enzyme (ACE) activity by the anthocyanins delphinidin- and cyanidin-3-O-sambubiosides from Hibiscus sabdariffa. J Ethnopharmacol 2010; 127:7–10.  Back to cited text no. 14
    
15.
Herrera-Arellano A, Flores-Romero S, Chávez-Soto MA, Tortoriello J. Effectiveness and tolerability of a standardized extract from Hibiscus sabdariffa in patients with mild to moderate hypertension: A controlled and randomized clinical trial. Phytomedicine 2004; 11:375–82.  Back to cited text no. 15
    
16.
McKay DL, Chen CYO, Saltzman E, Blumberg JB. Hibiscus sabdariffa L. tea (tisane) lowers blood pressure in prehypertensive and mildly hypertensive adults. J Nutr 2010; 140:298–303.  Back to cited text no. 16
    
17.
Ikeda H, Tsuruya K, Toyonaga J, Masutani K, Hayashida H, Hirakata H et al. Spironolactone suppresses inflammation and prevents l-NAME-induced renal injury in rats. Kidney Int 2009; 75:147–155.  Back to cited text no. 17
    
18.
Mason RP, Jacob RF, Kubant R, Walter MF, Bellamine A, Jacoby A et al. Effect of enhanced glycemic control with saxagliptin on endothelial nitric oxide release and CD40 levels in obese rats. J Atheroscler Thromb 2011; 18:774–783.  Back to cited text no. 18
    
19.
Mohan M, Khade B, Shinde A. Effect of A-HRS on blood pressure and metabolic alterations in fructose-induced hypertensive rats. Nat Prod Res 2012; 26:570–574.  Back to cited text no. 19
    
20.
Fawcett JK, Scott E. A rapid and precise method for the determination of urea. J Clin Pathol 1960; 13:156–159.  Back to cited text no. 20
    
21.
Fabiny DL, Ertingshausen G. Automated reaction rate method for determination of serum creatinine with CentrifiChem. Clin Chem 1971; 17:696–700.  Back to cited text no. 21
    
22.
Nishi HH, Elin RJ. Three turbidimetric methods for determining total protein compared. Clin Chem 1985;31: 1377–1380.  Back to cited text no. 22
    
23.
Zdenek AP, Cushman LL, Conner BJ. Estimation of product of lipid peroxidation (malondialdehyde) in biochemical systems. Anal Biochem 1966; 16:359–364.  Back to cited text no. 23
    
24.
Ellman GI. Tissue sulfhydryl group. Arch Biochem Biophys 1959; 82:70–77.  Back to cited text no. 24
    
25.
Flohé L, Gunzler WA. Assays of glutathione peroxidase. Methods Enzymol 1984; 105:114–121.  Back to cited text no. 25
    
26.
Gadola L, Noboa O, Marquez MN, Rodriguez MJ, Nin N, Boggia J et al. Calcium citrate ameliorates the progression of chronic renal injury. Kidney Int 2004; 65:1224–1230.  Back to cited text no. 26
    
27.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(T) (Delta C) method. Methods 2001; 25:402–408.  Back to cited text no. 27
    
28.
Majid DAS, Navar G. Suppression of blood flow auto-regulation plateau during nitric oxide blockage in canine kidney. Am J Physiol 1992; 262:F40–F46.  Back to cited text no. 28
    
29.
Mattson DL, Roman RJ, Cowley AW. Role of nitric oxide in renal papillary blood flow and sodium excretion. Hypertension 1992; 19:766–769.  Back to cited text no. 29
    
30.
Xavier F, Magalhães AMF, Gontijo JAR. Effect of inhibition of nitric oxide synthase on blood pressure and renal sodium handling in renal denervated rats. Braz J Med Biol Res 2000; 33:347–354.  Back to cited text no. 30
    
31.
Bernátová I, Kopincová J, Púzserová A, Janega P, Babál P. Chronic low-dose l-NAME treatment increases nitric oxide production and vasorelaxation in normotensive rats. Physiol Res 2007; 56:S17–S24.  Back to cited text no. 31
    
32.
Toba H, Nakagawa Y, Miki H, Shimizu T, Yoshimura A, Inoue R et al. Calcium channel blockage exhibits anti-inflammatory and anti-oxidative effects by augmentation of endothelial nitric oxide synthase and inhibition of angiotensin converting enzymes in the N-nitro-L arginine methyl ester-induced hypertensive rat aorta: vasoprotective effects beyond the blood pressure lowering effect of amlodipine and manidipine. Hypertens Res 2005; 28:689–700.  Back to cited text no. 32
    
33.
Kobayashi A, Ishikawa K, Mastsumoto H, Kimura S, Kamiyama Y, Maruyama Y. Synergetic antioxidant and vasodilatory action of carbon monoxide in angiotensin II-induced cardiac hypertrophy. Hypertension 2007; 50:1040–8.  Back to cited text no. 33
    
34.
Mistry GC, Maes AL, Lasseter KC, Davies MJ, Gottesdiener KM, Wagner JA et al. Effect of sitagliptin, a dipeptidyl peptidase-4 inhibitor, on blood pressure in nondiabetic patients with mild to moderate hypertension. J Clin Pharmacol 2008; 48:592–8.  Back to cited text no. 34
    
35.
Pacheco BP, Crajoinas RO, Couto GK, Davel AP, Lessa LM, Rossoni LV et al. Dipeptidyl peptidase IV inhibition attenuates blood pressure rising in young spontaneously hypertensive rats. J Hypertens 2011; 29:520–528.  Back to cited text no. 35
    
36.
Jackson EK, Dubinion JH, Mi Z. Effects of dipeptidyl peptidase IV inhibition on arterial blood pressure. Clin Exp Pharmacol Physiol 2008; 35:29–34.  Back to cited text no. 36
    
37.
Aliyu B, Oyeniyi YJ, Mojiminiyi FB, Isezuo SA, Alada AR. The aqueous calyx extract of Hibiscus sabdariffa lowers blood pressure and heart rate via sympathetic nervous system dependent mechanisms. Niger J Physiol Sci 2014; 29:131–136.  Back to cited text no. 37
    
38.
Nassar NN, Al-Shorbagy MY, Arab HH, Abdallah DM. Saxagliptin: a novel antiparkinsonian approach. Neuropharmacology 2015; 89:308–317.  Back to cited text no. 38
    
39.
Solini A, Rossi C, Duranti E, Taddei S, Natali A, Virdis A. Saxagliptin prevents vascular remodeling and oxidative stress in db/db mice. Role of endothelial nitric oxide synthase uncoupling and cyclooxygenase. Vascul Pharmacol 2016; 76:62–71.  Back to cited text no. 39
    
40.
Ochani PC, D’Mello P. Antioxidant and antihyperlipidemic activity of Hibiscus sabdariffa Linn. leaves and calyces extracts in rats. Indian J Exp Biol 2009; 47:276–282.  Back to cited text no. 40
    
41.
Chen JH, Wang CJ, Wang CP, Sheu JY, Lin CL, Lin HH. Hibiscus sabdariffa leaf polyphenolic extract inhibits LDL oxidation and foam cell formation involving up-regulation of LXRα/ABCA1 pathway. Food Chem 2013; 141:397–406.  Back to cited text no. 41
    
42.
Polichnowski AJ, Lu L, Cowley AW. Renal injury in angiotensin II+l-NAME-induced hypertensive rats is independent of elevated blood pressure. Am J Physiol Renal Physiol 2011; 300:F1008–F1016.  Back to cited text no. 42
    
43.
Sakai M, Uchii M, Myojo K, Kitayama T, Kunori S. Critical role of renal dipeptidyl peptidase-4 in ameliorating kidney injury induced by saxagliptin in Dahl salt-sensitive hypertensive rats. Eur J Pharmacol 2015; 761:109–115.  Back to cited text no. 43
    
44.
Birnbaum Y, Bajaj M, Qian J, Ye Y. Dipeptidyl peptidase-4 inhibition by saxagliptin prevents inflammation and renal injury by targeting the Nlrp3/ASC inflammasome. BMJ Open Diabetes Res Care 2016; 4:e000227.  Back to cited text no. 44
    
45.
Lee WC, Wang CJ, Chen YH, Hsu JD, Cheng SY, Chen HC et al. Polyphenol extracts from Hibiscus sabdariffa Linnaeus attenuate nephropathy in experimental type 1 diabetes. J Agric Food Chem 2009; 57:2206–2210.  Back to cited text no. 45
    
46.
Wang SC, Lee SF, Wang CJ, Lee CH, Lee WC, Lee HJ. Aqueous extract from Hibiscus sabdariffa Linnaeus ameliorate diabetic nephropathy via regulating oxidative status and Akt/Bad/14-3-3γ in an experimental animal model. Evid Based Complementary Altern Med 2011; 2011:938126.  Back to cited text no. 46
    
47.
Yang YS, Wang CJ, Huang CN, Chen ML, Chen MJ, Peng CH. Polyphenols of Hibiscus sabdariffa improved diabetic nephropathy via attenuating renal epithelial mesenchymal transition. J Agric Food Chem 2013; 61:7545–7551.  Back to cited text no. 47
    
48.
Kao ES, Hsu JD, Wang CJ, Yang SH, Cheng SY, Lee HJ. Polyphenols extracted from Hibiscus sabdariffa L. inhibited lipopolysaccharide-induced inflammation by improving antioxidative conditions and regulating cyclooxygenase-2 expression. Biosci Biotechnol Biochem 2009; 73:385–390.  Back to cited text no. 48
    
49.
Li J, Guan M, Li C, Lyv F, Zeng Y, Zheng Z et al. The dipeptidyl peptidase-4 inhibitor sitagliptin protects against dyslipidemia-related kidney injury in Apolipoprotein E knockout mice. Int J Mol Sci 2014;15:11416–11434.  Back to cited text no. 49
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Material and methods
Results
Discussion
Conclusion
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed51    
    Printed0    
    Emailed0    
    PDF Downloaded10    
    Comments [Add]    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]