Ahmed A. Shokeir, Professor of Urology, Vice Director of Urology and Nephrology Center, Mansoura University, 35516, Mansoura, Egypt. e-mail: email@example.com
What's known on the subject? and What does the study add?
It is known that the kidney damage continues even after release of ureteric obstruction. This study found that giving ferulic acid, antioxidant, after release of ureteric obstruction enhanced the recovery of kidney functions in solitary kidney.
• To evaluate the effect of ferulic acid (FA) on the recovery of renal function and renal damage after relief of partial ureteric obstruction (PUO) of a solitary kidney.
• Male mongrel dogs (n= 32) were classified into three groups: sham (eight), control (12) and study (12).
• A right nephrectomy was carried out and dogs in the study and control groups were subjected to 4 weeks of PUO.
• Serum creatinine, creatinine clearance (CrCl) and renographic clearance (RC) were measured at baseline, after 4 weeks of obstruction and 8 weeks after relief of obstruction.
• Markers of lipid peroxidation (malondialdehyde [MDA]), superoxide dismutase (SOD), and reduced glutathione (GSH), and immunostaining of markers of apoptosis (caspase 3 and Bcl2), cell proliferation (Ki67) and interstitial fibrosis in the kidney were evaluated at the end of experiment.
• Ferulic acid enhanced the recovery of serum creatinine, CrCl and RC by an extra 22%, 26% and 33.7%, respectively, of the basal values at 8 weeks, after relief of 4 weeks' obstruction.
• In addition, FA caused a significant decrease in MDA and a significant increase in GSH and SOD.
• Ferulic acid also significantly reduced the interstitial fibrosis, and caspase 3 expression, and significantly increased the expression of Bcl2 and Ki67 in kidney tissues at 8 weeks after relief of the obstruction.
• Ferulic acid enhances the recoverability of renal function and minimizes the renal damage through reduction of oxidative stress, tubular apoptosis and the interstitial fibrosis in the solitary kidney after relief of PUO.
Obstructive uropathy with resultant hydronephrosis is the eventual outcome of many urological disorders. It triggers tubular cell death by apoptosis and necrosis , interstitial inflammatory infiltration  and progressive fibrosis with loss of renal parenchyma, myofibroblast activation and extracellular matrix deposition [3,4]. Although, the definite treatment of obstructive uropathy is the relief of obstruction, some experimental studies have shown that the harmful effect of obstruction continue even after relief of the obstruction . Studies have examined the effects of relief of ureteric obstruction (UO) on the histopathological changes that accompany UO. Continuous progression of renal injuries, including interstitial fibrosis and tubular apoptosis, has been found in the previously obstructed kidney (POK) of neonatal and adult rats after relief of unilateral UO (UUO) [6–8]. Thus, relief of UUO by itself might not be sufficient to improve the function and histopathology of the kidney, but could require a concurrent pharmacological intervention. Effective intervention could minimize irreversible renal injury.
A few studies have investigated the effect of certain pharmacological agents, such as L-arginine, losartan and enalapril, on the recoverability of kidney functions after release of UUO [9–11]. However, these agents were given during the period of obstruction and continued after its relief [9–11]. Therefore, the improvement obtained at the end of the experiment could not be attributed solely to the effect of the agent on enhancement of recoverability of renal functions after relief of obstruction because the protective effect against obstruction could not be excluded. Moreover, these studies used animal models of UUO in the presence of contralateral normal kidney. This could cause difficulty in the estimation of renal functions in view of the compensatory hypertrophy that always occurs in the contralateral kidney in response to UUO of the corresponding one. These limitations were avoided in the present study by changing the model to one with solitary kidney and giving the agent under investigation after relief of obstruction.
Oxidative stress and reactive oxygen species (ROS) play a significant role in renal tubular and interstitial injury resulting from UUO , and causes overexpression of fibrogenic cytokines such as TGF-β and chemoattractants after relief of obstruction , which leads to interstitial fibrosis. Previous studies have shown the benefits of using antioxidants as renoprotective agents against renal injury during UUO [14–16]. Ferulic acid (FA) is a phytochemical compound that exhibits a wide range of therapeutic effects against various diseases, such as cancer, diabetes, cardiovascular and neurodegenerative disorders. FA is an scavenger of free radicals that effectively scavenges superoxide anion radicals and inhibits the lipid peroxidation . Moreover, Khanduja et al.  have recently reported that phenolic compounds like FA exhibit significant anti-apoptotic activity in normal peripheral blood mesenchymal cells (PBMCs) exposed to H2O2-induced oxidative stress. Nevertheless, to the best of our knowledge, no studies investigated the effect of FA on obstructive uropathy as well as its effect on the recoverability of renal functions after relief of obstruction. In the present study, we examined the effect of FA on the recoverability of renal functions after relief of ureteric obstruction in a canine model with a solitary kidney. In addition, we examined the effect of FA on interstitial fibrosis, tubular apoptosis, oxidative state and cellular proliferation in the kidney tissues after release of the obstruction in both a control and study groups.
MATERIALS AND METHODS
In all, 32 male mongrel dogs aged 2–3 years and weighing 18–25 kg were involved in the present study. The dogs were randomly divided into three groups: a sham group: eight dogs, right nephrectomy + left sham surgery + no medications; a control group: 12 dogs, right nephrectomy + left partial ureteric obstruction (PUO) + no medications; and a study (FA) group: 12 dogs, right nephrectomy + PUO + FA. The dogs were housed in individual boxes under habitual conditions in a temperature-controlled room (24°C). They received a balanced diet plus free access to water. Experiments were performed according to the Guide for the Care and Use of Laboratory Animals. All protocols were approved by our local Animal Care and Use Committee.
Dogs were anaesthetized by thiopental sodium (10 mg/kg) with endotracheal intubation and mechanical ventilation. Right nephrectomy was carried out. The model of unilateral left PUO was performed as described by Shokeir . The study and control groups were subjected to 4 weeks of left PUO. These were then reopened and subjected to Lich–Grigoir vesico-ureteric reimplantation. All dogs of the control and study groups were killed by the end of the eighth week after relief of obstruction.
For sham-operated animals, the abdomen was entered with a midline incision and right nephrectomy carried out. The bladder was opened, a 6 F ureteric catheter was inserted into the left ureteric orifice for 2 h for collection of urine samples and a blood sample was taken from the left renal vein. The catheter was then removed and the bladder and wound closed without induction of left PUO. Dogs in the sham group were subjected to sham surgery at basal condition, 4 and 8 weeks and killed thereafter.
Dogs of the study group were given FA (purchased from Sigma, St Louis, MO, USA) in their drinking water (or milk or bone soup) at a dose 70 mg/kg per day with the onset of relief of the 4-week obstruction and continued until they were killed.
For renal function measurements, blood and urine samples were collected from the corresponding kidney during surgery just before induction of obstruction, during relief of obstruction (at 4 weeks of obstruction) and at sacrifice of the dogs at the end of the eighth week after relief of obstruction. Two urine samples were collected, each for 2 h from the corresponding ureter and blood samples were obtained from the corresponding renal vein. Blood samples and the mean of two readings of urine samples were used for calculation of the creatinine clearance (CrCl) using the following equation :
Doppler ultrasonography with measurement of renal resistive index (RI) of the corresponding kidney were carried out in the basal condition before induction of obstruction, just before relief of obstruction (at the fourth week of obstruction) and at 4 and 8 weeks after relief of obstruction. Doppler ultrasonography was done by single expert radiologist, as previously described .
Radioisotope renography with calculation of the split function of the corresponding kidney was performed. Renograms were taken in the basal condition before induction of obstruction, just before relief of obstruction (at the fourth week of obstruction) and at 4 and 8 weeks after relief of obstruction, as previously described .
For morphometric evaluation of renal interstitial fibrosis, kidney tissues for the histological study were fixed in 10% formalin (pH 7.4) and embedded in paraffin. Sections (3 µm thick) were prepared, and stained with haematoxylin & eosin (H&E) and Masson's trichrome to evaluate the fibrosis of cortical interstitium. The sections were observed on an Olympus BX51 light microscope. Pictures were obtained by a PC-driven digital camera (Olympus E-620). The computer software (Cell*, Olympus Soft Imaging Solution GmbH) allowed morphometric analysis to be performed. The interstitial volume index was determined as previously described  by superposing a grid containing 100 (10 × 10) sampling points on pictures of 10 non-overlapping fields (×200) of Masson's trichrome-stained sections. The number of points overlying interstitial space were counted and expressed as a percentage of the total. Large arteries and glomeruli are excluded from the quantification.
For immunohistochemistry to assess the apoptotic index, anti-apoptotic activity and proliferative index, 3-µm-thick sections were prepared on coated slides and deparaffinized. All sections were incubated for 30 min with 0.3% hydrogen peroxide in methanol and microwave-heated in 10 mM citrate buffer, pH 6.0, for 10–20 min. Subsequently, an indirect immunoperoxidase technique was applied, using monoclonal antibodies for: anti-caspase 3 (Abcam, catalogue number: ab79123) cytoplasmic staining with human tonsils as positive control; anti-Bcl2 (Abcam, catalogue number: ab59348) cytoplasmic staining with human colon carcinoma tissue as positive control; anti-K67 (Abcam Cat.# ab86373) nuclear staining with human lymph node as positive control. Indirect immunoperoxidase was performed using ImmunoPure Ultra-Sensitive ABC Peroxidase (Thermo Scientific, catalogue number: 32052) with (DAB) as chromogen.
The apoptotic index and anti-apoptotic activity were assessed with a standard point-counting method for the percentage of labelled tubular cells in each of the 10 examined, non-overlapping, randomly selected ×400 fields of each slide. Labelling indices were expressed as the mean scores of the10 fields . The proliferation index was defined as the percentage of the counted immunoreactive nuclei per at least 1000 tubular cells .
To estimate the oxidative and antioxidative variables, kidney tissue was perfused with a PBS solution, pH 7.4, containing 0.16 mg/mL heparin to remove any red blood cells and clots. The kidney was then weighed, minced, homogenized in 5–10 mL cold buffer (i.e. 50 mM potassium phosphate, pH 7.5. 1 mM EDTA). Homogenates were centrifuged at 10 000g for 15 min at 4 °C and the supernatant was kept at −80°C until it was used for analysis of lipid peroxides (malondialdehyde, MDA), superoxide dismutase (SOD) and reduced glutathione (GSH). MDA, SOD and GSH were measured using a colorimetric kit (Bio-Diagnostics, Dokki, Giza, Egypt) according to the manufacturer's instructions.
For the statistical analysis, the data of dogs of the three groups were compared at different time points of assessment. Statistical analyses were carried out with the two-tailed Student's t-test and anova. P < 0.05 was considered to indicate statistical significance.
All animals survived the experiments without complications for sham surgery, after induction of PUO or after vesico-ureteric reimplantation. The non-obstructed pattern of the isotope renographic curve and the half time drainage (T1/2) <10 min showed the relief of obstruction after vesico-ureteric reimplantation in all dogs of the control and study groups.
At the basal conditions, the mean values of serum creatinine, CrCl and RC were similar among dogs of the three groups. The sham-operated group showed stable values of the three variables for the whole duration of the study. By the end of the fourth week of obstruction, control and study (FA) groups showed a significant increase in serum creatinine, a decrease in CrCl and a decrease of RC (Table 1). There were no significant differences in the mean values of the three variables among the FA and control groups (Table 1).
Table 1. Percentage recovery of renal function 8 weeks after relief of obstruction in the control and FA groups
a, significant with sham; b, significant with control.
All data are expressed as means ±sd. P= statistical significance among different groups (one-way anova with post-hoc Scheffe's test).
By the end of eighth week after relief of obstruction, there was a significant improvement of the variables in both the study and control groups compared with values before relief of obstruction (Table 1). The mean serum creatinine of the FA group was significantly lower, while CrCl and RC were significantly higher in the FA group than in controls. The ability of the kidney to regain its functions was significantly better in the FA group than in other groups (Table 1). Compared with the control group, FA enhanced the recovery of serum creatinine, CrCl and RC by an additional 22%, 26%, and 33.7% of the values at the fourth week of obstruction, respectively (Table 1).
There was no significant difference in the mean RI of the left kidney of the sham, control and FA groups in the basal condition (Table 1). The mean RI of the sham-operated group remained stable for the study duration (Table 1). After 4 weeks of obstruction, there was a significant rise in mean renal RI in the control and FA groups compared with basal values (P < 0.001 and P < 0.001, respectively). By the end of the fourth week of obstruction, there was no significant difference in the mean RI between the FA group and controls. There was a marked drop in the RI to near basal values at 4 weeks after relief of obstruction in both the control and FA groups. Follow-up of RI at 8 weeks after relief of obstruction showed almost stable values, similar to those at 4 weeks after relief of obstruction in both the control and FA groups (Table 1).
Compared with the sham-operated animals, the harvested kidneys of the control group exhibited a marked interstitial fibrosis, mostly in perivascular and intertubular areas, indicated by a positive blue colour in Masson's trichrome-stained sections. By contrast, harvested kidneys from FA-treated animals had significantly less sclerotic damage. The mean percentage of fibrosis in the FA-treated animals was significantly lower than in the control group (Table 2). FA reduced interstitial fibrosis by 34.81% in comparison with controls. Figure 1A represents one of the control group animals, while Fig. 1B represents one of the FA-treated animals.
Table 2. Score of interstitial fibrosis (%), Ki67 expression, Bcl2 expression and apoptosis (caspase 3) expression in different groups at the end of study
% Interstitial fibrosis
Apoptosis (caspase-positive cells)
a, significant with sham; b; significant with control.
All data are expressed as means ±sd. One-way anova with post-hoc Scheffe's test.
Percentage change of the mean FA compared with control group
There was significantly lower tubular apoptosis, higher expression of Bcl2, and higher expression of Ki67 in the FA group than in the control (Table 2). FA reduced apoptosis by 50.55%, increased expression of Bcl2 by 218.89%, and increased expression of Ki67 by 69.68% compared with the control group (Table 2). Figures 2A,B, 3A,B, and 4A,B are examples of the tubular apoptosis indices, Bcl2 and Ki67, expression in the control and FA groups, respectively.
In the assay of oxidants and antioxidants, there was significantly lower MDA, higher GSH and higher SOD in the FA group than in the control group (Table 3). FA reduced MDA by 64.43%, increased GSH by 165.57%, and increased SOD by 84.88% compared with the control group (Table 3).
Table 3. Markers of oxidative stress (MDA) and antioxidants (GSH and SOD) in different groups at the end of study
MDA, mmol/g tissue
GSH, mg/g tissue
SOD, % inhibition
a, significant with sham; b; significant with control.
All data are means ±sd. One-way anova with post-hoc Scheffe's test.
Percentage change of the mean in FA compared with control group
Partial ureteric obstruction is a frequent clinical finding in patients with ureteric stones under medical treatment, those passing stone fragments after ESWL, those awaiting treatment and in some patients with an equivocal diagnosis of PUO. The definite treatment for obstructive uropathy is the relief of obstruction, but some studies have shown that the harmful effects of renal obstruction might continue even after its relief . Therefore, a search for drugs that can enhance the recoverability of renal function after relieving UO would be invaluable, particularly in a solitary kidney. The present study is a controlled experiment aimed at evaluating the role of a new antioxidant (FA) in enhancing the recoverability of renal functions after relief of obstruction and investigating its effect on the markers of progressive renal damage in a solitary kidney. The present study, for the first time, demonstrated that at 8 weeks after relief of a 4 week-obstruction, FA enhanced recovery of the renal functions, through reduction of the renal interstitial fibrosis, tubular cell proliferation, the tubular apoptotic cell death and markers of oxidative stress.
Many studies have investigated the recovery of the kidney at the level of renal functions and/or renal damage [6,7,9–11,26,27]. Recently Soliman et al. [10,11] in a UUO model in dogs for 4 weeks, showed recovery of renal functions by ≈51% of baseline values at 8 weeks after relief of the 4-week UO. The present study showed recovery of the renal functions of solitary kidney by 40% at 8 weeks after relief of 4 weeks of UO. Previous studies have investigated the recovery of renal functions in models of UUO in the presence of a contralateral kidney. Compensatory hypertrophy of the contralateral kidney could cause difficulty in estimating renal functions. In the present experiment we examined the recovery of renal functions in a UO model of a solitary kidney, which gives a more accurate estimation of the functions of the corresponding kidney than in previous studies.
The renal damage after relief of UO was examined by several investigators [6,7,9]. In a neonatal rat model, at 28 days after relief of a 5-day UUO, Chevalier et al.  showed a dramatic rise in interstitial collagen, α-smooth muscle actin and TGF-β1 in the POK compared with sham-operated rats. In a similar study concerning an adult rat model of UUO for 3 days, Ito et al.  showed that the interstitial fibrosis was strongest at 28 days after relief of UO; the concentration of TGF-β was increased in POK 3 days after relief of UO, with apoptosis still evident at 28 days after relief of UO. In the present study, we examined several markers of renal damage after relief of UO. We found that renal interstitial fibrosis was strong at 8 weeks after relief of UO. Tubular cell apoptosis was still evident at 8 weeks after UO release. Furthermore, tubular cell proliferation was significantly increased in obstructed kidney compared with the sham group. Finally, the markers of oxidative stress were disturbed in the corresponding kidney, with a marked increase in MDA (marker of lipid peroxidation) and a significant reduction in the endogenous antioxidants such as SOD and GSH. This finding suggests the presence of an oxidative stress state in the corresponding kidney even 8 weeks after relief of UO.
Oxidative stress plays an important role in pathogenesis of tubulo-interstitial inflammation during obstructive nephropathy  and after relief of obstruction . Manucha et al.  and Sugiyama et al.  demonstrated an increase in the concentration of reactive oxygen species in obstructed kidney, together with decreased activities of the major protective antioxidant enzymes SOD, catalase and glutathione peroxidase. The down-regulation of antioxidant enzymes catalase, glutathione peroxidase and SOD from tubular cells from the obstructed kidney increases the vulnerability of the kidney to oxidative damage . The results of our study are consistent with the findings of previous studies. Localized oxidative stress could be cytotoxic to renal tubular epithelial cells [33,34], resulting in increased apoptosis . There is also some indication that oxidative stress plays a role in TGF-β1 expression and in production of extracellular matrix (ECM) in tubulo-interstitial fibrosis .
Many studies have investigated the use of antioxidants as renoprotective agents against structural and functional damage induced by UO [14,15,37]. Pat et al.  used vitamin E and N-acetylcysteine or fluvastatin as renoprotective antioxidant therapy. They found that both fluvastatin and N-acetylcysteine attenuated fibrosis (decreased α-SMA [Smooth Muscle Actin], fibronectin, tubular epithelial apoptosis) and fluvastatin significantly decreased expression of antioxidant enzymes after relief of UUO. Notably, all of the previous studies investigated the protective effect of antioxidant therapy against renal damage induced by UO. Nevertheless, none of these studies investigated the effect of antioxidants on the recovery of renal functions after relief of obstruction.
Pharmacological intervention during the recovery period is not well investigated. Few studies only examined the effect of some medications on renal functions and damage after relief of UO. Ito et al.  found, in 3-day rat model of UUO, that dietary arginine supplementation during and after relief of UO did not improve GFR and effective renal plasma flow. However, renal damage, including fibrosis, apoptosis and macrophage infiltration, was significantly improved by L-arginine treatment. Recently, Soliman et al.  showed, in a 4-week PUO canine model, that losartan (angiotensin II receptor 1 blocker) prevented reduction in CrCl by 11% and RC by 20% of the basal values by the end of the fourth week of obstruction. Moreover, losartan enhanced recovery of CrCl by 26% and RC by 26% of the basal values at 32 weeks after relief of UO obstruction. Soliman et al.  tried enalapril (angiotensin II receptor 1 blocker) on the recoverability of renal functions after PUO. They found that enalapril offset the reductions of CrCl and RC by an extra 11% and 12% of the basal values by the end of the fourth week of obstruction. Moreover, enalapril enhanced the recovery of CrCl by an extra 10% and of RC by an extra 23% of the basal values at 8 weeks after relief of the 4-week UO. Although these studies investigated the effect of some medications on the recovery of renal functions in UO, they started treatment early on during the obstruction, so it is not known whether their effects are due to their renoprotective effects or their enhancing effects on the recoverability of kidney functions after relief of UO.
Ferulic acid is a phytochemical compound commonly found in fruits and vegetables such as tomatoes, sweet corn and rice bran . The wide spectrum of beneficial effects of this phenolic compound have been advocated, at least in part, because of its strong antioxidant activity. FA, besides its strong antioxidant activity, exhibits anti-apoptotic effects in blood mesenchymal cells . In the present study, treatment with FA for 8 weeks after relief of 4 weeks of UO of a solitary kidney enhanced the recovery of renal functions as indicated by significant improvement of serum creatinine, CrCl and RC in the FA-treated group. FA also attenuated the renal damage induced by 4 weeks of UO. This is indicated by a significant reduction of interstitial fibrosis and tubular cell apoptosis in the FA-treated group. Moreover, FA enhanced tubular cell proliferation and anti-apoptotic markers. At the same time, FA attenuated the oxidative stress in obstructed solitary kidney as indicated by significant reduction in the lipid peroxidation product, MDA, and enhanced the endogenous antioxidants SOD and GSH. Although, the antioxidant effect of FA confirmed its pharmaceutical effect, it is better to measure the serum concentration of FA. Assay of serum FA was difficult because high-performance liquid chromatography is not available at our centre. We therefore consider this issue as one of the limitations of our study.
At the level of renal functions, the findings of the present study are in agreement with those of Soliman et al. [10,11], who used losartan and enalapril; however, the results of the present study are more conclusive because we used an animal model with a solitary kidney and administered FA after the relief of UO. As the degree of recovery of the damaged kidney is influenced by the functional status of the contralateral one, in the present study the role of the contralateral kidney in recovery of the obstructed kidney was avoided by using a solitary kidney model.
In conclusion, FA enhances the recoverability of renal function after relief of PUO in a solitary kidney. This effect could be due to its antioxidant, anti-apoptotic, antifibrotic and proliferative properties. Other studies should be conducted to consolidate the results of the present study before recommending the use of FA in clinical practice for enhancing the recoverability of renal functions after relief of obstruction.
CONFLICT OF INTEREST
None declared. Source of funding: This work was funded through the Science and Technology Development Fund (STDF), Egypt-grant no. 996.