Effects of Losartan on Cell Apoptosis and Expression of Caspase-3 and JNK Proteins in Kidney Tissue in Renal Interstitial Fibrosis Rats

Lu BIAN1, Chunhua LUO2*, Weifeng FENG

1. College of Traditional Chinese Medicine, Jinan University, Guangzhou 510632, China; 2. Newborn Intensive Care Unit, Guangzhou Women and Children’s Medical Center, Guangzhou 510623, China; 3. Department of Traditional Chinese Medicine, First Affiliated Hospital, Jinan University, Guangzhou 510632, China

Abstract [Objectives] To investigate the effects of losartan on cell apoptosis and the expression of caspase-3 and JNK proteins in kidney tissue in the adenine-induced renal fibrosis rats. [Methods] Thirty Wistar rats were randomly divided into three groups: control group (n=10), model group (n=10) and losartan group (n=10). The rats in the control group received saline, while those in the model group and losartan group both received adenine by gavage, for 21 d. After the renal interstitial fibrosis model was established, the rats in the losartan group were treated with losartan[10 mg/(kg·d)], while the rats in the control group and the model group rats were administered with the same amount of saline. The course of treatment was 30 d. Finally, the renal function, blood urea nitrogen (BUN), serum creatinine (Scr), creatinine clearance rate (Ccr) and the pathological morphology of the rats were detected. The apoptosis of renal tubular epithelial cells was tested by TUNEL. The caspase-3 and JNK protein expression was tested by Western blotting. [Results] After administering adenine for 21 d, the BUN, 24MTP and kidney/body weight in the model group were increased, significantly higher than the control group (P<0.01), and the Ccr was remarkably decreased (P<0.01), signifying that the renal interstitial fibrosis model was successfully built. After treating with losartan for 30 d, the Scr, BUN, and 24MTP were significantly decreased (P<0.01), and the Ccr was significantly increased in the losartan group (P<0.01). In addition, in comparison to the model group, renal tubular epithelial apoptosis was decreased and caspase-3 and JNK expression was downregulated in the losartan group (P<0.05). [Conclusions] Losartan can reduce the adenine-induced elevation of Scr, BUN and 24hMPT, increase Ccr, improve general condition of renal interstitial fibrosis in rats and ameliorate the progression of chronic kidney failure (CKD). The effectiveness of losartan is probably due to its ability to regulate caspase-3, JNK protein expression and attenuate renal cell apoptosis.

Key words Losartan, Renal interstitial fibrosis, Cell apoptosis, Caspase-3, JNK

1 Introduction

Renal interstitial fibrosis (RIF) is the excessive deposition of extracellular matrix (ECM) in the renal interstitium due to various reasons[1], and is a common occurrence in a variety of chronic kidney diseases. The incidence mechanism involves interstitial fibroblast activation, apoptosis and phenotypic transformation of the renal tubular epithelial cells, extracellular matrix transformation imbalance and a series of relevant processes. The severity of RIF does not only affect the renal function, but also have a close relation with the prognosis of renal disease[2].

Losartan is non-peptide vascular angiotensin II (Ang II) receptor antagonist that has been listed and used in clinics since 1994. It is a new type of antihypertensive drug, and works through being a competitive antagonist for AngII type 1 receptors (AT1), thus blocking the Ang II-induced pathological effect. At present, ARBs is the first-line treatment used for patients with hypertension and nephropathy in the clinic[3-4]. The AngII type 1 receptor (AT1R) finds expression in kidney[5], and plays a key role in the renal function, the long-term blood pressure regulation and the progression of disease[6].

In this study, after inducing renal interstitial fibrosis with adenine in the rat model, the rats were administered with losartan, while concurrently establishing a normal group and a model control group. The rats were examined for 24-h urine volume, 24-h urine protein, serum creatinine, urea nitrogen and the glomerular filtration rate. In addition, the pathological changes of the renal tissue were observed through pathological sections, the apoptotic cells were stained with TUNEL assay, and the tissue protein levels of caspase 3 and JNK were also detected. Overall, this study was devised in order to observe the effects of losartan on renal function and its possible mechanism of action.

2 Materials and methods

2.1 Experimental materialsThe Losartan Potassium Tablets (100 mg) were produced by the Hangzhou MSD Pharmaceutical Co. Ltd. The Adenine (C5H5N5) was produced by the BBI Life Science Corporation in Canada. The protein extraction, quantification kit, and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis kit were purchased from the Nanjing KeyGen Biotech Co., Ltd. The JNK and caspase-3 monoclonal antibodies were purchased from the Cell Signaling Technology Inc.

2.2 Animal grouping and modelingPrior to experiment, 30 male Wistar rats of SPF grade were purchased at the Experimental Animal Center of the Southern Medical University. They were weighing approximately 160-180 g, and were aged around 6 to 8 weeks. The rats were reared in the animal room of the School of Medicine, Jinan University, in a single cage, and freely provided with food and water for 1 week.

The rats were then randomly divided into a control group (n=10) and a model group (n=20). For establishing a reference model[7], the model group was provided with freshly dissolved saline in physiological saline at concentration of 2.5%, maintaining a dosage of 250 mg/(kg·d) of drug content. The molding cycle was 21 d. On the 22nd d, blood was collected from the inner canthus. A 24-h urine collection tested the serum creatinine (Scr) and blood urea nitrogen (BUN). By measuring the 24-h urinary protein excretion, serum creatinine, urea nitrogen, it became possible to calculate the glomerular filtration rate and prove that the model was successfully reproduced (3 rats died during the molding process in the normal group).

2.3 Drug treatmentAfter the modeling success, the normal group received physiological saline, and the rats were randomly divided into a model group (n=10) and a losartan group (n=10). The losartan group was given 10 mg/kg per day via gavage. The duration of the drug intervention was 30 d (4 rats in the losartan group, and 3 rats in the model control group died during the course of the treatment).

2.4 Specimen collectionAfter the 30-d drug intervention, for the first 24 h, the cages were weighed, the metabolism rate was recorded, and also the urine was collected. On the second day, anesthesia was produced via intraperitoneal injection of sodium pentobarbital (45 mg/kg). Subsequently, 3 mL of blood was collected from the abdominal aorta of each rat without anticoagulant. After standing for 40 min, the blood samples were centrifuged for 20 min at 4 000 rpm. Then, the separated serum was stored in Eppendorf tubes for later measurement of the kidney function and electrolytes. Meanwhile, the kidneys were quickly removed, decapsulated, washed with saline at 4 ℃, and weighed. One-fourth of the left kidney was fixed with 4% paraformaldehyde. The right kidney and three-fourths of the left kidney were stored at -80 ℃ for later use.

2.5 Detection of biochemical indexesAfter the collection of serum and the 24-h urine sample, the materials were immediately sent to the inspection center of the First Affiliated Hospital of Jinan University, to determine the levels of urine protein, urine creatinine, serum creatinine (Scr) and blood urea nitrogen (BUN). Then, the creatinine clearance rate (eGFR) was calculated according to the following formula:

Ccr=24-h urinary creatinine × 24-h urine volume/Creatinine concentration

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2.6 Detection of apoptosis by HE, Masson staining and TUNEL assayFor the HE, Masson staining, tissue fixation, dehydration, paraffin embedding and slicing were performed in success. Apoptotic cells were stained according to the TUNEL kit operation steps, observed under the microscope light, photographed with the camera, counted using computer image analysis software, and finally analyzed statistically.

2.7 Protein detectionRenal tissue was homogenized for extraction of protein, and the protein concentration was determined by BCA. A certain amount (30 μg) of total protein was taken together with the buffer sample for protein electrophoresis. Then it was transferred to a nitrocellulose membrane and the PVDF membrane rinsed. After it was sealed, diluted monoclonal antibody (1∶1 000) was added, and incubation was conducted at 4 ℃ overnight. Afterwards, it was added with horseradish peroxidase IgG (1∶12 000), and incubated at 37 ℃ for 1h. Then, it was washed by TBST. Diaminobenzidine was then added for color development. Based on the absorbance of each band in the image analyzer (A), the values for quantitative analysis were determined.

2.8 StatisticsAll data were normally distributed and presented as the mean ± standard deviation. In cases of multiple mean comparisons, the data were analyzed by one-way analysis of variance (ANOVA). Difference was considered statistically significant atP<0.05.

3 Results and analysis

3.1 Establishment and evaluation of the renal interstitial fibrosis modelAfter establishing the adenine model, using metabolic cages to collect 24 hour urine, and procuring the blood from the inner canthus, all the samples were sent to the inspection center of the First Affiliated Hospital of the Jinan University. The laboratory inspection aimed to examine the amount of urine protein, urine creatinine, Scr, BUN, 24hMTP and calculate the eGFR. The results for the model group show that the Scr, BUN, and the 24hMTP were significantly higher than those of the normal group, while the eGFR decreased significantly (P<0.05,P<0.01) (Table 1).

Table1 Comparisons of Scr, BUN, 24MTP and eGFR among groups after the modeling(mean±SD)

GroupnScr∥μmol/LBUN∥mmol/L24 h MTP∥mg/deGFR∥mL/minNormal725.0±1.877.11±0.97113.09±28.5398.64±4.69Model20309.73±121.97∗∗52.39±17.72∗517.30±47.80∗∗7.05±2.73∗∗

Note:*P<0.05,**P<0.01 compared with normal group.

3.2 Comparisons of kidney weight and indexThe kidney weight of the rats in the losartan group and the model group was significantly higher than that of the normal group (P<0.01). No significant difference was observed in kidney weight between the model group and the losartan group (P>0.05). At the same time, the kidney index (which was calculated as follows: kidney index=kidney weight/body weight×1 000‰) of the model group was significantly increased, in comparison with that of the normal group (P<0.01). Furthermore, the kidney index of the losartan group was significantly decreased, when compared with that of the model group (P<0.01) (Table 2).

3.3 Effects of losartan on BUN, SCr, 24hMT and eGFR

Compared with the normal group, the levels of Scr, BUN and 24MTP were significantly increased (P<0.01) in the losartan

Table 2 Comparisons of kidney weight and index among groups(mean±SD)

GroupnKidney weight∥gKidney index∥‰Normal71.58±0.15 5.30±0.24Model83.37±0.20∗∗18.70±1.93∗∗Losartan63.40±0.15∗∗14.77±2.29∗∗##

Note:**P<0.01 compared with normal group;#P<0.05,##P<0.01 compared with model group.

group, while the Ccr was significantly decreased (P<0.01). In comparison to the model group, the levels of Scr, BUN, 24MTP were significantly decreased (P<0.01) in the losartan group, while the Ccr was significantly increased (P<0.01) (Table 3).

3.4 Renal pathological changes

3.4.1Kidney observation. The rat kidneys were visually inspected. In the normal group, the rat kidneys were bean shaped, red-

Table 3 Comparisons of BUN, SCr, 24hMTP and eGFR among groups(mean±SD)

GroupnScr∥μmol/LBUN∥mmol/L24hMTP∥mg/deGFR∥mL/minNormal734.83±4.126.10±1.01115.00±5.7198.2±9.99Model8340.00±22.99∗23.74±2.51∗675.86±74.58∗19.3±2.48∗Losartan6174.67±11.00∗▲▲14.83±2.08∗▲▲303.37±52.8∗▲▲51.17±3.54∗▲▲

Note:*P<0.01 compared with normal group;▲P<0.05,▲▲P<0.01 compared with model group.

dish-brown, firm, elastic and difficult to decapsulate. The kidneyshad a smooth surface without spots, they were of a relatively small size, and had a clear boundary between the cortex and medulla on the front section. The rat kidneys of the model and the losartan groups were increased at different degrees. More specifically, the model group increased much more obviously when compared to the losartan group. The kidney was white, the surface was visible, containing dense particles ranging in size, that could be described as human like "white kidneys", having a solid texture, and lack of flexibility (Fig.1).

Fig.1 Pictures of kidneys in rats after treatment

3.4.2HE staining. The rats in the normal group had no significant hypertrophy, contracted or distended glomerular capsule, basement membrane hyperplasia, or inflammatory cell infiltration within the glomeruli. The juxtaglomerular tubules and medullary tubules were tightly packed and of a regular shape. There was only a small amount of interstitium. In the model group, the rats were observed to have contracted glomeruli and distended glomerular capsules without deposition of adenine metabolites; atrophy of the proximal tubule, distal tubule, medullary loop, and collecting duct; and stenosis or dilatation of the lumen or even disappearance of the tubules. The epithelial cells had a reduced size and a heavily stained nucleus. A heavy deposition of adenine metabolites was found in the lumen. Interstitial inflammatory cell and fibroblast infiltration, as well as interstitial deposition of adenine metabolites, was also observed. In the losartan group, the glomeruli were slightly contracted, and the glomerular capsules were relatively distended and had no adenine deposition. The tubules were of a regular shape, with a small amount of adenine deposition. Interstitial inflammatory cell and fibroblast infiltration was at a level lower than the model group (Fig.2).

Fig.2 Pathological pictures of the interstitial inflammatory cells and fibroblast infiltration with HE staining

3.4.3Masson staining. In the normal group, the kidney medulla skin had clear boundaries, its glomerular and tubular structure was normal, and the renal interstitial had almost no blue fiber. Under the microscope, the model group showed evidence of tubular atrophy or dilatation. There were many brown granules deposited in the renal tubules and a small amount of glomeruli. The renal interstitium showed increased amounts of collagen fibers, full of blue. In comparison to the model group, the losartan group showed significant decrease of blue collagen fibers, a reduced degree of tubular atrophy or dilatation, and reduced deposition of the brown yellow particles in the renal tubules (Fig.3).

Fig.3 Pathological pictures of the interstitial inflammatory cells and fibroblast infiltration with Masson staining

Fig.4 Results of TUNEL staining

3.4.4Detection of tubular cell apoptosis by TUNEL assay. The TUNEL results revealed that mainly the tubular cells and rarely the glomerular cells were apoptotic in each group. A small number of apoptotic tubular cells were detected in the normal group. The largest number of apoptotic tubular cells was observed in the model group. Additionally, a relatively small number of apoptotic tubular cells were in the losartan group. Pairwise comparisons between each group were statistically significant (P<0.01) (Fig.4 and Table 4).

Table 4 Comparison of the number of apoptotic cells among groups(mean±SD)

GroupnNumber of apoptotic cellsNormal 75.59±1.71Model 840.14±4.60∗Losartan 611.76±1.50∗▲▲

Note:*P<0.01 compared with normal group;▲P<0.05,▲▲P<0.01 compared with model group.

3.4.5Detection of expression of caspase-3 and JNK proteins. As the experimental results show, after the renal interstitial fibrosis, the caspase-3 and the JNK protein expression was up-regulated in comparison to the normal group (P<0.05, Fig.5). After the losartan treatment, in comparison to the model group, the caspase-3, and the JNK protein expression was down-regulated (P<0.05, Fig.5).

Note:*P<0.05 compared with normal group;△P<0.05 compared with model group.

Fig.5 Expression of caspase-3 and JNK proteins in renal tissue of rats

4 Discussion

Clinically, CRF is a primary or secondary chronic kidney disease caused by a progressive renal damage characterized by the emergence of a series of symptoms as well as a metabolic disorder syndrome, and is usually the final stage of the development of a variety of chronic kidney diseases. Recent research suggests that renal interstitial fibrosis is a major cause of chronic renal failure[8]; and studies have found that renal interstitial fibrosis and the pathogenesis of chronic renal failure are related with a variety of cytokines and apoptosis signal mechanism.

Apoptosis is described as a physiological programmed death in the regulation of a gene, started by the endogenous DNA enzyme activity, leading to the natural death of the cell. Normally, through apoptosis the body succeeds in removing damaged, aging and mutant cells, in order to maintain physiological balance. However, in excessive apoptosis induced by the renal parenchyma, the cell numbers were reduced. As studies have demonstrated cells apoptosis, glomerulosclerosis, and tubular atrophy, are closely related to the occurrence in renal interstitial fibrosis[9]. Caspase (cysteine aspartic acid specific protease, a cysteine protease) is the central link in the apoptotic regulation, which upon activation leads to irreversible apoptosis[10]. Research thinks that renal interstitial fibrosis is associated with the cell apoptosis process. The mitochondrial pathway of apoptosis is mediated by caspase including the activation of p38 MAPK, and the inhibition of ERK MAPK signal pathway[11]. JNK belongs to a member of MAPKs. It has 3 kinds of isozymes, and may through the phosphorylation of Bcl-2 and Bcl-xL, it promotes the release of cytochrome C from mitochondria and activates the caspase reaction, ultimately leading to cell apoptosis. Experimental evidence shows that, the JNK mediated signal pathway is involved in the regulation of the proliferation, apoptosis, extensive exercise, metabolism and DNA damage repair of cells during the life process. Any imbalance of the JNK signal is closely related to the development of renal fibrosis[12]. Recent studies have found that losartan has the efficacy for renal interstitial fibrosis, but the action mechanism of losartan still remains unclear.

This study has discovered the existence of a large number of apoptotic cells due to interstitial fibrosis in the adenine induced chronic renal rat model. In the renal interstitial fibrosis, the levels of caspase-3 and JNK were significantly increased, suggesting that they are involved in the process of cell apoptosis. In renal interstitial fibrogenesis, the cell apoptosis in the kidney produces a large number of products, and these products further worsen the degree of renal injury. The administration of losartan after drug treatment, can reduce the 24MTP, serum Scr, BUN levels, and inhibit the expression of caspase-3 and JNK proteins in the rats with renal interstitial fibrosis, thereby inhibiting excess apoptosis to a certain extent and improving the renal interstitial fibrosis, thus suggesting that losartan can protect renal function in certain cases, and delay renal injury. Finally, evidence suggests that its mechanism might be related to the inhibition of caspase-3 and JNK protein expression and the decrease of renal cell apoptosis.