Histopathological study of diclofenac induced acute renal failure under lipoic acid and bosentan therapy in male albino rats

: Abstract: Acute kidney injury (AKI), formly known as acute renal failure (ARF), is an abrupt and reversible decrease in kidney function as indicated by the glomerular filtration rate (GFR). Diclofenac-induced AKI is due to toxic effect of it on renal glomeruli, resulting in glomerular lesions. Furthermore, diclofenac causes autolysis, which increase renal intracellular osmolarity that leads to proximal renal tubular dilatations. Lipoic acid (LA) has antioxidant and anti-inflammatory activities. Bosentan is a competitive endothelin A (ETA) and endothelin B (ETB) receptors antagonist. In this study, the evaluation of effectiveness of lipoic acid and bosentan against diclofenac-induced AKI was done by histopathological examination. The results showed that diclofenac caused histopathological changes include; retracted glomerulus, tubular cast, tubule-interstitial inflammation and tubular necrosis. Lipoic acid or bosentan alone could not reduce the histopathological alterations caused by diclofenac. Meanwhile, the combination therapy was able to reduce the histopathological changes significantly ( p > 0.05 ). Therefore, the combination therapy of lipoic acid and bosentan showed promising ameliorative effect against diclofenac-induced AKI.


Introduction
Acute renal failure (ARF), also known as acute kidney injury (AKI) is a complex health condition related to significant mortality and morbidity [1] . In hospitalized patients, around 19-33 percent of AKI episodes are linked to nephrotoxic drugs. Diclofenac (2-[(2,6-diclorophenyl) amino] phenyl acetate) is phenyl acetic acid derivate that inhibits prostaglandin biosynthesis. It has antipyretic, painrelieving, anti-rheumatic and antiinflammatory activities [2] . It also has many uses include: gout, ureteric colic, as well as rheumatoid arthritis, and osteoarthritis [3] . Diclofenac-induced AKI is due to toxic effect of it on renal glomeruli, resulting in glomerular lesions. Furthermore, diclofenac causes autolysis, which increase renal intracellular osmolarity that leads to proximal renal tubule dilatations [4] . Diclofenac causes ischemia by inhibiting renal prostaglandin production, limiting renal afferent arteriole vasodilation, increasing afferent resistance; thus decreasing the glomerular capillary pressure below normal values and GFR will decrease resulting in AKI [5] . In the last few decades, many animal studies and molecular experiments have been done and revealed that endothelial injury, leukocytemediated inflammation and decrease microvascular blood flow are central mechanisms for AKI induced by ischemia [6] .
Lipoic acid (LA) is a naturally occurring micronutrient, synthesized in small amounts by plants and animals [7] . LA can act as antioxidant in lipophilic and hydrophilic environments (8) . In addition, LA has anti-inflammatory action by inhibiting nuclear factor kappa beta (NFkappaβ), which responsible for regulation of gene expression of many pro-inflammatory cytokines e.g. tumor necrosis factor alpha (TNF-α), interlukine-1 (IL-1), interlukine-6 (IL-6) [9] . Bosentan is a derivative of non-peptide pyrimidine that acts on both endothelin A (ETA) and endothelin B (ETB) receptors as a competitive, unique antagonist [10] . It was approved in patients for treatment of pulmonary arterial hypertension (PAH) [11] . Endothelin-1 (ET-1) is a 21 amino acid peptide [12] . It has two structurally related G protein-coupled receptors, endothelin type A receptor (ETAR) and endothelin type B receptor (ETBR) [13] . ET-1 is involved in many transcription factors activation such as NF-κB and expression of proinflammatory cytokines including TNF-α, IL-1, and IL-6 [14] . ET-1 is also a potent proliferative and mitogenic peptide [15] .

Animals
In this study, thirty mature male albino rat weighing between 200 and 250g were included. The animals were provided by the Iraqi center for cancer research and medical inheritance/Mustansiriyah University. The study was approved by the ethical committee for animal experimentation of college of pharmacy / Mustansiriyah University, where the work was done. Each six animals were placed in disinfected cage, with artificial light cycle of 12/12 and a temperature of (22±2°C). They were provided unrestricted access to water and normal chow pellets and left for 14 days to acclimate.

Chemicals
The chemical substances with their sources are clarified in the table (1): and bosentan (100mg/kg p.o.) for 11 days, and on the 5 th day they were given an intraperitoneal injection of diclofenac sodium (100mg/kg). On 12 th day, the rats were anesthetized with ketamine (alfasan Woerden-Holland) (90mg/kg) and xylazine (Kepro-Holland) (10mg/kg). The rat's abdominal cavity was opened using forceps and scissors in order to harvest and preserve the kidneys in 10% formalin for histopathological examinations.

Kidney tissues processing for histopathological examination
For light microscopy, there are many techniques can be used such as paraffin sections, semithin sections, and frozen sections techniques.
The paraffin technique has been used in present study. It can be summarized in the following steps (16) :

1.
Fixation: The whole kidney of the male rat was fixed by adding chemical fixative agent (formaldehyde 10%). 2. Dehydration and clearing: The tissue was embedded in paraffin wax and then cut into sections. Because the wax is not soluble in alcohol or water and soluble in xylene (paraffin wax solvent). Xylene was used to replace the water in the sample with alcohol. This was accomplished by exposing the tissue to increasing concentrations of ethanol (from 0 to 100%).Finally, alcohol was replaced with xylene, which is miscible with alcohol. The final stage is known as clearing. 3. Embedding: The tissue was dipped in paraffin wax, which fills the spaces that usually have water in them. The tissue hardens after cooling. 4. Sectioning: The cooled tissue was trimmed and placed on a microtome. Thin pieces of tissue are sliced into 4 µm and stained before being placed on microscope slide. 5. Staining and mounting: The wax has to be dissolved and replaced with water (rehydration) since most staining solutions are aqueous. The sections are processed through xylene, and then decreasing strengths of ethanol (100% to 0%) and finally water. The staining reagents that used in this study are hematoxylin and eosin (H&E). The section is dehydrated and placed in xylene after it has been stained. It is then placed on microscope slide in mounting medium dissolved in xylene. To keep the sample secure, a coverslip put on top. Evaporation of xylene that surrounding the edges of the coverslip dries the mounting medium and securely adheres the coverslip to the slide. The histological changes in current study were scored by EGTI (endothelial, glomerular, tubular, and interstitial) scoring system as clarified in table (2) [17] . Table (2): EGTI histology scoring system (17) Tissue type Histopathological changes score

Results
The kidney tissue sections that were stained with hematoxylin and eosin as previously described were placed under light microscope using x400 magnification for histopathological evaluation. Histopathological changes were assessed by experienced pathologist using the EGTI scoring system as mentioned in table (1). Figure (1) showed kidney section of control rat with apparently normal renal tissues (glomerulus, tubules) and EGTI scores: tubular: 0, endothelial: 0, glomerular: 0, tubule-interstitial: 0 In present study, Kruskal-Wallis H test was used for general comparison among the groups for significant histopathological changes and Mann-whitney U test was also applied to determine which group exactly has a significant difference with other as shown in table (3). Histopathological changes including endothelial, glomerular, and tubuleinterstitial damages in the induction group showed a significant damage (p>0.01) when compared with the control and combination groups. On the other hand, these changes in lipoic acid group showed non-significant difference (p=1) when compared with that in the induction and bosentan groups. Finally, the combination group showed a nonsignificant difference (p=1) in morphological appearance when compared to the control group. For tubular damages, the induction group showed a significant damage (p>0.01) in comparison to control and combination groups. Lipoic acid group showed a nonsignificant difference (p=0.127) when compared with the induction group. Meanwhile, the bosentan group showed a significant damage (p>0.01) in comparison to all other groups. Finally, the combination group has a non-significant difference (p=1) when compared to control group.

Discussion
The histopathological results in present study showed significant detrimental effects of diclofenac on renal tissue architecture, where retracted glomeruli, tubular cast, tubule-interstitial inflammation, and tubular necrosis appeared in the induction group compared to control group. These results resembled two previous studies that showed diclofenac induced renal glomerular proliferation, significant interstitial inflammation and cast formation (18) (19). Histopathological results in lipoic acid group showed no protective effect aganist diclofenac induced acute kidney injury. In fact, there is more damage occur in this group compared to the induction group, this finding is in line with research done by Grdović N et al (2021), that found ALA contribute to the profibrotic processes and collagen formation. In which, treatment with ALA was linked with development of glomerulosclerosis, tubulointerstitial fibrosis and decrease in renal function (20). On the other hand, the present study results partially agreed with previous study mentioning that lipoic acid attenuate histopathological changes significantly following methotrexate administration. This could be due to different induction mechanism that caused by methotrexate (21). Bosentan group showed a non-significant reduction in renal injury following diclofenac administration when compared to the induction group, this result partially agreed with a previous study that found bosentan could attenuate significantly renal injury induced by arsenic, in which a positive correlation between high level of endothelin and renal dysfunction was noticed (22). The disagreement might be due to different induction models used and longer treatment duration. Finally, the rat's kidney in the combination group appeared to be almost normal, this finding suggests that lipoic acid and bosentan have additive effects giving promising role in protecting renal tissues against detrimental effect of diclofenac.

Conclusion
The study showed a promising protective effect of combination therapy of lipoic acid and bosentan against diclofenac-induced AKI.