|Year : 2017 | Volume
| 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
Omaima M Abd Allah1, Abeer A Shoman2
1 Department of Pharmacology, Faculty of Medicine, Benha University, Benha, Egypt
2 Department of Physiology, Faculty of Medicine, Benha University, Benha, Egypt
|Date of Submission||14-Nov-2016|
|Date of Acceptance||20-Dec-2016|
|Date of Web Publication||24-May-2017|
Omaima M Abd Allah
Department of Pharmacology, Faculty of Medicine, Benha University, Al-Sahaa Street, Diverted from Farid Nada Street, Benha, Qalyubia Governorate, 13518
Source of Support: None, Conflict of Interest: None
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.
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.
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 2018 Mar 17];34:17-27. Available from: http://www.bmfj.eg.net/text.asp?2017/34/1/17/206902
| Introduction|| |
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 . Glomerular sclerosis (GS), tubular interstitial fibrosis, and vascular sclerosis have been found to be important factors in the progression of chronic kidney injury . 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 . TGF-β1 induces renal fibrosis by activating interstitial fibroblasts to become myofibroblasts, which produce large amounts of matrix components . 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 ,.
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 . 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 . 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 . In addition to lowering blood glucose, DPP4 inhibitors have been shown to attenuate hepatic fibrosis in rats . 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 .
Hibiscus sabdariffa Linn extract (HSE) has been reported to possess antioxidative and antihypertensive activities in animal and human studies . 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 ,. Inhibition of angiotensin-converting enzyme (ACE) by HSE was also reported  and this could be another important antihypertensive mechanism of action. Several clinical trials confirmed the antihypertensive effectiveness and safety of HSE ,.
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|| |
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.
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.
Animals were randomly allocated into five equal groups of eight rats each:
- Group 1: served as control group in which animals received distilled water orally for 8 weeks without any drugs.
- 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 .
- Group 3 (SAX+l-NAME-treated group): SAX was received orally at a dose of 10 mg/kg/day  in concomitant with l-NAME treatment for 8 weeks.
- Group 4 (HSE+l-NAME-treated group): HSE was given orally at a dose of 100 mg/kg/day  concomitantly with l-NAME treatment for 8 weeks.
- 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.
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.
Blood urea nitrogen, serum creatinine, and urinary creatinine were estimated colorimetrically as previously described ,. In addition, total protein contents in 24-h urine samples were determined  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 ; glutathione (GSH) was determined in μmol/g protein in renal tissues ; and glutathione peroxidase (GPx) activity was estimated in IU/G protein , 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 : 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 . 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  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|
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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|| |
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).|
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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|
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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).|
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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|
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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.|
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|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.|
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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.|
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|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.|
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|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.|
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|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.|
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|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.|
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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.|
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|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.|
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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.|
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| Discussion|| |
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 . 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 . Xavier et al.  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.  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.  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.  demonstrated that l-NAME administration increased oxidative stress, vascular inflammation, and ACE activity. Kobayashi et al.  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.  reported that SAX reduces blood pressure and inflammation in hypertensive rats and increases NO bioavailability. These results also go along with Mistry et al.  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.  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.  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 . In addition, diuretic activity due to the modulation of the aldosterone action was found to be involved in the hypotensive action of HSE . Besides, ACE inhibition was proved to be induced by HSE leading to reduction in blood pressure . Another mechanism of the antihypertensive effect of HSE has been discussed by Aliyu et al.  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.  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. , 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 ,. 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.  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.  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.  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.  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 . Our results also go along with Yang et al.  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.  stated that treatment of HSE recovered morphological changes of cell junction and basement membrane. Furthermore, Kao et al.  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.  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.  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.  concluded that HSE treatment reduced renal TGF-β1 evoked by high glucose in diabetic nephropathy.
| Conclusion|| |
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.
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
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12]
[Table 1], [Table 2], [Table 3]