Annals of the College of Medicine, Mosul

ddiction is a growing public and health problem widespread in spite of all hard work to avoid and switch it; painkillers are amid the most common medications which abused. ¹ Among   the opioids that frequently prescribed include morphine, tramadol, methadone, diamorphine, and codeine. ²

   Tramadol is a opioid pain reliever that acting centrally which is mostly in use for combating pain ranging from moderate to severe.³ It is analgesic effect has a complex mechanism; as many  reports suggested that opioid and non-opioid mechanisms  of action is responsible for it's painkilling effects and other  medical properties. ⁴

   The absorption of tramadol is 95-100% happen in small intestine namely in its upper part with bioavailability is 70% and when tramadol used in frequent doses, it is bioavailability amplified reaching to 100%.⁵

   The metabolized of tramadol done in the liver via cytochrome p450 enzyme system and its bio transformed products are expelled by the kidneys by urine  accordingly; this creates the kidney is a main goal organ of tramadol toxicity particularly in misuse and over dose cases. ⁶ About 30% of the dose is expelled in the urine as unaffected drug, and 60% of the dosage is eliminated as metabolites while the residual drug is evacuated with the feces. ⁷

MATERIAL AND METHODS

In the current study both tramadol hydrochloride  and  vitamin C ampoules are used and  are obtained from local pharmacies in Iraq. Thirty healthy Wister albino male rats weighting about (220-250) gm. and aged three months are used in this study. These animals were kept at controlled room temperature (23-25Cº) with a 12 hours light/ dark cycle and placed in plastic cages using homogenized wood shaving as bedding for acclimatization for one week before start of the experiment. The animals had allowed access of both water ad libitum and packed optimized food. The animals were divided into three groups.

   Group A (control group(: are daily injected with distilled water intraperitoneally (I.P). Group B (tramadol treated group) (TGI):are injected with tramadol in dose 50mg/kg I.P. daily for four weeks. Group C (treated with tramadol + Vitamin C) (TGII): are injected with 100 mg/kg b.wt. of vitamin C  I.P. daily half hour prior to tramadol injection in a dose 50 mg /kg b.wt. I.P.  for four weeks.

   After finishing of the fourth weeks of experiment, the animals were sacrificed and kidneys were removed and utilized for histopathological  examinations, in which  10% neutral buffered formalin was used as fixator for the specimens which then processed to get paraffin sections of 5μm thickness, that stained with Haematoxylin and Eosin to be examined by  light microscopic.

RESULT

Group A (Control group):Light microscopic study showed no deviaton in histological findings from those seen in other normal tissues, in which kidney sections consist of renal cortex and medulla. In the cortex there is renal corpuscle, proximal convoluted tubules (PCT) along with distal convoluted tubules (DCT). Every renal corpuscle consists of blood capillaries tuft, named as glomerulus bounded by Bowman's capsule. The latter is a double walled cup formed of two layers of simple squamous epithelium, an outer parietal layer and an inner visceral one separated by a capsular space. The PCT is lined by cuboidal epithelium with spherical and basally located nuclei. The DCT is lined by cuboidal epithelium and spherical and apically located nuclei and their cytoplasm is lighter than those of PCT (Fig. 1).

   Group B (TGI): Shows atrophy and congestion of glomeruli, others showed segmentation and lobulation along with widening of bowman's space (Fig. 2). The most severe changes are observed in the tubules and mainly PCT as some of tubules are dilated, others showed marked hydropic degeneration and cytoplasmic vacuolization of tubular epithelial cells (Fig. 3). Patches of mononuclear cells (MNCs) infiltration are noticed around blood vessels and  within the necrotic tubules (Fig. 4). Hyaline cast is seen within the cortex mainly inside the degenerated tubules along with   hemorrhage in the interstitisum (Fig. 5).

Figure 1: Photomicrograph of control group showing glomerulus (black arrow), bowman's space (arrow head), PCT (blue arrow) and DCT (green arrow) (H & E X 400).

Figure 2: Photomicrograph of TGI showing glomerular atrophy with segmentation (red arrow) and widening of bowman's space (arrow head)(H & E X 400).

   Group C (TGII): shows more preserved kidney architecture, most glomeruli regained their normal appaerance (Fig. 6) Some glomeruli showed mild atrophy with mild dilatation of bowman's space (Fig. 7). Few tubules showed vacoulation in their epithelial cytoplasm (Fig. 8). Interstitial bleeding is still present but mild(Fig. 9) and little MNCs infiltration in the interstitisum of renal cortex (Fig. 10).

Figure 3: Photomicrograph of TGI showing extensive hydropic  degeneration (black arrows), with interstitial bleeding (arrow head) (H &E X 400).

Figure 4: Photomicrograph of TGI showing MNCs infiltration within the interstitisum (black arrows) (H &E X 400).

Figure 5: Photomicrograph of TGI showing hyaline casts within the degenerated tubules (black arrows), extensive vacuolar degeneration (red arrow) and hemorrhage (green arrow)(H &E X 400).

Figure 6: Photograph of kidney rat of TGII showing  normal architecture of renal cortex with nearly normal looking glomeruli (black arrows ) (H&EX100).

Figure 7: Photomicrograph of TG II showing  mild glomerular atrophy (black arrow) with mild dilatation of bowman's space (arrow head) (H & E X 400).

Figure 8: Photomicrograph of TGII showing nearly normal looking renal corpuscle (red arrow) while few tubules showed vacuolar degeneration (green arrow) (H&EX400).

Figure 9: Photomicrograph of TG II showing interstitial bleeding (green arrow) with scattered vacuolar degeneration (red  arrows) with (H & E  400).

Figure 10: Photomicrograph of TG II showing mild MNCs infiltration (red arrow)(H & E  400).

DISCUSSION

As the kidney is the main excretory organ for drugs and chemicals, so it is susceptible to toxicity which requests for its continuous clinical assessment throughout treatment with drugs to defense against toxicity.⁸

   In the current study there is many microscopical deviations in the tramadol treated group TGI if compared to the group A (control group), as glomerular atrophy, congested glomeruli and widening of bowman's space are in agreements with El-khateeb et al,2015⁹ along with Youssef and Zidan, 2016 ¹⁰ findings. Some glomeruli in TGI group showed segmentation and lobulation and this run with similar finding reported by Zaghlol et al., 2018. ¹¹

   The mechanism of expansion of the glomerular chamber and swelling of their lining epithelium and consequently glomerular atrophy result from drop in aerobic metabolism due to decrease in O2 level, as renal cells consume O₂ at high rate and in need to aerobic metabolism for ATP generation.¹² The cells must rely more on glycolysis in order to maintain such ATP levels, as  glycolysis can leads to the accumulation of lactic acid, that causes drop in the intracellular pH which induce cell dysfunction of the Na+/K+ ATPase followed by an influx of sodium and H₂O with consequent cell swelling. ¹³

   Another important finding of the current study is the  degenerative injuries in the renal tubules particularly in  PCT. A similar dose i.e. 50mg/kg b.wt produced the same degree of degeneration after treating animal   for only two weeks as reported by Hafezet al.,2015.¹⁴  Also such tubular epithelial injuries induced by tramadol was reported by many studies. ^9-11 The mechanism of tubular damage of the renal tubules is particularly due to sensitivity of renal tubules to toxic effects, in part since they have high consumption of O₂ and weak enzyme system. ¹³ On the other hand a study done by Atici et al., 2005¹⁵ indicated that the risk of increased level of lipid peroxidation and renal damage after prolonged use of opioids, particularly morphine in the rat's kidney.

   Other observation in the current study is MNCs infiltration in the renal cortex around degenerating tubules giving  a picture of interstitial nephritis, this finding is in agreement with previous  studies ^9,10,15, and this  finding in addition to the other findings can be reflected as a sign of renal damage as described by Muslim Z,2018¹⁶ while is in conflicts with this finding by Zaghlol et al., 2018 ¹¹and Ali et al., 2018¹⁷ and this might be due to small dose or small dose plus short period, respectively.

   The hyaline cast seen within the degenerating tubules is another finding in the present study which ascribed to the  solidification of Tamm-Horsfall mucoprotein, that  secreted by the renal tubular cells.¹⁸

   Many researchers proposed that vitamins, mineral selenium,  ascorbic acid,  quercetin and melatonin fractions in date fruit may be held responsible for renal protecting action. ¹⁹ However, yet now there is no previous study  about the role of vitamin C against nephrotoxicity  induced by tramadol. In the current study using of vitamin C, result in mild improvement in many microscopical  changes induced by tramadol and  showed more preserved renal architecture, with decrease in glomerular atrophy, bowman's space dilatation with degenerated tubules and  mild to moderate MNCs infiltration, but congestions of interstitisum and blood vessels is still present.

   Many researchers proved the protective effect of vitamin C against histological changes induced by formaldehyde.^20,21 Also vitamin C as chemo protective agent was proved by  Al-jammas S.,2011 after he injected rats with vitamin C prior to cisplatineum result in improvements in the microscopical changes induced by cisplatineum. ²²

   The protection produced by vitamin C in renal tissue in concomitant use with tramadol was recognized due to antioxidant property of vitamin C that capable to reduce lipid peroxidation which results in alleviate kidney damage by enrichment of scavenging capability of antioxidant defense organization. ²³ But such improvement is not bring the renal tissues to complete normality which might be due to short period of treatment with antioxidant vitamin C or might need a larger doses vitamin C.

CONCLUSION

Tramadol had toxic effects on kidney structure as glomerular atrophy, widening bowman's space and degenerating tubules with vacoulation in their epithelial lining, all are improved after the addition of vitamin C.

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