Efficacy of jamun Syzygium cumini seed and orange Citrus sinensis peel extracts against microcystin LR induced histological damage in the kidney of rat

. In this study we evaluated the protective effects of jamun Syzygium cumini seed and orange peel extracts on renotoxicity of microcystin LR in male rats. Groups A-F were given daily treatments for 30 days. Group A (Control): No treatment was given; Group B: microcystin (10 µg/kg body wt); Group C: microcystin (10 µg/kg body wt) and jamun seed extract (200 mg/kg body wt); Group D: microcystin (10 µg/kg body wt) and orange peel extract (200 mg/kg body wt; Group E: orange peel extract (200 mg/kg body wt); Group F: jamun seed were not seen in tubular lumina. In OPE (group E) and JSE (group F) treated rats the kidney exhibited no histological changes.


Introduction
In many parts of the world an increasing environmental hazard has been reported due to toxic cyanobacteria which are found in freshwater, eutrophic and municipal water supplies (Filipic et al., 2007). Cyanobacteria produce hepatotoxins, neurotoxins and lipopolysaccharide endotoxins (Srivastava and Srivastav, 2017). Ingestion of cyanobacteria adversely affects domestic and aquatic animals as well as humans (Elleman et al., 1978;Zin and Edwards, 1979;Bell and Codd, 1994;Negri et al., 1995;Benson et al., 2005;Shahi et al., 2012;Srivastava and Srivastav, 2017).
The present study was aimed to evaluate the protective effects of extracts of jamun (Syzygium cumini) seeds and orange (Citrus sinensis) peels against microcystin LR (MCLR) induced histological changes in the kidney of male rats. Prior to this study no report exists regarding protective effects of extracts of seed of Syzygium cumini (JSE) and peels of Citrus sinensis (OPE) on renotoxicity induced by microcystin LR.

Materials and methods
Male Wistar rats (70-90 g) were housed in polypropylene cages and acclimatized for 2 weeks under laboratory conditions prior to different treatments. During entire experimental period rats were maintained on the standard laboratory feed and water ad libitum. Animal handling and sacrifice were carried out following the guidelines provided by Ethics Committee of the University.
After acclimation rats were divided into six groups -A, B, C, D, E, and F, each consisting of 20 animals. Following treatments were orally given daily to these groups at 08:00 each day throughout the experiment: Rats from all the groups (10 from each group) were sacrificed 24 h after last dose on 15th and 30th day after initiation of the experiment under light ether anesthesia. Animals were fasted overnight before sacrifice.
For use in experiment purified Microcystin LR (Enzo Life Sciences, Inc.) was dissolved in 0.9% NaCl. The preparation of jamun seed and orange peel extracts have been described in detail by Srivastava et al. (2018).
Kidney of control and treated rats were fixed in Bouin's fluid. These fixed tissues (kidney) were dehydrated in an ethanol gradient, treated with a clearing agent, infiltrated and embedded in paraffin, sectioned at 6 μm, floated on a heated water bath and mounted to glass slides. After drying overnight, paraffin was removed with a clearing agent, tissue was rehydrated in an ethanol gradient and then stained with hematoxylin and eosin (HE) for light microscopic examination.
For electron microscopic studies, small kidney pieces were fixed in paraformaldehyde and glutaraldehyde mixture for 4 h at 4 °C, washed with phosphate buffer and stored at 4 °C. These tissues were processed at Sophisticated Analytical Instrument Facility, All India Institute of Medical Sciences, New Delhi, India.

Results
Kidney of control rats contain numerous nephrons, each nephron consist of a dilated portion, the renal corpuscle; the proximal tubule; loop of Henle and the distal tubule. The renal corpuscles contain a tuft of capillaries, the glomerulus which is surrounded by the Bowman's capsule (Figure 1). There is present the urinary space between the glomerulus and Bowman's capsule. The proximal tubule is lined by simple cuboidal (Figure 2) or columnar epithelium whereas the distal tubule is lined by simple cuboidal epithelium (Figure 1).  In 15 day MCLR treated rats (group B) the glomeruli at few places are noticed to be shrunken thus more space is visible between the Bowman's capsule and glomerulus ( Figure 2). The proximal and distal tubules show hypertrophy of epithelial cells, vacuolation of cytoplasm and exhibit obliterated lumina (Figures 3 and 4). In MCLR+JSE (group C) and MCLR+OPE (group D) treated rats almost similar changes are noticed as seen in MCLR treated rats. In OPE (group D) and JSE (group F) treated rats no visible morphological alterations are seen.  Following 30 day MCLR treatment (group B), the cellularity of glomeruli is increased at several places thus there is no space between the Bowman's capsule and glomerulus ( Figure 5). However, at some places glomeruli are seen degenerating ( Figure 6). The proximal and distal tubules are dilated (Figure 7). Tubules also display separation of epithelial cells from underlying basement membrane, vacuolization and degeneration having necrotic nuclei in lumina (Figures 7, 8 and 9). Large deposits of eosinpositive material appear in the tubules (Figures 8 and 10). In MCLR+JSE (group C) and MCLR+OPE (group D) treated rats the glomeruli are swollen showing increased cellularity. No degeneration is noticed in glomeruli. Tubules are dilated, however, at few places few epithelial cells are degenerating. Necrotic nuclei are not seen in tubular lumina. In OPE (group E) and JSE (group F) treated rats the kidney architecture is almost similar to control rats.

Figure 5.
Glomerulus exhibiting hypercellularity after 30 day microcystin treatment. Note the absence of space between the glomerulus and Bowman's capsule. Hematoxylin-eosin X 500.
In the foregoing study shrinkage of glomerulus has been observed in MCLR treated rats. Shrinkage of glomerulus concordant with this study has also been reported in rats after exposure to paraquat (Abdel-Mageid, 1994), chlorpyrifos (Tripathi and Srivastav, 2010) and cadmium (Tripathi and Srivastav, 2011). The degeneration of glomerulus in MCLR treated rats may cause decreased GFR as Barrouillet et al. (1999) have noticed decrease in GFR in their in vitro studies of cadmium exposure.
In MCLR treated rats degenerated cells have been encountered in the tubular lumina. Hosser et al. (1989) and have reported eosinophilic to basophilic material within the lumina of renal cortical tubules after MCLR treatment to rats. After microcystin treatment Suput (2011 -in rats) and Xiping et al. (2019 -in mice) have noticed dilated tubules filled with eosinophilic material. Brzoska et al. (2003) and Shashi et al. (2002) have also reported occurrence of degenerated cells in tubular lumina of rats (exposed to cadmium) and rabbit (exposed to fluoride), respectively.
Kidney alterations such as tubular vacuolization, dilatation and degeneration caused by toxicants might be due to hydrolic changes in the renal tissue. These changes clearly indicates that toxicants provoke renal failure in pump transport of tubular cells and also cause functional disturbances in kidney. In past, it has been suggested that dilatation may be a compensatory mechanism after the loss of renal excretory function (Sanchez-Chardi et al., 2009).

Conclusion
We can conclude that exposure to microcystin adversely affected the kidney structure of the rats. The structural damage caused by microcystin to this vital organ could be protected by supplementation of extracts of jamun seed and orange peel. It is suggested that the microcystin exposed organisms should be given dietary supplement of these botanical extracts which would ease the toxic symptoms.