ISSN 2277 758X International Journal of Advanced Life Sciences (IJALS) Hussein M. Ageely et al., IJALS, Volume (8) Issue (2) May - 2015. RESEARCH ARTICLE Morphological and apoptotic changes in the kidney of rats after Khat extract administration Hussein M. Ageely* and Alaa S. Abou-Elhamd**, *** *Department of Internal Medicine, Faculty of Medicine, Jazan University, Saudi Arabia. **Department of Anatomy and Histology, Faculty of Veterinary Medicine, Assiut University, Egypt. ***Department of Medical Biology, Faculty of Medicine, Jazan University. E-mail : hageely@me.com Corresponding Author Hussein M. Ageely Department of Internal Medicine, Faculty of Medicine, Jazan University, Saudi Arabia. Email : hageely@me.com, Article History Received on 20 March, 2015; Received in revised form 22 April, 2015; Accepted 12 May, 2015 Abstract The habit of Khat-chewing has prevailed for centuries among populations in the horn of Africa and the Arabian Peninsula. It represents a major socio-economic problem in many countries. The biological effects of Khat on the kidney are inadequately investigated. The aim of this study is to investigate the histological and morphological changes in kidney of rats after crude Khat extract treatment. The present study was carried out on 60 male rats for one month. They were divided into two groups (Control and treated groups). Half of each group was sacrificed after two weeks from the beginning of the experiment. Khat treated rats showed non-significant differences (P< 0.01) in their kidney weights (gm/ 100gm BW). Hematoxylin and Eosin stained sections of the kidney parenchyma of Khat treated group showed abnormal architecture of the kidney parenchyma, congestion and atypical tubules. Degenerative changes in the renal tubules were also observed. Immunohistochemistry by using TACS® 2 TdT DAB apoptosis detection kit indicates many apoptotic cells in the parenchyma of the kidney of Khat treated group compared to the control one; specially in the Malpighian corpuscles. In conclusion, Khat consumption could be associated with histopathological changes in the kidney and renal diseases. Keywords : Khat, Kidney, rat, apoptosis, parenchyma, congestion and atypical tubules. Introduction Khat (Catha edulis) is an evergreen shrub growing in East Africa and Southern Arabia. In these regions, people chew fresh Khat leaves for their psychostimulant properties (Cox and Rampes, 2003). Since only fresh Khat leaves produced the desired stimulant effect, until recently their use remained virtually unknown beyond the areas of plantation (Kalix, 1992 and Al-Mamary et al., 2002). The extract of fresh leaves of Khat contains a number of pharmacologically-active compounds (Kite et al., 2003) such as nor-pseudoephedrine (cathine) and alphaaminopropiophenone (cathinone). The latter causing the major pharmacological effects (Kalix, 1992). Cathine and Cathinone are similar in structure and pharmacological activity to amphetamines (Wagner et al., 1982). Several literature indicated that repeated Khat consumption induces not only cytotoxic effects in cells (Al-Ahdal et al., 1988; Al-Mamary et al., 2002; Al-Meshal et al., 1991; Dimba et al., 2003; Al-Habori, 2005), but also it is associated with an increase in micronuclei frequencies in oral mucosa and urothelial cells, suggesting genotoxic effects (Kassie et al., 2001). Khat extract was shown to contain the major alkaloid compounds cathinone and cathine. The compounds Int. j. Adv. Lif. Sci., Available online on at www. Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 145 ISSN 2277 758X International Journal of Advanced Life Sciences (IJALS) Hussein M. Ageely et al., IJALS, Volume (8) alone and in combination also induced apoptosis (Dimba et al., 2004). In vitro, Khat induced apoptotic cell death in primary normal keratinocytes and fibroblasts (Lukandu et al., 2008) as well as in L02 cells (Abid et al., 2013). Administration of the Catha edulis extract induced cytotoxic effects in the liver and kidney (Al-Habori, 2005). In addition, acute tubular nephrosis in kidney of laboratory animals was recorded (Al-Habori et al., 2002; Al-Mamary et al., 2002). Moreover, Khat extract increased oxidative stress parameters and impair renal and hepatic functions in rats (Al-Hashem et al., 2011b). The aim of the present work is to study the effects of Khat extract on the kidney of rats by using morphological, histological and immunohistochemical methods. Material and methods Khat extraction The processes of Khat extraction were performed as previously described by Ageely et al. (2014). Fresh Khat bundles were purchased from local Yemeni markets and transferred to the biology department of the faculty of medicine for processing. The fresh leaves were washed thoroughly with distilled water, blotted neatly, then chopped and finally crushed. Crushed material was immersed in a conical flask containing sufficient amounts of methanol (Sigma-Aldrich) and kept on rotary shaker for 18 hrs. Filtration of the previous mixture was carried in two steps; firstly using the gauze roll to separate the larger particles and secondly with Whatman No. 1 filter paper. The resultant filtrate was evaporated in a vacuum at 60 65 °C to remove all traces of methanol (Kimani et al., 2008), thus leaving semi-solid material (i.e. the extract) kept as a powder in the refrigerator. The fresh solution Khat extract was prepared by dissolving the powder of Khat extract in distilled water, just prior to its oral administration to the rats daily throughout the experiment for 30 days. Issue (2) May - 2015. RESEARCH ARTICLE Experimental Animals After Ageely et al. (2014), 60 Sprague-Dawley (SD) male rats weighting between 100 - 150 gm was used for the study. The animals were housed in group cages and given free access of food and water. The rats were housed at a 24˚C room temperature and 12-h: 12h light and dark cycle. They were acclimatized for one week before starting the experiment. Experimental design The rats were assigned randomly into two main groups, each consisting of 30 animals: the Khat extract - treated groups and the control-untreated group. The Khat treated rats groups were given orally 700 mg/kg body weight Khat extract (Al-Qirim et al., 2002) daily throughout the period of the experiment (30 days). While the control rats group were given orally normal saline (volume equal to that of the dose of extract in the previous group) daily throughout the period of the experiment. After 15 days from the beginning of the experiment, half of the animals from the control and treated groups were sacrificed. They represented as control I and treated I groups and the other animals were sacrificed at the end of the experiment after 30 days; were denoted as control II and treated II groups. The body weights of the animals were recorded at the beginning and at the end of the experiment just before sacrificing. Tissue preparation and examination Fixation and embedding For histological examination, kidney tissue pieces of appropriate dimensions were fixed in 10% neutral buffered formalin and processed for embedding in paraffin. Samples for immunohistochemistry were fixed in 4% neutral buffered formalin. Sectioning, staining and examination Paraffin-embedded tissue sections were cut by Int. j. Adv. Lif. Sci., Available online on at www. Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 146 ISSN 2277 758X International Journal of Advanced Life Sciences (IJALS) Hussein M. Ageely et al., IJALS, Volume (8) microtome into 7 - 5 µm thick sections. Sections were stained by routine histological stain (Hematoxylin and Eosin, (Harris, 1900) for general examination and identification of the stages of apoptotic cells morphologically. The sections were examined and photographed on an Olympus microscope with DP72 camera and cell software. Images were imported into adobe Photoshop for labelling. Immunohistochemistry To study the effect of Khat extract on apoptosis; 4% neutral buffered formalin fixed samples were processed and embedded in paraffin. Sections of 5 µm thickness were prepared and processed for immuno- staining by using TACS® 2 TdT DAB in situ Apoptosis Detection Kit (Trevigen, USA) according to the manufacturer’s instructions (Ageely et al., 2014). The number of apoptotic cells was estimated by counting the number of apoptotic cells in five microscopic fields in each animals of the control and treated groups. The counting process was performed at high power (X400). Statistical analysis The parameters measured, the body weights, the ratios of the kidney weight relative to body weight, numbers of the apoptotic cells are expressed as mean ± SEM for the control I and II, Khat-treated I and II groups. The one-way analysis of variance (ANOVA) and post hoc by the Scheffe test were used to statistically analyze and compare the means of control groups with the correspondent Khat extract-treated groups using SPSS version 17. P <0.05 was considered significant. Results Issue (2) May - 2015. RESEARCH ARTICLE As well as in this report, we found that the relative rat kidneys weight was nonsignificantly (ANOVA, p ≥ 0.05) different between groups (Table - 1 and Fig.- 1). Histological and immunohistochemical observations Microscopic examination of kidney sections of control rats showed normal renal parenchyma with normal histoarchitecture. Typical and normal histological features of the Malpighian corpuscles, glomerular capillaries, Bowman’s capsule and renal tubules were observed (Fig. 3A). Kidneys of Khat - treated rats showed abnormal architecture of the kidney parenchyma such as amorphous Malpighian corpuscles, hypertrophied glomerular capillaries, dilated Bowman’s capsule, congestion, typical tubules and infiltrative inflammatory cells (Fig. 3B, C and 4). Furthermore, degenerative changes in the renal tubules as well as cytoplasmic vacuolization of tubular cells and foamy appearance in the tubular epithelial cells. These changes were well marked in the Khat-treated II group (Fig. - 5). Immunohistochemical studies were performed by using TACS® 2 TdT DAB for the detection of apoptotic cells, which showed that the kidney of Khat - treated I and II rats had significant increment in the numbers of the apoptotic cells (8 ± 1.08, 12.83 ± 1.53, respectively) when compared to the corresponding control I and II groups (5.25 ± 1.65, 6± 1.47, respectively) (Fig. - 6). Most of the apoptotic cells were observed in the Malpighian corpuscles (Fig. 7A - D). Discussion The present results are consistent with Physical parameters Shewamene and Engidawork (2014) who found that Khat- treated rats showed a non-significant increment In our previous study (Ageely et al., 2014) we observed that there is no significant difference between the mean final body weights of the control- and Khat treated rats groups (Supplementary data, Fig -2). in their relative kidney weights. Shewamene and Engidawork (2014) added that a dose of crude Khat up to 400mg/ kg produced a significant increase in normalized kidney weight compared to the control. Int. j. Adv. Lif. Sci., Available online on at www. Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 147 ISSN 2277 758X International Journal of Advanced Life Sciences (IJALS) Hussein M. Ageely et al., IJALS, Volume (8) Issue (2) May - 2015. RESEARCH ARTICLE Table - 1. Kidney weights of the control and Khat-treated rats relative to their final body weights (represented in gm/ 100gm of BW). Groups Parameters Control I Mean Body weight(gm) Mean Right Kidney Wight(gm) Right Kidney weight (gm/100 gm of BW) Mean Left Kidney weight (gm) Left Kidney weight (gm/100 gm of BW) Treated I Control II Treated II 144.2 ± 15.56278 0.62 ± 0.069693 151.428 ± 11.712 0.54571 ± 0.101353 143.66 ± 13.17941 0.60666 ± 0.07101 144.25 ± 13.04096 0.6625 ±0.061482 0.43419 ± 0.020162 0.35928 ± 0.021362 0.42250 ± 0.014468 0.46093 ± 0.014863 0.646 ± 0.099688 0.58428 ± 0.098739 0.65166 ± 0.099346 0.70375 ± 0.063653 0.44720 ± 0.016290 0.38541 ± 0.022145 0.45289 ± 0.03515 0.489641 ± 0.01514 Data are presented as mean ± SEM Right Kidney 0.6 Left Kidney 0.4 0.2 0 Control I Treated I Control II Treated II Fig. -1. Histogram showed that no significant differences in the mean kidneys rats weights (gm/ 100gm of BW) of the Khat treated groups (treated I and II) comparing to corresponding control ones (Control I and II). Error bars represent standard errors (Kidney weight (gm/100 gm of BW) 180 160 140 120 100 80 60 40 20 0 Initial body weight (gm) Control I TreatedI Final body weight (gm) Control II Treated II Fig.-2. Histogram showing the initial and final mean body weight (gm/ 100gm BW) of the control and Khat treated rats groups (Ageely et al., 214). Int. j. Adv. Lif. Sci., Available online on at www. Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 148 ISSN 2277 758X International Journal of Advanced Life Sciences (IJALS) Hussein M. Ageely et al., IJALS, LS, Volume (8) Issue (2) May - 2015. RES ESEARCH ARTICLE Fig.- 3. Khat-treated rats showed abnor abnormal architecture of the kidney parenchyma. a. Kidneys K of Khat-treated rats (I& II) showed abnormal archite itecture of the kidney parenchyma in the form off amorphous am Malpighian corpuscles, hypertrophied glomerular ular capillaries, dilated Bowman’s capsule (B& C,, asterisks) as compared to the control group (A). Invasiv vasive inflammatory cells (B, arrowheads) and degene enerative changes in the renal tubules. Fig.-4. Photomicrograph showing wing abnormal architectures of kidney parenchym yma, congestion (asterisks) and infiltrative inflammator tory cells (arrows). Haematoxylin and Eosin stain, X 200. 00. 20 Fig.-5. Administration of Khat extract led to degenerative changes in rat at kidneys ki tubules. A typical tubules, cytoplasmic vacuoli uolization of tubular cells (arrows) and foamy appear ppearance in the tubular epithelial cells of the Khathat- treated II group were observed. Haematoxylin and nd Eosin E stain, X 1000. Number of apoptetic cells 15 10 5 0 Control I Treated I Control II Treated T II Fig. - 6. Histogram showing a signif gnificant (p<0.01) increment in the number of apoptotic apoptot cells in the Khat treated II group (Mean + SE) when n ccompared to their control group. Error bars repre present standard errors. Int. j. Adv. Lif. Sci., Available online on at www. Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 149 International Journal of Advanced Life Sciences (IJALS) Hussein M. Ageely et al., IJALS, LS, Volume (8) Issue (2) May - 2015. ISSN 2277 758X RES ESEARCH ARTICLE Fig. - 7. Photomicrograph showing ng h high number of apoptotic cells (arrows) in the kidney kidne parenchyma of the Khat treated (I & II) group of rats ts ((B & D) when compared to their control groups oups (A ( & C). TACS® 2 TdT DABapopt apoptosis detection kit, A, B & C X1000, D: X 400. They argue this by the acute tubular necr necrotic changes caused by Khat consumption (Erdem et al., 2000). In agreement with Al-Hashem et al. (2011a); Alsalahi et al. (2012); Shewamene and nd Engidawork (2014) the present study showed thatt aadministration of Khat led to an abnormal architecturee of the kidney parenchyma, typical tubules and infilt nfiltrative inflammatory cells, furthermore, degenerat rative changes in the renal tubules. Theses degenerat rative changes support the earlier findings in histop histopathological changes in the liver (Ageely et al., 2014 14) and kidney (Shewamene and Engidawork, 2014) ass associated with administration of Khat. Although the m mechanism by which Khat produces nephrotoxicity is not clearly known, it is thought that they result from rom a decrease in the blood supply to the kidney, possi possibly from the vasoconstrictive effect of Khat (Al-M l-Motarreb and Broadley, 2003; Shewamene and Engidawo dawork, 2014). By using TACS® 2 TdT DAB (a marker for apoptosis), the present study showed ed that Khat induced apoptosis in kidney cells lining ng tthe glomeruli and tubules. Thos in agreement with Abid id et al. (2013); who reported that Khat induced apoptosi osis in primary normal oral keratinocytes and fibroblas lasts and L02 cells, respectively. Furthermore, it induc duced apoptosis and exacerbated acute cerebral infarct rction through LO expression (Alsharafi modulating Smac/ DIABLO et al., 2014). Khat extract has cytotoxic effects in cells (Al-Mamary et al., 2002 and Dimba et al.,2003). ound morphological effects Moreover, it induced profound loid leukemia cells (Dimba in HL-60 human acute myeloi nism of the cytotoxic effect et al., 2004). The mechanism ertain. Dimba et al. (2004) of Khat on the liver is uncert hat extract was dependent found that cell death by Khat nthesis. Furthermore, the on de novo protein synthe ha edulis extract showed a administration the of Catha deranged systemic capacity to handle oxidative radicles and induced cytotoxic effect ects in cells of liver and kidney. Moreover, it induced uced cell death in various human leukemia cell lines and a in peripheral human blood leukocytes (Al-Habori bori, 2005) In vitro, Abid et al. (2013) demonstrated ed that Khat trigger the generation of intracellular reactive r oxygen species (ROS) and sequentially induced indu the sustainable activation of c-Jun NH2-termi rminal kinase (JNK), which in turn resulted in a decrease se in cell viability and an increase in cell apoptosis. Conclusion It could be concluded ded that the Khat had a deleterious effect on kidney ey tissue of rats, such as amorphous Malpighian corpuscles, cor hypertrophied glomerular capillaries, dilate lated Bowman’s capsule, Int. j. Adv. Lif. Sci., Available online on at www. Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 150 International Journal of Advanced Life Sciences (IJALS) Hussein M. Ageely et al., IJALS, Volume (8) congestion and atypical tubules. Moreover, Khat produced a significant increment in the number of apoptotic cells in the kidney. Acknowledgements We would like to thank Dr. Moustafa El-Nagar for constructive discussions, and critical reading of the manuscript. The project funded by the Medical research center, Jazan University project grant number 3048. [ References Abid, M.D., Chen, J., Xiang, M., Zhou, J., Chen, X. and Gong, F. 2013. Khat (Catha edulis) generates reactive oxygen species and promotes hepatic cell apoptosis via MAPK activation. Int. J. Mol. Med., 32: 389 - 95. Ageely, H.M., El-Nagar, M.M., Abouelmagd, A., Abou-Elhamd, A.S., Kelany, M.E. and Pati, B.R. 2014. Khat extract mediated morphological and histochemical alterations in rat liver. International Journal of Advanced Research., 2: 971 - 980. Al-Ahdal, M.N., Mcgarry, T.J. and Hannan, M.A. 1988. Cytotoxicity of Khat (Catha edulis) extract on cultured mammalian cells: effects on macromolecule biosynthesis. Mutat. Res., 204: 317-22. Al-Habori, M. 2005. The potential adverse effects of habitual use of Catha edulis (khat). Expert Opin. Drug Saf., 4: 1145 - 54. Al-Habori, M., Al-Aghbari, A., Al-Mamary, M. and Baker, M. 2002. Toxicological evaluation of Catha edulis leaves: a long term feeding experiment in animals. J. Ethnopharmacol., 83: 209 - 17. Al-Hashem, F.H., Bin-Jaliah, I., Dallak, M.A., Nwoye, L.O., Al-Khateeb, M., Sakr, H.F., Eid, R.A., AlGelban, K.S., Al-Amri, H.S. and Adly, M.A. 2011a. Khat (Catha edulis) Extract Increases Issue (2) May - 2015. ISSN 2277 758X RESEARCH ARTICLE Oxidative Stress Parameters and Impairs Renal and Hepatic Functions in Rats. Bahrain Medical Bulletin., 33. Al-Hashem, F.H., Bin-Jaliah, I., Dallak, M.A., Nwoye, L.O., Al-Khateeb, M., Sakr, H.F., Eid, R.A., AlGelban, K.S., Al-Amri, H.S. and Adly, M.A. 2011b. Khat (Catha edulis) Extract Increases Oxidative Stress Parameters and Impairs Renal and Hepatic Functions in Rats. Bahrain Medical Bulletin., 33: 1-9. Al-Mamary, M., Al-Habori, M., Al-Aghbari, A.M. and Baker, M. M. 2002. Investigation into the toxicological effects of Catha edulis leaves: a short term study in animals. Pytother. Res., 16 : 127 - 132. Al-Meshal, I.A., Qureshi, S., Ageel, A.M. and Tariq, M. 1991. The toxicity of Catha edulis (khat) in mice. J. Subst. Abuse., 3: 107-15. Al-Motarreb, A.L. and Broadley, K.J. 2003. Coronary and aortic vasoconstriction by cathinone, the active constituent of khat. Auton Autacoid Pharmacol., 23: 319 - 26. Al-Qirim, T.M., Shahwan, M., Zaidi, K.R., Uddin, Q. and Banu, N. 2002. Effect of khat, its constituents and restraint stress on free radical metabolism of rats. J. Ethnopharmacol., 83: 24550. Alsalahi, A., Abdulla, M.A., Al-Mamary, M., Noordin, M.I., Abdelwahab, S.I., Alabsi, A.M., Shwter, A. and Alshawsh, M.A. 2012. Toxicological Features of Catha edulis (Khat) on Livers and Kidneys of Male and Female Sprague - Dawley Rats: A Subchronic Study. Evid. Based Complement Alternat. Med., 829401. Alsharafi, W.A., Bi, F.F., Hu, Y.Q., Mujlli, H.M. and Xiao, B. 2014. Effect of Khat on apoptosis and related gene Smac/DIABLO expression in the Int. j. Adv. Lif. Sci., Available online on at www. Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 151 International Journal of Advanced Life Sciences (IJALS) Hussein M. Ageely et al., IJALS, Volume (8) cerebral cortex of rats following transient focal ischemia. Environ. Toxicol. Pharmacol., 39: 424 - 432. Cox, G. and Rampes, R. 2003. Adverse effects of khat: a review. Adv. Psychiatric. Treat., 9: 456 - 463. Dimba, E., Gjertsen, B.T., Francis, G.W., Johannessen, A.C. and Vintermyr, O. K. 2003. Catha edulis (Khat) induces cell death by apoptosis in leukemia cell lines. Ann. N Y Acad. Sci., 1010 : 384 - 8. Dimba, E.A., Gjertsen, B.T., Bredholt, T., Fossan, K. O., Costea, D.E., Francis, G.W., Johannessen, A.C. and Vintermyr, O.K. 2004. Khat (Catha edulis) induced apoptosis is inhibited by antagonists of caspase-1 and -8 in human leukaemia cells. Br. J. Cancer., 91: 1726 - 34. Erdem, A., Gundogan, N.U., Usubutun, A., Kilinc, K., Erdem, S.R., Kara, A. and Bozkurt, A. 2000. The protective effect of taurine against gentamicin induced acute tubular necrosis in rats. Nephrol. Dial. Transplant., 15: 1175 - 82. Harris, H.F. 1900. On the rapid conversion of haematoxylin into haematin in staining reactions. J. Appl. Microsc. Lab. Methods., 3: 777. Issue (2) May - 2015. ISSN 2277 758X RESEARCH ARTICLE Kimani, S.T., Patel, N.B. and Kioy, P.G. 2008. Effect of single and daily khat (Catha edulis) extract on locomotor behaviour in CBA mice. Scientific Research and Essay., 3: 187 - 196. Kite, G.C., Ismail, M., Simmonds, M.S. and Houghton, P. J. 2003. Use of doubly protonated molecules in the analysis of cathedulins in crude extracts of khat (Catha edulis) by liquid chromatography / serial mass spectrometry. Rapid. Commun. Mass. Spectrom., 17: 1553 - 64. Lukandu, O.M., Costea, D.E., Neppelberg, E., Johannessen, A.C. and Vintermyr, O.K. 2008. Khat (Catha edulis) induces reactive oxygen species and apoptosis in normal human oral keratinocytes and fibroblasts. Toxicol. Sci., 103 : 311 - 24. Shewamene, Z. and Engidawork, E. 2014. Subacute administration of crude khat (Catha edulis F.) extract induces mild to moderate nephrotoxicity in rats. BMC Complement Altern. Med., 14: 66. Wagner, G.C., Preston, K., Ricaurte, G.A., Schuster, C. R. and Seiden, L.S. 1982. Neurochemical similarities between d,l-cathinone and d-amphetamine. Drug Alcohol Depend., 9: 279 - 84. Kalix, P. 1992. Cathinone, a natural amphetamine. Pharmacol Toxicol., 70: 77 - 86. Kassie, F., Darroudi, F., Kundi, M., Schulte-Hermann, R. and Knasmuller, S. 2001. Khat (Catha edulis) consumption causes genotoxic effects in humans. Int. J. Cancer., 92: 329 - 32. Corresponding Author : Hussein M. Ageely, Department of Internal Medicine, Faculty of Medicine, Jazan University, Saudi Arabia. Email : hageely@me.com, © 2015, IJALS. All Rights Reserved. Int. j. Adv. Lif. Sci., Available online on at www. Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 152