Effects of nicorandil on renal function and histopathology in rats with partial unilateral ureteral obstruction

Abstract

To evaluate the effects of nicorandil in a rat kidney model of partial unilateral ureteral obstruction (PUUO). Thirty male rats were randomly divided into three groups as follows: (1) Group 1 (Sham‐control), ureters of the rats were manipulated but not ligated; (2) Group 2 (PUUO‐untreated), PUUO was performed with two‐thirds of the left ureter embedded in the psoas muscle; and (3) Group 3 (PUUO‐nicorandil treated). After PUUO was established, nicorandil (15 mg/kg/day) was administered by gastric lavage for 21 days to determine its effects on PUUO‐induced histopathological‐, functional‐, and oxidative stress‐induced changes. The serum levels of blood urea nitrogen and creatinine were reduced in Group 3. The level of urinary albumin and the ratio of urinary protein/creatinine were increased in the kidneys of Group 2 but decreased in Group 3. Malondialdehyde value was decreased in Group 3 compared with Group 2. Antioxidant enzyme activities (catalase, superoxide dismutase, and glutathione peroxidase) were decreased in Group 2. Nicorandil treatment caused an increase in these enzyme activities. In Group 3, leukocyte infiltration and tubular dilatation were significantly reduced. Other parameters, such as degeneration of tubular epithelium and fibrosis, also showed a marked improvement in Group 3. Expression of inducible nitric oxide synthase in Group 2 and expression of endothelial nitric oxide synthase in Group 3 were significantly elevated. Nicorandil can inhibit renal tubular damage and tubulointerstitial fibrosis by reducing the effects of oxidative stress after PUUO.

Introduction

Obstructive uropathy (OU) is a major cause of chronic renal insufficiency, particularly in infants and children [[1], [2]]. It affects renal morphogenesis, maturation, and growth because of congenital renal maldevelopment [3]. In the most severe cases, this type of renal injury ultimately results in progressive renal tubular atrophy and interstitial fibrosis, with loss of nephrons [4]. Hence, OU accounts for 23% of pediatric cases of renal insufficiency and 50% of pediatric patients with end‐stage renal disease undergoing renal transplantation [5].

Although the therapeutic focus has shifted to regenerative cell‐based agents, the lack of a comprehensive understanding of the pathogenesis of renal scar formation following injury remains a major challenge to the development of effective therapeutic strategies. Known factors in the pathophysiology of renal obstructive parenchymal injury include renal blood flow impairment, intrapelvic pressure elevation, and vasoactive and inflammatory mediators [[6], [7], [8]]. Recently, it has been suggested that reactive oxygen species (ROS), which are formed during ureteral obstruction, may play a role in this process [[9], [10]].

Nicorandil [N‐(2‐hydroxyethyl)‐nicotinamide nitrate], a vasodilator that acts as a potassium channel opener, is thought to inhibit superoxide anion production by canine neutrophils, which are activated by either phorbol myristate acetate or opsonized zymogen [11]. In a liver perfusion experiment, Naito et al [12] verified that nicorandil had antioxidative action. Recent studies also reported that nicorandil was beneficial in experimental renal disease, including preventing renal injury induced by ischemia–reperfusion [13] and glomerulonephritis [14] in rats. In humans, the pharmacokinetics of nicorandil have been examined in healthy volunteers and patients with impaired renal function [[15], [16]]. However, no studies have examined whether nicorandil could ameliorate partial unilateral ureteral obstruction (PUUO). Furthermore, the specific mechanisms by which nicorandil confers renoprotection are not known. To our knowledge, the protective effects of nicorandil have not been studied in PUUO induced in a rat model. The aim of this study was to evaluate the effects of nicorandil on antioxidant enzyme levels, renal function, and renal histopathology in a rat kidney model of PUUO.

Materials and methods

All surgical and experimental procedures were approved by the Institutional Animal Care and Use Committee of Abant Izzet Baysal University (Bolu, Turkey). Thirty male (age, 8–10 weeks) Sprague‐Dawley rats (body weight, 190–210 g) were enrolled. The animals were provided by Experimental Animal Department of Abant Izzet Baysal University. The procedures were performed according to routine animal care guidelines, and all experimental procedures complied with the Guide for the Care and Use of Laboratory Animals (1996). All the animals were housed under the same environmental conditions and fed the same diets. The rats were kept in sawdust‐lined cages (47 × 34 × 18 cm; 4 animals/cage) in one room at a constant temperature (22 ± 2°C), with light from 7:00 AM to 7:00 PM and water and food ad libitum.

PUUO surgery

Briefly, the rats were anesthetized by an intraperitoneal injection of xylazine (10 mg/kg) and ketamine hydrochloride (100 mg/kg) and placed on a homeothermic table to maintain a core body temperature of 37°C. Following catheterization of the right femoral vein, fluid replacement was performed with 3 mL/kg/hour of lactated Ringer's solution using an infusion pump.

Surgical PUUO was performed according to the method previously described in the literature [[6], [8]]. Briefly, under anesthesia, a midline longitudinal abdominal incision was made to permit access to the left kidney, ureter, and psoas muscle. Then, a 10‐mm groove was created in the psoas muscle. A ureter was placed in the muscle groove, and the muscle edges were fitted together by suturing three points with 6/0 polydioxanone sutures. Thus, the ureter lay in a tunnel with proximally and distally acute angles. The abdomen was then closed with 4/0 silk sutures. The rats were randomly divided into three groups.

Group 1 (Sham‐control, n   =  10): The rats underwent laparotomy through the abdominal midline incision, and their ureters were manipulated but not ligated. The rats in this group were used to determine basal values for biochemical and tissue evaluation.

Group 2 (PUUO‐untreated, n   =  10): PUUO was performed, with two‐thirds of the left ureter embedded in the psoas muscle through a midline abdominal incision using 6/0 polydioxanone sutures, as described above.

Group 3 (PUUO‐nicorandil treated, n   =  10): After PUUO was established, nicorandil (15 mg/kg/day) was administered by gastric lavage for 21 days to determine its effects on PUUO‐induced histopathological‐, functional‐, and oxidative stress‐induced changes.

At the end of the experiment, 24‐hour urine samples were collected using metabolic cages. The animals were anesthetized again, blood samples were collected from the abdominal aorta, and the left kidneys were removed for later analysis. All the rats were sacrificed after the experimental procedures.

Preservation of kidneys

The left kidney was removed under fully maintained anesthesia. After removal, the kidney was fixed in 10% phosphate‐buffered formalin or immediately frozen and stored at −80°C for subsequent analyses.

Assessment of renal function

Serum and urinary supernatants were stored at −80°C until used in the laboratory analysis. The samples were thawed before the analysis. Serum blood urea nitrogen (BUN) and creatinine and urinary creatinine, albumin, and protein analyses were performed using an autoanalyzer (Architect c 8000, Abbot Laboratories, State, U.S.A.).

Measurements of serum antioxidant enzyme activities and lipid peroxidation

Kidney tissues were placed in labeled glass tubes. The samples were washed with phosphate‐buffered saline (PBS) once and stored at −80°C until biochemical analysis. The samples were thawed before the analysis. All the tissues were homogenized once in 1 mL of PBS. The samples were frozen and thawed twice to facilitate cell membrane disintegration. The homogenate was centrifuged at 4°C at 5000g for 5 minutes, and the supernatant was removed. Catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH‐Px) activities were measured using commercially available enzyme‐linked immunosorbent assay kits (Cusabio Biotech, Wuhan, PRC), according to the manufacturer's instructions. Malondialdehyde (MDA) concentrations were measured using commercially available colorimetric assay kits (Cayman Chemical Company, State, U.S.A.), according to the manufacturer's instructions. The bicinchoninic acid (BCA) protein assay was used for quantitation of the total protein in the tissue (Thermo Fisher Scientific Inc., Rockford, State, U.S.A.).

Histopathological evaluation

All histologic analyses were performed in routinely processed formalin‐fixed, paraffin‐embedded tissue sections (3‐μm thick), which were stained with hematoxylin‐eosin and Masson's trichrome. For the evaluation of leukocyte infiltration, the widening of interstitial spaces, in addition to focal leukocyte infiltration, was assessed in each histology sample. The number of leukocytes per 0.28 mm² in 20 nonoverlapping microscopic fields (×400) was calculated. To estimate the degree of interstitial fibrosis, the interstitial area stained with Masson's trichrome was evaluated as a percentage of the area of the total examined area using image analyzer software (Nicon NIS‐Elements Documentation Software, Nicon Instruments Europe BV, Amsterdam, Netherlands). In each slide, the widening of interstitial spaces, focal leukocyte infiltration, and interstitial fibrosis were assessed under three to five high‐power fields (×400), and the results were quantitated. The fixed tissue segments were then embedded in paraffin according to standard procedures, and serial sections (5‐μm thick) were prepared.

Degenerative findings (e.g., cell swelling without cytoplasmic vacuolation and pyknosis of the nuclei) of the tubular epithelium and tubular dilatation in the groups were then semi‐quantitatively evaluated. Briefly, both were separately observed in 20 randomly chosen high‐power (×400) fields and graded from 0 to 5+, according to the area of alteration, with 0 denoting no lesions, 1+ denoting < 10%, 2+ denoting 10–25%, 3+ denoting 25–50%, 4+ denoting 50–75%, and 5+ denoting > 75% in each left kidney [17]. Additionally, desquamation and loss of tubular epithelium were also evaluated. For the quantification of the fibrotic areas, 20 high‐power fields in each left kidney, excluding glomeruli, were randomly selected, stained, and the fibrotic area was calculated using an image analysis system (Nicon NIS‐Elements Documentation Software, Nicon Instruments Europe BV, Amsterdam, Netherlands).

Immunohistochemical study

All the renal specimens were embedded in paraffin blocks and stored at 4°C. The specimens were later deparaffinized, rehydrated, and sectioned at 5 μm. The sections were microwave pretreated in 10 mM citrate buffer for 20 minutes, followed by cooling at room temperature for 20 minutes and washing with buffer solution for 10 minutes. Sections were incubated with a hydrogen peroxide block for 10 minutes and washed twice in PBS. Protein block were applied to sections and incubated for 10 minutes at room temperature to block nonspecific background staining and washed once in buffer. The expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) was evaluated using anti‐eNOS antibody (Abcam, ab66127) and anti‐iNOS antibody (Abcam, ab 15323) (1/100 dilutions, 1 hour at room temperature), respectively. Both the antibodies were rabbit polyclonal antibodies and the EXPOSE Rabbit specific HRP/DAB detection IHC kit (Abcam, ab94727) was used as secondary antibody kit. Sections were washed thrice in buffer and incubated in rabbit‐specific horseradish peroxidase conjugate for 15 minutes in room temperature. Tissues were rinsed four times in buffer, incubated in DAB solution for 5 minutes, and then washed. The sections were counter‐stained with hematoxylin for 1 minute and then rinsed in tap water. In each group, the expression of eNOS and iNOS was calculated by counting the number of positively stained cells in 20 randomly chosen high‐power (×400) fields.

Statistical analysis

All values were expressed as means ± standard error. The differences among the groups were compared by one‐way analysis of variance and post hoc Tukey tests. A p value of < 0.05 was considered to indicate statistical significance.

Results

Left hydroureteronephrosis was observed in Groups 2 and 3. Mortality was not observed either in the preoperative period or in the postoperative 21 days. Complications, such as infection, allergic reaction, urination problems, and urinary retention, were not observed in any of the groups. The levels of serum BUN and creatinine were increased in Groups 2 and 3 compared with Group 1 (p < 0.0001 and p < 0.0001, respectively). The serum levels of BUN and creatinine were reduced in Group 3 compared with Group 2 (p < 0.0001 and p < 0.0001, respectively), as shown in Figure 1A and B. Additionally, the level of urinary albumin was increased in the kidneys of Group 2 (p < 0.0001) but decreased in Group 3 (p < 0.0001). As shown in Figure 1C and D, the ratio of urinary protein/creatinine was also elevated in Group 2 (p < 0.0001) but decreased in Group 3 (p < 0.002). The MDA value was decreased in Group 3 (p<0.0001) compared with Group 2. In addition, SOD, CAT, and GSH‐Px levels were increased in Group 3 compared with Group 2 (p < 0.05, p < 0.0001, and p < 0.000, respectively), as shown in Table 1.

Figure 1.

(A) Serum creatinine, (B) plasma urea, (C) urinary albumin, and (D) urinary protein/creatinine ratio were measured to assess the renoprotective effect of nicorandil treatment following PUUO in groups. Group 1: Sham‐control; Group 2: PUUO‐untreated; Group 3: PUUO‐nicorandil treated. BUN = blood urea nitrogen; Cr = creatinine; PUUO = partial unilateral ureteral obstruction. *p < 0.0001 (Group 2 vs. Group 1); †p < 0.0001 (Group 2 vs. Group 3).

Table 1.

Effects of nicorandil treatment on antioxidant enzyme and lipid peroxidation in PUUO.

Groups	Antioxidant enzyme activities			Lipid peroxidation MDA (pmol/μg protein)
	GSH‐PX (mIU/μg protein)	SOD (pg/μg protein)	CAT (mIU/μg protein)
Sham‐control (Group 1, n = 10)	0.31 ± 0.002	0.30 ± 0.002	2.26 ± 0.010	5.1 ± 0.30
PUUO‐untreated (Group 2, n = 10)	0.28 ± 0.004*	0.27 ± 0.003*	2.08 ± 0.020*	12.9 ± 0.41
PUUO‐nicorandil (Group 3, n = 10)	0.36 ± 0.011†	0.34 ± 0.010†	2.65 ± 0.088†	7.6 ± 0.32

CAT = catalase; GSH‐Px = glutathione peroxidase; MDA = malondialdehyde; PUUO = partial unilateral ureteral obstruction; SOD = superoxide dismutase.

*Group 2 versus Group 1, p < 0.05; Group 2 versus Group 3, p < 0.0001.

†SOD, CAT, and GSH‐Px levels were increased in Group 3. Group 2 versus Group 3, p < 0.0001.

Group 2 versus Group 1, p < 0.05; Group 2 versus Group 3, p < 0.0001.

The MDA levels were decreased in Group 3. Group 2 versus Group 3, p < 0.0001.

The effects of nicorandil on the renal damage in the PUUO model are shown in Figure 2A–F. In Group 1, no histopathological changes were observed in the kidney (Figure 2D). In Group 2, desquamation, in addition to loss of varying degrees of tubular epithelium, was detected, accompanied by spindle‐ or star‐shaped mononuclear cellular infiltration in the interstitium, tubular dilation, peritubular congestion, and tubulointerstitial fibrosis (Figure 2E). Tubular dilatation and leukocyte infiltration were significantly reduced in Group 3. In particular, regenerative changes (nuclear enlargement) were also observed in the tubular epithelium of the renal cortical region (Figure 2F). Tubulointerstitial fibrosis was significantly lower in Group 3 than in Group 2, indicating the inhibitory effects of nicorandil on tubulointerstitial fibrosis (Figure 3A–C).

Figure 2.

The effects of nicorandil on renal damage in the PUUO model. (A), (B), and (C) upper row: H&E stain, magnification: ×40, bar 100 μm. (D), (E), and (F) lower row: H&E stain, magnification: ×200, bar 100 μm. (D) In the sham‐control group, no significant tissue change was seen in kidney. (E) In the PUUO‐untreated group, there were desquamation and loss of tubular epithelium (d) of varying degrees, several focal dense () and very small foci (►) leukocyte infiltration in the periglomerular and peritubular interstitium, tubular dilation (t), interstitial widening (i), and peritubular congestion (*). (F) PUUO‐nicorandil treated, tubular dilatation and leukocyte infiltration was significantly reduced. In particular, regenerative changes (nuclear enlargement) (►) were observed in the tubular epithelium of the renal cortical region. H&E = hematoxylin and eosin; PUUO = partial unilateral ureteral obstruction.

Figure 3.

Representative photomicrography of Masson's trichrome stain (bar 100 μm). (A) Sham‐control group. (B) There is an increase of collagen (blue staining, black arrows) within the glomerular and in the peritubular area in PUUO rat kidneys. (C) Nicorandil treatment significantly reduced the development of fibrosis. PUUO = partial unilateral ureteral obstruction.

The quantitative evaluation revealed significantly high levels of leukocyte infiltration, tubular dilatation, degeneration of tubular epithelium, and fibrosis in Group 2 compared with Groups 1 and 3 (p < 0.0001 and p < 0.0001, respectively), as shown in Table 2. In Group 3, leukocyte infiltration and tubular dilatation were significantly reduced (p < 0.05 and p < 0.0001, respectively) (Table 2). Other parameters, such as degeneration of tubular epithelium and fibrosis, also showed a marked improvement in Group 3 (p < 0.0001 and p < 0.0001, respectively).

Table 2.

Comparison of the groups according to histopathological parameters in PUUO.

Groups	Leukocyte infiltration	Tubular dilatation	Tubular epithelial degeneration	Fibrosis	iNOS	eNOS
Sham‐control (Group 1, n = 10)	0.1 ± 0.1	0.1 ± 0.1	0.1 ± 0.1	0.1 ± 0.1	3.2 ± 0.1	2.3 ± 0.1
PUUO‐Untreated (Group 2, n = 10)	1.6 ± 0.3*	2 ± 0.2*	1.5 ± 0.1*	1.3 ± 0.1*	7.6 ± 0.4	5.3 ± 0.2
PUUO‐nicorandil (Group 3, n = 10)	0.4 ± 0.2†	0.4 ± 0.1†	0.4 ± 0.1†	0.3 ± 0.1†	4.2 ± 0.1	8.1 ± 0.3

eNOS = endothelial nitric oxide synthase; iNOS = inducible nitric oxide synthase; PUUO = partial unilateral ureteral obstruction.

*Group 2 versus Group 1, p < 0.0001; Group 2 versus Group 3, p < 0.0001.

†Leukocyte infiltration, tubular dilatation, degeneration of tubular epithelium, and fibrosis were decreased in Group 3. Group 2 versus Group 3, p < 0.0001.

In Group 3, iNOS expression was decreased and eNOS expression was increased. Group 2 versus Group 3, p < 0.0001 & Group 3 versus Group 2, p < 0.0001.

In Group 2, iNOS expression was significantly elevated compared with that in Groups 1 and 3 (p < 0.0001 and p < 0.0001, respectively), as shown in Table 2 and Figure 4A–D. In contrast, in Group 3, the mean value of eNOS expression in tubular and glomerular cells was significantly increased compared with that in Groups 1 and 2 (p < 0.0001 and p < 0.0001, respectively), as shown in Table 2 and Figure 5A–C.

Figure 4.

The appearance of iNOS by immunohistochemical staining in the kidney tissue (bar 100 μm). (A) In the control group, mild staining in tubule cells surrounding the glomerulus. (B) and (C) In the PUUO rats, the intensity of staining apparent in tubular epithelial cells and in the collector tubule was higher. The presence of a large number of colored granules in tubular epithelial cell in the inner cortex (→). (D) Expression of iNOS decreased in the treatment group. ct = collector tubule; dt = distal tubule; iNOS = inducible nitric oxide synthase; pt = proximal tubule; PUUO = partial unilateral ureteral obstruction.

Figure 5.

(A) Endothelial nitric oxide synthase (eNOS) expression was found minimal in the sham‐control group and (B) eNOS expression in Group 2 were increased compared with those in Group 1. (C) However, eNOS expression in the nicorandil‐treated group was significantly higher in the glomeruli and tubules cells. Bar 100 μm.

Discussion

In the pediatric population, pelvic ureteral junction obstruction is a common cause of hydronephrosis, and the obstruction is mostly partial and congenital [18]. Unilateral ureteral obstructions are also common and usually reversible. Urinary tract obstructions can occur secondary to calculi, tumors, strictures, ureterovesical junction obstruction, ectopic ureters, ureteroceles, megaureters, and posterior urethral valves [19]. Despite advances in understanding pathogenesis, knowledge on the natural history of congenital hydronephrosis has been still incompletely described [20]. Nonetheless, obstructive uropathy is known to lead to functional and morphological changes in the kidney. If the ureteral obstruction persists, tubular atrophy, interstitial fibrosis, ischemia, and necrosis may occur [8]. As mentioned above, urinary tract obstruction in children is almost always partial in nature; therefore, in this experimental study, we used a partial ureteric obstruction model in which the ureter was placed into the psoas muscle groove to investigate the effects of hydronephrosis secondary to PUUO. This method was used in the dog by Ulm and Miller [21] first time. In the present study, obvious dilatation of the renal pelvis and proximal ureter and deterioration in histopathological parameters were apparent in all the rats 3 weeks after PUUO as observed in other studies using the same method in rats [[6], [8], [10], [22]]. Therefore, we also think that this method is easy, practical, and suitable for creating partial ureteral obstruction in rats. The PUUO groups also showed a significant increase in creatinine and BUN levels, reflecting decreased renal function. Nicorandil treatment hindered the progression of PUUO‐induced changes in histopathology, renal function, and oxidative stress parameters. Thus, nicorandil appears to be useful in the prevention renal damage caused by PUUO.

In the pathogenesis of urinary tract obstructions, recent studies asserted that vasoconstriction and decreased renal blood flow, in addition to pressure increases and ischemic atrophy, were important factors [[19], [23]]. ROS (e.g., superoxide, hydrogen peroxide, and hydroxyl radicals) are intermediary substances that are normally produced during oxygen metabolism. Research showed that diminished antioxidant expression and increased levels of ROS in obstructed kidneys were primarily responsible for tubulointerstitial injury and fibrogenesis [24]. ROS have been shown to damage proteins, lipids, nucleic acids, carbohydrates, and other molecules, resulting in the development of inflammation, apoptosis, fibrosis, and cell proliferation [9]. In addition, research demonstrated that ROS caused severe injury of the cell membrane by lipid peroxidation reactions and contributed to tubulointerstitial damage and fibrosis in rat models of PUUO. MDA is reportedly a reliable indicator of ROS‐induced ischemia–reperfusion tissue damage [[9], [25]]. Yasar et al [8] demonstrated that serum and tissue levels of MDA and nitric oxide (NO) were significantly increased in a rat model of PUUO compared with a control group. Demirbilek et al [26] showed that oxidative stress products, antioxidants, and lipid peroxidation levels in a PUUO group were significantly different from those of a control group. Kawada et al [27] studied Nepsilon‐carboxymethyl‐lysine, an integrative biomarker of accumulated oxidative stress, in kidneys after unilateral ureteral obstruction. Their study suggested that suppression of renal antioxidant enzymes in the setting of ureteral obstruction contributed to the progression of renal fibrosis in the interstitium of the kidney. Other authors have reported similar findings in studies of rat models of PUUO [[28], [29]]. The present study showed that MDA levels increased and those of SOD, CAT, and GSH‐Px decreased in obstructed kidneys in response to PUUO. Nicorandil administration significantly reduced the elevation in MDA levels, resulting in an increase in SOD, CAT, and GSH‐Px activities in the PUUO group. These results indicated that oxidative stress played an important role in renal damage in a PUUO animal model and that nicorandil treatment protected the renal tissue against PUUO‐induced oxidative stress and inflammatory responses.

The renoprotective effects of nicorandil have been reported in several models, such as renal ischemia–reperfusion, diabetic nephropathy, and remnant kidney [30]. Previous research reported that nicorandil acted as both a nitrovasodilator and potassium channel opener. Furthermore, research suggested that nicorandil might act as a direct scavenger of hydroxyl radicals and inhibitor of free radical production in both human and canine leukocytes. Additional research indicated that it might function as an antioxidant via the formation of NO, which can interfere with free radical production, or other mechanisms [31].

Nitric oxide synthase, an enzyme responsible for the production of NO, has three major isoforms, namely, neuronal, eNOS, and iNOS. Although eNOS is calcium dependent and expressed in many tissues, iNOS is calcium independent and induced in tissues after exposure to inflammatory cytokines or ischemia [32]. Some in vivo studies suggested that nicorandil increased endothelium‐derived NO production by activation of eNOS, inhibited endothelial cell death, and exerted anti‐inflammatory and antioxidative effects [[33], [34]]. In addition, nicorandil was shown to activate cytoplasmic guanylate cyclase, leading to an increase in cellular levels of cyclic guanosine monophosphate, a reduction in cytosolic calcium, and thus the relaxation of vascular smooth muscle [35]. Tashiro et al [30] demonstrated that nicorandil not only increased total eNOS expression but also the state of eNOS coupling in high‐salt‐loaded Dahl salt‐sensitive hypertensive rats. They concluded that the increase in eNOS activity may contribute to the renoprotective effect of nicorandil. However, NO synthesized by iNOS under pathological conditions was shown to be toxic [36]. In general, iNOS is activated in response to infection and inflammation as a part of the defense response, and its expression is minimal under physiological conditions. When iNOS is upregulated in response to proinflammatory cytokines, it generates 100–1000‐fold more NO than eNOS. Excessive NO production can exert detrimental effects on cardiovascular function. The activation of iNOS within vascular smooth muscle cells is a major cause of hypotension in septic shock [37]. In the present study, the mean value of eNOS expression in tubular and glomerular cells was significantly increased in the PUUO nicorandil‐treated group compared with the PUUO‐untreated group. Additionally, nicorandil treatment decreased iNOS expression. The decrease in the iNOS expression in Group 3 may be due to antioxidant and anti‐inflammatory effects of nicorandil. As stated in the study of Tashiro et al [30], nicorandil increases eNOS expression, and the increase in eNOS activity may also contribute to the renoprotective effect of nicorandil.

Previous studies reported that nicorandil could potently reduce urinary albumin excretion in diabetic eNOS‐deficient mice and proteinuria in hypertensive subjects, thereby blocking albuminuria [[38], [39], [40]]. In the present study, the level of urinary albumin was increased in the kidneys exposed to PUUO but decreased by nicorandil treatment. Additionally, the ratio of urinary protein/creatinine was further increased by PUUO but decreased by nicorandil treatment. Tamura et al [40] suggested that nicorandil is a podocyte protectant that preserves podocyte number and reduces urine albumin excretion in a rat model of chronic kidney disease. The protective mechanism involves a reduction in oxidative stress, likely stimulated via ATP‐sensitive potassium channel.

The lack of cardiovascular data and a study group of nicorandil without PUUO were considered as limitations of the current study. We believe that an integrated assessment can be made with further studies that include these limitations.

In conclusion, the present study is the first to evaluate the potential protective effects of nicorandil against PUUO‐induced oxidative stress in rat kidneys. The findings suggest that the administration of nicorandil may attenuate the effects of oxidative stress through an increase in serum antioxidant enzyme activities, reduction in lipid peroxidation, and changes in concentrations of NOS in renal tubular tissues after PUUO. In addition, it can be speculated that nicorandil may be used clinically as an antioxidant agent in ureteral obstruction to minimize or prevent parenchymal damage in the kidney.