INTRODUCTION
Earlier, acute kidney injury (AKI) was named acute renal failure (ARF)1, a disease in which the renal function is abruptly impaired2 due to a rapid reduction in the glomerular filtration rate3 and severe tubular damage.2 This condition is manifested by an increased level of urea and creatinine in the blood, oliguria, or both.1,3 The etiological factors of AKI are infections, septicaemia, nephrotoxic drugs, failure of renal cell repair2, surgery, trauma, heart diseases, acute glomerulonephritis4, ischemia, hypoxia, etc.5 Studies have reported that during the last 50 years, the mortality rate due to AKI has been found to be higher than that due to diabetes, heart failure, and breast cancer. This higher prevalence might be due to either community or hospital-related infections.4 Another report revealed that nearly 20% of AKI cases in hospitalized patients are associated with serious complications like electrolyte imbalance, volume overload, uremic disorders, etc.3, and are further responsible for the deaths of 9.5% of patients.2 Moreover, some recent epidemiological studies have reported that the incidence of AKI has dramatically increased, particularly in hospitalized patients. The data obtained from 49 million hospitalized patients suffering from acute diseases has suggested that one in five adults and one in three children are more susceptible to developing acute kidney injury.6 In the present scenario, the nephrotoxicity caused by pharmaceutical agents is considered a major challenge for medical practice, hence, their misuse or overuse may be avoidable.7 In this regard, gentamicin, which belongs to the aminoglycoside group of antimicrobial agents, is more responsible for producing renal toxicities. After the invention of many newer, highly efficacious antibiotics, gentamicin is still preferred, especially by clinicians in developing countries, to treat several infectious diseases8 owing to its availability, cost-effectiveness, and potential nature.9 Around 3-5% of the concentration of gentamicin is reabsorbed through cells of the proximal tubules of the kidney following its administration and is responsible for necrosis, particularly in the S1 and S2 segments.8 Some studies have reported that such necrosis is caused by over-production of reactive oxygen species (ROS), reactive nitrogen species (RNS), low concentration of antioxidant enzymes, induced inflammatory pathway, and decreased blood supply to the kidney. Hence, it is clear that nearly 30% of gentamicin users develop renal failure.9
In the Unani system of medicine, renal failure or renal insufficiency is referred to as du’f al-kulya10, which is treated by using muqawwī-i-gurda (nephroprotective) drugs. According to the Unani concept, the kidney has four faculties viz; (i) quwwat-i-hādima (digestive faculty), (ii) quwwat-i-jādhiba (absorptive faculty), (iii) quwwat-i-māsika (retentive faculty), and (iv) quwwat-i-dāfiʼa (expulsive faculty). These faculties play an important role in the normal functioning of the kidney. A derangement in any one of these faculties is responsible for renal diseases.11 Ibn Sina (980-1037 AD), a versatile Unani scholar, stated that muqawwī drugs (tonics) maintain the homeostasis of all the vital organs of the body, including the kidney.12 Moreover, drugs used in the treatment of kidney diseases should have the potential to strengthen all the above mentioned faculties.11 Kasondi (Cassia occidentalis L.) (Synonym: Sennaoccidentalis (L.)38, an important medicinal plant belonging to the Caesalpiniaceae family13, is used in Unani14,15; Ayurveda16,17,Siddha17, Asian and African traditional medicines for the treatment of various diseases.18 It is a common weed, straight, smooth, semi-woody, somewhat branched, foetid herb, 0.8-1.5 m in height, hard, stout, with few lateral roots on its mid section.19 This medicinal plant is found growing in India20, the tropical and subtropical regions of America, Africa, Asia19,and Australia.16 The roots of Cassia occidentalis L. are of the tapered type, with 15-25 cm of length and 1-15 cm of diameter. The shape of the root is cylindrical, with a tapering at the upper extremity. The surface of the root is rough and dark brown externally and creamy internally.19 According to Unani medicine, its temperament is hār-yābis (hot-dry).21,22 It is therapeutically used as muhallil (resolvent), mudirr-i-bawl (diuretic), and jālī (detergent)14,21 for the treatment of istisqāʼ (ascites), wajaʻal-mafāsil (arthralgia), dīq al-nafas (bronchial asthma), yaraqān (jaundice)14,21-23 and renal diseases. A study revealed that the aqueous extract of the leaves of Cassia occidentalis L. shows a significant diuretic effect as compared to furosemide and hydrochlorothiazide in rats.18 On the basis of such evidence, the present study was designed to evaluate the nephroprotective activity of HAE obtained from the root of Kasondi (Cassia occidentalis L.) against gentamicin-induced nephrotoxicity in albino Wistar rats. The hypothesis of the study was to introduce a nephroprotective herbal drug that can be used as an adjuvant therapy while treating infectious diseases with aminoglycoside antibiotics, particularly gentamicin.
MATERIAL & METHOD
1. Collection and authentication of the plant material
The root of Kasondi (Cassia occidentalis L.) was collected from the campus of Aligarh Muslim University, Aligarh, Uttar Pradesh, India, during the month of September 2019 and was authenticated by Dr. Akhtar H. Malik, Centre for Biodiversity and Taxonomy (CBT), Dept. of Botany, University of Kashmir, Jammu & Kashmir. The voucher specimen was deposited with reference number 3058 KASH for future reference.
2. Chemicals and other agents
Gentamicin was procured from Nitin Life Sciences Ltd. (marketed by Abbott Healthcare Pvt. Ltd.), Unit III, Rampur Ghat Road, Ponta Sahib, District Sirmour, Himachal Pradesh, India (173025). The batch number of gentamicin was NFF 0041; it was manufactured and expired in January 2020 and December 2021, respectively. Reagents for evaluation of blood urea, serum creatinine, and serum uric acid were purchased from Erba Mannheim, Transasia Bio-Medicals Ltd., Nalagarh Road, Village Malpur, Baddi, District Solan, Nalagarh Road, Village Malpur, Baddi, District Solan, Himachal Pradesh, India (173205). Eosin was procured from Merk Life Science Pvt. Ltd., Godrej One, 8th floor, Mumbai, India (400079) and haematoxylin was procured from HiMedia Laboratories Pvt. Ltd., 23 Vadhani Ind. Est., LBS Marg, Mumbai, India (400086).
3. Preparation of hydro-alcoholic extract
The roots of Kasondi (Cassia occidentalis L.) were first dried in the shade, followed by coarse powdering, which was done by using an electric grinder. The extraction was carried out at the Drug Standardization Research Unit (DSRU) of the Regional Research Institute of Unani Medicine, Srinagar. For extraction, the sample was first defatted with petroleum ether at the boiling point (60 ℃ – 80 ℃) in a continuous extraction apparatus using Soxhlet for 6 hours. The extract was quantitatively filtered into a tarred evaporating dish, and the solvent was evaporated on a water-bath. The residue was dried at 105 ℃. The yield of pet-ether soluble extract was calculated. After defatting, the sample was subjected to hydro-alcoholic extraction (1:1) for 72 hours. The liquid extract was cooled and filtered through Whattman filter paper (40). The extract was concentrated under reduced pressure using a rotary vacuum evaporator. Thereafter, the extract was weighed and the yield percentage was calculated with reference to the weight of the crude drug, and it was preserved in an airtight glass container at 4℃ for further use.39
4. Dosage and route of administration
In Unani literature, the therapeutic dose of Kasondi (Cassia occidentalis L.) for humans is mentioned as 10 g, which was further calculated by multiplying with a conversion factor of 7 for rats24 and it was found to be 1000 mg/ kg b. w. Since the test drug was used in the form of an extract, the dose was further calculated according to the yield obtained in extraction, and it was found to be 67 mg/kg b. w., which was administered by oral route. The dose of gentamicin was given as 100 mg/kg b. w. through a subcutaneous route.
5. Experimental animals
A total of 18 albino Wistar rats of either sex weighing 150-200 g were procured from IIIM, Jammu. They were housed in the animal house of the Regional Research Institute of Unani Medicine, Srinagar, following all the CPCSEA guidelines. They were housed in polypropylene cages under standard laboratory conditions at 23℃ ± 2℃ and 12 h light and dark cycles. The rats were fed standard commercial food pellets and RO water ad libitum. The research protocol was approved by the Institutional Animal Ethics Committee (IAEC) of RRIUM, Srinagar, India, with Reg. No. 927/GO/Re/S/2006/CPCSEA.
6. Experimental design
The study was carried out according to the method described by Fahamiya et al., 2012 with minor modifications.25 The animals were randomly divided into three groups of six animals each. The study design was as follows:
Group I (Plain control): This group was treated with 1 ml of normal saline per os for 8 days.
Group II (Disease control): This group was administered gentamicin at 100 mg/ kg b. w. s. c. in the neck region daily from the 4th to the 8th day.
Group III (Treatment group): This group was treated with gentamicin at 100 mg/ kg b. w. s. c. in the neck region daily from the 4th to the 8th day, along with the hydro-alcoholic extract of the root of Cassia occidentalis L. at 67 mg /kg b. w. per os daily for 8 days.
7. Collection of blood sample and kidney specimen
At the end of the experiment, all the animals were sacrificed by administering thiopentone sodium (50 mg/kg b. w.) i. p., after an overnight fast. The blood sample was collected through cardiac puncture and centrifuged using a centrifuge machine at 3000 rpm for 10 minutes24 to separate the serum for biochemical estimation. The kidneys of all the rats were collected and preserved in a 10% phosphate-buffered formalin solution for histopathological studies.24,26
8. Biochemical evaluation
The kidney function tests, including blood urea (GLDH-Urease method)27, serum creatinine (Jaffe’s method)28, and uric acid (Modified Trinder method, End point)29, were analyzed using an automatic biochemistry analyzer.
9. Histopathological examination of kidney specimens
After the collection of the kidney specimens, a gross examination was done regarding their size, shape, colour, and any other abnormal findings. The tissue sections were also examined microscopically for histopathological changes. The sliced formalin-preserved kidney tissue was processed for routine paraffin embedding, and 3-5 μ sections were stained with Mayer’s haematoxylin and eosin stains, and a blinded histopathological assessment was made.30,31 Nikon Eclipse E 200 microscopic camera was used to take photomicrographs in this study. It is pertinent to mention that the histopathological examination was done by a histopathologist, Prof. Pankaj Goswami, who was associated with this study and is one of the co-authors of this publication.
10. Statistical analysis
The data were analyzed using the data editor of SPSS version 20.0 and GraphPad Prism software. All the data collected from the biochemical analysis are expressed as Mean ± Standard Error of Mean (SEM). A one-way ANOVA was employed for the analysis of various parameters among different groups, followed by a post hoc multiple comparison test. The p-value < 0.05 was considered statistically significant.
DISCUSSION
The hydro-alcoholic extract of the root of Cassia occidentalis L. at 67 mg/kg b. w. showed significant nephroprotective effects against gentamicin-induced nephrotoxicity in albino Wistar rats. The rats treated with gentamicin along with the test drug did not show any abnormal signs during physical examination. Their gait, skin, fur, mucus membranes, and general behaviour were found to be normal. The biomarkers of kidney function such as s. creatinine, b. urea, and s. uric acid levels were found to be promisingly increased in the rats of disease control as compared to those of the plain control, which indicates that gentamicin produced renal toxicity at the dose level of 100 mg/kg b. w. The histological changes of the kidney include a markedly distorted nephron with degenerative glomeruli, severe tubular necrosis along with peritubular congestion, cortical necrosis, marked congestion in tubular structure with marked oedematous stroma, and desquamated epithelium of convoluted tubules, which also supported the evidence of renal toxicity in the rats of disease control. The same biomarkers of kidney function were found to be significantly reduced in the rats of the treatment group as compared to those of the disease control group. This evidence is also supported by the improvement in the histological changes of the kidneys of rats belonging to the treatment group, like a healthy cortico-medullary junction with minimal congestion of blood vessels in the cortex with mild interstitial oedema and the normal position of the glomerular tuft with mild tubular degenerative changes.
According to Unani principles, abnormal temperament, structural anomalies, obstructions, deformities, rupture, trauma, wounds of the kidney, etc. are important causes of renal failure.32 Moreover, Ibn Rushd (1126-1198 AD) advocates that an imbalance or derangement in any one of the faculties of the kidney, viz; quwwat-i-hādima (digestive faculty), quwwat-i-jādiba (absorptive faculty), quwwat-i-māsika (retentive faculty), quwwat-i-dāfiʻa (expulsive faculty), and quwwat-i-mumayyzah (distinguishing faculty), leads to renal dysfunction.32,33 In Unani medicine, mudirr-i-bawl (diuretics), mufattit-i-ḥasāh (lithotryptic), and muqawwī-i-gurda (nephroprotective) drugs are prescribed in the treatment of renal diseases.34 Kasondi (Cassia occidentalis L.) possesses muqawwi (tonics), mudirr-i-bawl (diuretics), and muqawwī-i-gurda (nephroprotective) pharmacological properties and is responsible for the moderation of pathological changes and maintenance of the homeostasis of all vital organs12, including the kidney. However, this important medicinal plant had not been scientifically studied previously. The present study has reported that the HAE of the root of Cassia occidentalis L. promisingly reduced the kidney biomarkers and improved the histological changes of nephrotoxic rats induced by gentamicin. Various mechanisms are involved in gentamicin-induced renal toxicity, but the most important one is the excessive generation of ROS, especially hydroxide and hydrogen peroxide, which further causes damage in the nephrons. Moreover, the production of antioxidant enzymatic activity in the kidney is also found to be decreased in the recipients of gentamicin, which is further responsible for lipid peroxidation production and derangement in the morphological and functional cells of the kidney.35 A study has reported that the aqueous leaf extract of Cassia occidentalis L. potentially produces antioxidant activity against nitric oxide scavenging activity, β-carotene-linoleic acid model system, hydroxyl radical scavenging activity, reducing power, metal chelating activity, and superoxide radical scavenging activity. This pharmacological activity might be due to the presence of phenolic compounds in the test substance.36 Another study revealed that the hydro-alcoholic extract of the leaf of Cassia occidentalis L. shows significant nephroprotective effects against gentamicin-induced renal toxicity in rats.37 In Unani medicine, drugs obtained from natural sources are used for the treatment of several diseases.38 The data of the present study showed that the HEA of the root of Cassia occidentalis L. significantly produces nephroprotective effects against gentamicin-induced renal toxicity in albino Wistar rats, as evident by reductions in kidney biomarkers like s. creatinine, b. urea, and s. uric acid and a promising improvement in the histological changes of the diseased kidney. Furthermore, the result of the present study also validated the claims of the healers of Unani and other traditional medicines about the use of the root of Kasondi (Cassiaoccidentalis L.) in the treatment of kidney diseases.