April 2019, Volume 69, Issue 8

Animal Research

Reno-protective effect of berberine

Authors: Nawar Raad Hussien  ( Department of Clinical Pharmacology, Medicine and Therapeutic, Al- Mustansiriya University, Baghdad, Iraq )
Hayder M. Al-kuraishy  ( Department of Clinical Pharmacology, Medicine and Therapeutic, Al- Mustansiriya University, Baghdad, Iraq )
Ali Ismail Al-Gareeb  ( Department of Clinical Pharmacology, Medicine and Therapeutic, Al- Mustansiriya University, Baghdad, Iraq )


Objectives: To assess the reno-protective effect of berberine on diclofenac-induced acute kidney injury in rats.
Methods: The experimental study was conducted at the College of Medicine, Mustansiriya University, Baghdad, Iraq, from January to March 2018, and comprised Sprague Dawley male rats which were divided into 3 equal groups. Group1 rats were treated with distilled water plus normal saline for 14 days, Group2 rats were treated with distilled water plus diclofenac for 14 days and Group3 rats were treated with berberine plus diclofenac for 14 days. Parameters measured were blood urea, serum creatinine, serum malondialdehyde, superoxide dismutase, glutathione reductase, neutrophil gelatinase associated lipocalin, kidney injury molecules-1, Interleukin-18and cystatin-c. Anthropometric measurements and estimated glomerular filtration rate were also noted. SPSS 20 was used for data analysis.
Results: Of the 30 rats, the three groups had 10(33.3%) each. Berberine reduced blood urea, serum creatinine, malondialdehyde, neutrophil gelatinase associated lipocalin, kidney injury molecules-1 and Interleukin-18 significantly compared to diclofenac-induced acute kidney injury (p<0.01 each). Berberine improved anti-oxidant capacity through significant elevation of superoxide dismutase and glutathione reductase sera levels (p<0.01 each).
Conclusions: Berberine was found to be an effective agent in the attenuation of diclofenac-induced acute kidney injury through the modulation of pro-inflammatory and oxidative stress biomarkers.
Keywords: Berberine, Diclofenac, Acute kidney injury. (JPMA 69: S-83 (Suppl. 3); 2019)


Acute kidney injury (AKI) is a renal-specific condition in which the excretion of toxic metabolites does not depart proficiently due to toxic agents and drugs.1 Diclofenac-induced AKI is due to the toxic effect of diclofenac on renal glomeruli and tubules. Diclofenac provokes tubular dilatation and loss of brush border. 2 Long-term therapies with diclofenac causes dosedependent diminution of renal prostaglandins, leading to disturbances of glomerular function and reduction in glomerular filtration rate (GFR). 3 It has been postulated that diclofenac may exert its toxic effects depending on its dose and kidney-related parameters like plasma coagulation parameters, oxidative damage, and adenosine deaminase enzyme activity. Moreover, diclofenac may cause exhaustion of adenosine triphosphate (ATP), lipid peroxidase, mitochondrial dysfunction and reduction of intracellular calcium concentrations. 4 The kidney injury caused by diclofenac is mainly due to the induction of renal vasoconstriction. Besides, oxidative damage induced by diclofenac toxicity causes note worthy lipid peroxidation of cellular membranes. 5 Additionally, diclofenac provokes renal mitochondrial dysfunction through the inhibition of mitochondrial complex, causing a reduction in ATP generation and apoptosis. Therefore, anti-oxidants are part of the problem in diclofenac-induced AKI. 6 Similarly, diclofenac-induced AKI is linked to the induction of pro-inflammatory cytokines, including interleukin-6 (IL-6), IL-1, IL-33 and tumour necrosis factor-alpha (TNF-α) that induce renal nuclear fragmentation and condensation. 7 Berberine belongs to the family of isoquinoline alkaloids derived from berberis plants. It has anti-oxidant and anti-inflammatory effects. 8 Berberine improves mitochondrial function via reduction of oxygen consumption rate and by increasing mitochondrial membrane potential to protect against oxidative damage. 9 The reno-protective effect of berberine was thought to happen through a mitochondrial-dependent pathway in rats with renal ischaemia-reperfusion model. Besides, berberine re-organises renal interstitial fibrosis in an  obstructed kidney and protects against the renal damage caused by hypertension in rats. 10 The current study was planned to assess the renoprotective effect of berberine on diclofenac-induced AKI in rats.

Materials and Methods

The experimental study was conducted in the Department of Pharmacology, College of Medicine, Mustansiriya University, Baghdad, Iraq from January to March, 2018, and comprised Sprague Dawley male rats aged 2-3 months and weighing 200-350g. The animals were kept at a suitable room temperature with artificial 12/12hrs light-dark cycle. After acclimatisation period of one week, the rats were randomly divided into three equal groups. The experimental procedures were done according to the ethics for the care and use of laboratory animals in research and permission was obtained from the institutional ethical committee. All care and efforts were done to reduce animal suffering. The study procedure and method for induction of AKI was in line with literature. 11 Group 1 rats were treated with distilled water 5ml/kg for 14 days and on days 6-12 they received daily intraperitoneal injection of normal saline 5ml/kg. Group 2 rats were treated with distilled water 5ml/kg for 14 days and on days 6-12 they received daily intra-peritoneal injection of diclofenac 15ml/kg (voltarin 15mg, Medopharm, Czech Republic). Group 3 rats were treated with berberine 100mg/kg (Natureganics, USA) for 14 days and on days 6- 12 they received daily intra-peritoneal injection of diclofenac 15ml/kg. Length was measured by graduated tape measure from nose to the anus (naso-anal length in cm). Rat bodyweight was measured by specific digital balance in gram. Body mass index (BMI) was calculated as per the standard procedure. 2,12

Estimated GFR (eGFR) was measured according to Schwartz formula,  eGFR =k × height (cm)/serum creatinine (mg/dl), K=0.55.

13 On day 14, chloroform was used to anesthetise the rats and sharp scissors were used to exudate the rats. The blood sample was centrifuged for 10 minutes at 5000rpm at room temperature after which the supernatant layer was isolated as the serum sample and kept in freezer at - 20°C to be assessed later. Blood urea and serum creatinine were estimated by using an auto-analyser (ILab-300-Biomerieux Diagnostic, Milano, Italy) and they were expressed as mg/dL. Serum malondialdehyde (MDA), superoxide dismutase (SOD), glutathione reductase (GSH), Neutrophil Gelatinase Associated Lipocalin (NGAL), kidney injury molecules-1 (KIM-1), interleukin-18 (IL-18), and cystatin-c (Cyst-c) were measured by enzyme-linked immunosorbent assay (ELISA) kit method according to the instructions given by the kit manufacturer (Myo-bio source, USA). Data was analysed using SPSS 20 and was presented as mean } standard deviation (SD). Unpaired or paired student t test was used to determine the differences. Analysis of variance (ANOVA) followed by Benferroni post-hoc test were used to compare the variables in different groups. The level of significance was set at p<0.05.


Of the 30 rats, the three groups had 10(33.3%) each. During AKI induction, the bodyweight increased non-  significantly compared to the controls (p>0.05). BMI increased significantly in the diclofenac group compared to the controls (p=0.0003). Blood urea and serum creatinine were elevated significantly in the diclofenac group compared to the control p<0.01). The estimated eGFR reduced significantly in the diclofenac group (6.12}1.7ml/min/1.73) compared to the controls (16.89}4.21ml/min/1.73 (p=0.0001). There was significant increase in the MDA serum levels in the diclofenac group compared to the controls (p=0.0001), while both SOD and GSH serum levels were reduced in the diclofenac group compared to the controls (p=0.0.0018). IL-18 and KIM-1 serum levels increased significantly in the diclofenac group compared to the controls (p=0.0001 each). Co-administration of berberine significantly reduced biochemical changes, inflammatory and oxidative stress through the reduction of blood urea, serum creatinine, MDA, IL-18, KIM-1 and NGAL sera levels (p<0.01 each). Besides, berberine improved renal function through the elevation of eGFR compared to the controls (p<0.0001). Also, berberine improved the anti-oxidant potential through elevation of SOD and GSH sera levels significantly compared to the AKI model (Table).

Cys-c serum levels were elevated during the induction of AKI to 0.0288}0.005ng/ml compared to the controls0.024}0.0005ng/ml (p=0.018), while berberine significantly reduced Cys-c serum levels to 0.0248}0.001 compared to the AKI model (p=0.02). There was insignificant difference in Cys-c serum level compared to the controls (p=0.05). During AKI, blood urea and serum creatinine were significantly correlated with Cys-c serum levels (Figure-1).

Moreover, high blood urea was significantly correlated with renal tubular injury and pro-inflammatory biomarkers during diclofenac-induced AKI (p<0.05). The correlation of blood urea with KIM-1, NGAL and IL-18 was significant (p<0.05 each) (Figure-2).


The present study illustrated that diclofenac led to significant AKI through elevation of blood urea and serum creatinine compared to the controls in the experimental rats as supported by Alabi et al. study which illustrated that diclofenac leads to significant renal tubular necrosis. 14 The induced AKI in the present study also showed significant reduction in eGFR due to the development of acute tubular necrosis and glomerular damage. Tomohiro et al. confirmed that short-term administration of diclofenac leads to acute glomerular damage due to potential inhibition of renal prostaglandins which reduces the renal blood flow and causes renal ischaemia. 15 BMI of the experimental rats were high in AKI compared to the controls which was perhaps due to daily bodyweight gain or fluid retention caused by renal damage. Moreover, reduced GFR also takes part in raised BMI via reduction of urine output and development of peripheral oedema as a number of rats developed ascites. An observational study suggested that fluid retention is the main element of AKI. 16 Oxidative stress and diminution of anti-oxidant capacity are the chief mechanisms of diclofenac-induced AKI since high doses of diclofenac administration lead to significant lipid peroxidation which reflect through the elevation of MDA serum levels and reduction of SOD. 17 Moreover, in diclofenac-induced AKI, the inflammatory process in the renal tissues is increased, so IL-18 serum levels significantly increase within a short period of renal injury 18 as revealed in our study. Likewise, diclofenac in the present study increased serum levels of NGAL significantly as NGAL level is correlated with renal tubular damage in diabetic nephropathy. 19 Furthermore, KIM-1 was significantly elevated in the present study compared to the controls. Lan et al. confirmed significant elevation of KIM-1in cadmiuminduced AKI. KIM-1 serum levels are elevated during the initiation of AKI as it increases in both serum and urine as it sheds from the renal proximal tubules. KIM-1 serum is significantly correlated with different grades of renal damage in AKI because it is highly sensitive and specific. 20 Cys-c is a sensitive biomarker for the detection of acute renal damage as it is more sensitive than blood urea and serum creatinine. Harman et al. confirmed that Cys-c is a surrogate biomarker for AKI and should be incorporated in the estimation of GFR. 21 Therefore, Cys-c serum levels in diclofenac-induced AKI of the present study were elevated significantly compared to the controls. Berberine, when combined with diclofenac, produced significant reduction in the rats' bodyweight and BMI compared to the diclofenac group due to the attenuation of diclofenac-induced nephrotoxicity and amelioration of oedematous status through the improvement of rat GFR, given that berberine improves renal functions with significant protection of renal tubules against diclofenacinduced renal tubules injury. 22 In addition, berberine reduced MDA serum levels and improved the endogenous anti-oxidant capacity via elevation of GSH serum levels, as supported by a study which confirmed significant reno-protective effect of berberine against nephrotoxicity in rats due to antioxidant and anti-inflammatory properties. 23Therefore, anti-oxidant effect is the chief mechanism of berberine in the attenuation of diclofenac-induced AKI. Different berberine derivatives have phenolic groups which activate and up-regulate SOD and GSH in a dosedependent manner. 24 Berberine attenuates renal injury and inflammation via significant reduction of KIM-1since it plays a potential role in the attenuation of gentamicin-induced nephrotoxicity through the reduction of oxidative and inflammatory pathways. 25 Likewise, berberine was successful in the reduction of Cys-c serum levels compared to the diclofenac group which might have been due to significant nephronprotective effect. In sum, berberine attenuates diclofenac-induced AKI through the inhibition of inflammatory and oxidative stress mediated pathways with potential augmentation of endogenous anti-oxidant capacity.


Berberine was found to be an effective agent in the attenuation of diclofenac-induced AKI through the modulation of pro-inflammatory and oxidative stress biomarkers.

Acknowledgment: We are grateful to Prof. Dr. Sadiq M. Al-Hamash for his great support.
Disclaimer: None.
Conflicts of Interest: None.
Source of Funding: None.


1. Al-Kuraishy HM, Al-Gareeb AI, Sadek Al-Naimi MS. Pomegranate attenuates acute gentamicin-induced nephrotoxicity in spraguedawley rats: the potential antioxidant and anti-inflammatory effects. Asian J Pharm Clin Res 2019;12:484-6.
2. Al-Kuraishy HM, Al-Gareeb AI, Hussien NR. Betterment of diclofenac-induced nephrotoxicity by pentoxifylline through modulation of inflammatory biomarkers. Asian J Pharm Clin Res 2019;12:433-7.

3. Yasmeen T, Qureshi GS, Perveen S. Adverse effects of diclofenac sodium on renal parenchyma of adult albino rats. J Pak Med Assoc 2007;57:349-51.

4. Aydin G, Gokcimen A, Oncu M, Cicek E, Karahan N, Gokalp O. Histopathologic changes in liver and renal tissues induced by different doses of diclofenac sodium in rats. Turk J Vet Anim Sci 2003;27:1131-40.

5. Al-Kuraishy HM, Al-Gareeb AI. Eustress and malondialdehyde (MDA): role of panax ginseng: randomized placebo controlled study. Iran J Psychiatry 2017;12:194-200.

6. Santos-Alves E, Marques-Aleixo I, Coxito P, Balca MM, Rizo-Roca D, Rocha-Rodrigues S, et al. Exercise mitigates diclofenac-induced liver mitochondrial dysfunction. Eur J Clin Invest 2014;44:668-77.

7. Barcelos RP, Bresciani G, Cuevas MJ, Martinez-Florez S, Soares FAA, Gonzalez-Gallego J. Diclofenac pretreatment modulates exerciseinduced inflammation in skeletal muscle of rats through the TLR4/NF-?B pathway. Appl PhysiolNutrMetab2017;42:757-64.
8. Zhang S, Zhang B, Dai W, Zhang X. Oxidative damage and antioxidant responses in Microcystis aeruginosa exposed to the allelochemical berberine isolated from golden thread. J Plant Physiol2011;168:639-43.

9. Hsu YY, Chen CS, Wu SN, Jong YJ, Lo YC. Berberine activates Nrf2 nuclear translocation and protects against oxidative damage via a phosphatidylinositol 3-kinase/Akt-dependent mechanism in NSC34 motor neuron-like cells. Eur J Pharm Sci 2012;46:415-25.
10. Wang FM, Yang YJ, Ma LL, Tian XJ, He YQ. Berberine ameliorates renal interstitial fibrosis induced by unilateral ureteral obstruction in rats. Nephrology (Carlton) 2014;19:542-51.

11. Singh AP, Junemann A, Muthuraman A, Jaggi AS, Singh N, Grover K, et al. Animal models of acute renal failure. Pharmacol Rep 2012;64:31-44.

12. Novelli EL, Diniz YS, Galhardi CM, Ebaid GM, Rodrigues HG, Mani F, et al. Anthropometrical parameters and markers of obesity in rats. Lab Anim2007;41:111-9.

13. Gacka E, ?yczkowski M, Bogacki R, Paradysz A, Hyla-Klekot L. The Usefulness of Determining Neutrophil Gelatinase-Associated Lipocalin Concentration Excreted in the Urine in the Evaluation of Cyclosporine A Nephrotoxicity in Children with Nephrotic Syndrome. Dis Markers 2016;2016:e6872149.

14. Alabi QK, Akomolafe RO, Adefisayo MA, Olukiran OS, Nafiu AO, Fasanya MK, et al. Kolaviron attenuates diclofenac-induced nephrotoxicity in male Wistar rats. Appl PhysiolNutrMetab2018;43:956-68.

15. Mizuno T, Ito K, Miyagawa Y, Ishikawa K, Suzuki Y, Mizuno M, et al. Short-term administration of diclofenac sodium affects renal function after laparoscopic radical nephrectomy in elderly patients. Jpn J Clin Oncol 2012;42:1073-8.
16. Al-Kuraishy HM, Al-Gareeb AI, Rasheed HA. Antioxidant and anti-inflammatory effects of curcumin contribute into attenuation of acute gentamicin-induced nephrotoxicity in rats. Asian J Pharm Clin Res 2019;12:466-8.

17. Al-Kuraishy HM, Al-Gareeb AI, Al-Maiahy TJ. Concept and connotation of oxidative stress in preeclampsia. J Lab Physicians 2018;10:276-82.

18. Bunel V, Tournay Y, Baudoux T, De Prez E, Marchand M, Mekinda Z, et al. Early detection of acute cisplatin nephrotoxicity: interest of urinary monitoring of proximal tubular biomarkers. Clin Kidney J 2017;10:639-47.

19. Kim SY, Jeong TD, Lee W, Chun S, Sunwoo S, Kim SB, et al. Plasma Neutrophil Gelatinase-Associated Lipocalin as a Marker of Tubular Damage in Diabetic Nephropathy. Ann Lab Med 2018;38:524-29.

20. Lan Z, Bi KS, Chen XH. Ligustrazine attenuates elevated levels of indoxyl sulfate, kidney injury molecule-1 and clusterin in rats exposed to cadmium. Food Chem Toxicol2014;63:62-8.

21. Harman G, Akbari A, Hiremath S, White CA, Ramsay T, Kokolo MB,

et al. Accuracy of cystatin C-based estimates of glomerular filtration rate in kidney transplant recipients: a systematic review. Nephrol Dial Transplant 2013;28:741-57.

22. Wu U, Cha Y, Huang X, Liu J, Chen Z, Wang F, et al. Protective effects of berberine on high fat-induced kidney damage by increasing serum adiponectin and promoting insulin sensitivity. Int J Clin Exp Pathol2015;8:14486-92.

23. Domitrovi? R, Cvijanovi? O, Pernjak-Pugel E, Skoda M, Mikeli? L, Crn?evi?-Orli? Z. Berberine exerts nephroprotective effect against cisplatin-induced kidney damage through inhibition of oxidative/nitrosative stress, inflammation, autophagy and apoptosis. Food Chem Toxicol2013;62:397-406.

24. Pongkittiphan V, Chavasiri W, Supabphol R. Antioxidant Effect of Berberine and its Phenolic Derivatives Against Human Fibrosarcoma Cells. Asian Pac J Cancer Prev2015;16:5371-6.

25. Adil M, Kandhare AD, Dalvi G, Ghosh P, Venkata S, Raygude KS, et al. Ameliorative effect of berberine against gentamicin-induced nephrotoxicity in rats via attenuation of oxidative stress, inflammation, apoptosis and mitochondrial dysfunction. Ren Fail 2016;38:996-1006.

Journal of the Pakistan Medical Association has agreed to receive and publish manuscripts in accordance with the principles of the following committees: