Nephroprotective Effects of Aqueous Extracts of Hibiscus sabdariffa Calyces and Jatropha curcas Leaves against Lead Toxicity

Background: Lead is a persistent heavy metal which has been found to be a multi-organ toxicant. In recent times, plants containing natural compounds with both chelating and antioxidant activities are considered as promising candidates for managing lead toxicity. Objectives: The present investigation was undertaken to evaluate the ameliorative potentials of aqueous extracts of Jatropha curcas (JC) leaves and Hibiscus sabdariffa (HS) calyces against renal toxicity resulting from lead acetate exposure. Materials and Methods: Ten groups of male Wistar rats were taken: The first and second groups represented the normal and lead acetate (PbA) (50mg/kg b.w) control groups respectively while groups 3 and 4 were given 100mg/kg JC and 200 mg/kg JC respectively. Groups 5 and 6 received 250 mg/kg HS and 500 mg/kg HS respectively. Groups 7-10 were given PbA (50mg/kg) and 100mg/kg JC, 200 mg/kg JC, 250mg/kg HS, 500mg/kg HS respectively. Rats were orally administered their relevant doses for 28 days. Blood samples were collected from heart puncture at the end of the experiment (28 days) for renal function analysis. Result: Serum creatinine and urea concentrations were significantly higher (p < 0.05) in the lead acetate group compared to the normal control. The renal morphology of the lead-exposed rats also revealed the degeneration and necrosis in tubules and swelling in the glomerulus. The significant increase in renal markers and morphological alterations after lead toxicity induction were moderately normalized by the administration of aqueous extracts of Jatropha curcas and Hibiscus sabdariffa. Conclusions: Results demonstrated moderate beneficial effects of J. curcas and H. sabdariffa treatment in managing Pb-induced tissue damage in the kidney.


Introduction
As a toxin, lead induces a wide range of physiological, biochemical and behavioural dysfunction; in particular, the kidney, liver, spleen, testes have been identified as key target organs for lead toxicity [1][2][3]. Lead toxicity occurs when lead accumulates in the body over a period of months or years. Elevated blood lead levels of 0.3 mg/ml and above have been found to cause toxic effects in humans and animals [4]. There is growing concern that a significant number of people especially children remain at risk from lead exposure in different parts of the world. Common environmental lead exposure sources to the general population include leaded water supply pipes, lead based paints and ceramics, gasoline, contaminated food, drinks, cosmetics and medicine [5].
The kidney plays a principal role in the excretion of metabolic wastes and in the regulation of intracellular fluid volume, electrolyte composition, and acid-base balance [6]. A toxic insult to the kidney, therefore, could disrupt any or all of these functions and could have a profound effect on total body metabolism. Tiwari et al [7] demonstrated that lead was nephrotoxic even at low doses. Renal tissue damage such as tubular atrophy, inflammation and necrosis have also been observed in lead treated mice. Generally, most metals including lead are concentrated by the kidney and this tends to produce a spectrum of biochemical injuries to the nephron. The extent of lead-induced nephrotoxicity is determined by factors such as the presence of high-affinity metal-binding proteins, cell type, exposure frequency and dietary status [8].
Consequently, there is now resurged interest in seeking strategies to reduce the toxicity associated with lead exposure. Several plants have been screened for their protective efficacy against lead toxicity in animal model [9][10][11][12]. Plants with appreciable levels of antioxidant and chelating activities are considered as promising candidates for mitigating lead toxicity [13]. Jatropha curcas and Hibiscus sabdariffa are two Nigerian plants with well-established neutraceutical properties. Asuk et al. [14] reported that J. curcas leaves were rich sources of both macro and micro-nutrients. They observed that the polyphenol contents of the J. curcas leaves were high. Polyphenols are phytochemicals that exercise anti-oxidative activities [15]. H. sabdariffa is a medicinal and food plant rich in phytochemical compounds which are the source of its biological properties [16]. Although from literature, both plants have been reported to have demonstrated significant antioxidant activities, there is limited information on their efficacy in lead-induced oxidative stress in the kidney of rats. Hence the present investigation was designed to study the protective efficacy of aqueous leaf extracts of J. curcas and H. sabdariffa respectively in the kidney upon exposure to lead.

Chemicals
Lead acetate Pb(CH 3 CO 2 ) 2 and all other chemicals used in the study were obtained from Sigma-Aldrich Corporation (St. Louis, Missouri, USA).

Plant material
The leaves of J. curcas were obtained from Abuja park of the University of Port Harcourt, Rivers state, Nigeria while the H. sabdariffa calyces were obtained from a local market in Obio/Akpor Local Government Area of Rivers State, Southern Nigeria. The plant material was washed using double distilled water and then dried at room temperature for 48 to 92 h and then grounded into powder and stored at room temperature until use.

Animals care handling and Experiment Design
A total of 60 male adult albino rats of varying weights (90 -200g), respectively bred in the animal house of the Department of Physiology, University of Port Harcourt was obtained for this study. The rats were divided into ten (10) groups and housed in polypropylene cages were maintained under standard conditions prescribed by the committee for control and supervision on experiments on animals (CPCSEA). Before the initiation of the experiment, the rats were left to acclimatize for a period of seven (7) days to natural environmental conditions of 12 hours light and 12 hours dark cycle with an ambient temperature of (25 ± 2) ºC. During the period of acclimatization and through the experiment the rats were fed with rat feed pellets and clean drinking water ad libitium. The research lasted for 28 days. At the end of the experiment, the animals were fasted overnight. The blood samples were collected, some of which was centrifuged to obtain the serum which was kept frozen at -20 ºC until used for biochemical analysis of electrolytes, urea and creatinine. Pieces of tissues from kidney were immediately kept in 10% of formalin fixative to study histological alterations.

Serum Biochemical Parameters
Serum urea and creatinine concentration were estimated using an automatic clinical chemistry analyzer (Mindray BS800, Shenzen, China) while the serum electrolytes were determined using ISO 4000 Automated electrolyte analyzer. SFRI, France.

Histological Examinations
For light microscopy examination, the formalin-fixed tissues were dehydrated through ascending grades of alcohol, cleared in three changes of xylene, and were embedded in paraffin. Serial sections, each of 4-micron thickness, were cut and stained with H and E as per standard protocols [17].

Statistical Analysis
The data were subjected to statistical analysis by using statistical package for social sciences (SPSS) version. Differences between means were tested using Student t-tests and significance was set at P < 0.05 and the results are expressed as Mean ± Standard Error of Mean (SEM).

Biochemical Parameters
The results as presented in Table 1 showed that the serum urea and creatinine increased significantly (p < 0.05) in the lead acetate exposed group compared to the control group. On the other hand, there was no significant difference for the serum electrolytes levels in both control and experimental groups (Table 2). Our results further showed significant decreases (p < 0.05) in the serum creatinine and urea in the groups that received concomitant treatment of lead acetate and 100 mg/kg JC, 200 mg/kg JC and 500 mg/kg HS extracts respectively compared to the lead acetate group. Values are mean ± SE of triplicate determinations. Superscript a and b indicates significant difference from normal and PbA control group respectively (p < 0.05) JC-Jatropha curcas HS-Hibiscus sabdariffa. Values are mean ± SE of triplicate determinations. Superscript a and b indicates significant difference from normal and PbA control group respectively (p < 0.05) JC-Jatropha curcas HS-Hibiscus sabdariffa.

Histopathological Examination
The examination of the photomicrographic section of the kidney of the normal control rats (Figure 1) and the groups that received the plant extracts only (Figures 3-6) showed histological normal kidney architecture with renal tubules, glomeruli and Bowman's capsular space appearing normal. The histological sections of the PbA exposed rats revealed distorted kidney with glomerulus having obliterated Bowman's capsule (Figure 2). Concomitant treatment of PbA exposed rats with 200 mg/kg J. curcas extract for 28 days induced a moderate restoration of the kidney architecture ( Figure 8). A similar trend was also observed for the PbA exposed rat group that received 500 mg/kg H. sabdariffa extract (Figure 10).

Discussions
Lead is the most abundant of all the nephrotoxic elements, and there have been both extensive clinical studies of lead-induced nephropathy and corroborative studies in experimental animal models which have yielded extensive insight into both the mechanisms of cellular toxicity and factors regulating these processes [8,18]. Oxidative damage has been identified as the main mechanism by which lead induces nephrotoxicity [19]. J. curcas and H. sabdariffa are two plants that have been used in traditional medicine to treat conditions such as anaemia, inflammation, and microbial activities. It has also been reported that both plants are sources of potent antioxidants [20][21][22]. The current investigation was designed to assess the nephroprotective potentials of J. curcas and H. sabdariffa against lead acetate-induced toxicity in the kidney. In this study, we observed that treatment with 50 mg/kg body weight of lead acetate for 28 days had a significant effect on serum renal markers and was associated with changes in kidney tissue including degeneration, vacuolization and debris.
In lead-induced nephrotoxicity, creatinine and blood urea levels become elevated in the blood. The biomarkers serum creatinine and blood urea are recommended for the assessment of kidney injury in preclinical studies as they are generally considered as more specific sensitive indicators of kidney damage. Low levels of creatinine and urea are normally found in the blood, but when the kidney is damaged or diseased, creatinine and urea accumulate in the bloodstream [23]. Asgharian et al. [24] reported a positive correlation between elevated blood lead levels and serum creatinine concentrations in animal models. Gennart et al. [25] similarly found that serum creatinine and blood urea concentrations were elevated in lead-exposed subjects. In agreement with these findings, our results revealed that lead administration resulted in increased serum creatinine and blood urea concentration in the lead acetate exposed group compared to the normal group. Creatinine directly relates to the muscle mass, and its serum level is used to evaluate the glomerular filtration rate because it is readily filtered and not subjected to any significant tubular reabsorption [26]. The elevated creatinine levels observed in the PbA treated group suggests possible impaired renal function confirmed by inadequate clearance of creatinine by the kidney.
The kidney performs the major function of excreting drugs and metabolites from the system, regulation of extracellular pH, and electrolyte contents, hence, plays a central role in homeostasis [28]. In this study, concomitant administration of PbA and J. curcas resulted in decreased serum creatinine and urea concentrations in a dose-dependent manner. A similar trend was also observed in the experimental groups that received PbA and H. sabdariffa. However, J. curcas administration was more effective in restoring the urea and creatinine levels. Our results also revealed non-significant changes in the electrolytes (Na + , K + , Cl -, and HCO3 -) levels in the PbA treated rats compared to the normal control. Mouw et al. [29] had previously reported that renal tissue electrolytes were unaltered by lead. Our results were consistent with his findings.
Histological examination of renal sections in lead acetate (50 mg/kg) treated group showed the degeneration and necrosis in tubules and swelling in the glomerulus, as compared to control negative group. Glomerular and tubular epithelial changes were mild in the group that received co-administration of PbA and aqueous extract of the 100 mg/kg J. curcas showed mild tubular epithelial changes while in case of animal treated with aqueous extracts of H. sabdariffa showed regeneration in tubular epithelial cells. We think that morphological changes in kidneys were because of lead acetate, but these changes tended to be considered mild in lead acetate plus plant extract treated groups. The restoration pattern was dose-dependent and showing different levels of protection in the rat tissues treated with H. sabdariffa aqueous extract. The present study reveals that the protective action of J. curcas and H. sabdariffa might be due to the interaction of the various vital phytoconstituents, such as flavonoids and polyphenolic compounds present in the plants.

Conclusions
The results suggest that the aqueous extract of J. curcas and H. sabdariffa might reduce lead acetate-induced nephrotoxicity by their antioxidant activities. Further experiments are however needed to determine their exact mechanism of protection.