The use of illicit drugs has become a worldwide health problem. It leads to numerous consequences at the health, economic, social and legal levels. These consequences interfere with the development of the countries and their efforts to respond to the needs of their populations. Apart from its economic cost, drug abuse leads to social problems among family members, abnormal behavior, crime, health and psychological problems as well as economic difficulties1.

Tramadol (Tr) is a synthetic opioid from the aminocyclohexanol group and is commonly used in clinical practice as analgesic for treatment of moderate to severe pain2. Tramadol is free of side effects of traditional opioid such as the risk for respiratory depression3 and drug dependence4. So, the abuse potential of tramadol is considered low or absent5, which is in contrast to other opioids.

Cannabis was used a long time ago for both recreational and medicinal purposes. The plant Cannabis sativa (Indian hemp) is the source of different psychoactive products. Marijuana comes from leaves, stems, and dried flower buds of the plant. Hashish is a resin obtained from flowering buds of the hemp plant6. Δ9-tetrahydrocannabinol (Δ9-THC) is the primary psychoactive compound and contributes to the behavioral toxicity ofcannabis7.

Calcium (Ca) is the most abundant divalent ion (Ca2+) in the body8. Calcium is reabsorbed along all segments of the nephron. The proximal tubule is the primary tubular segment where most of the filtered calcium is reabsorbed. Under normal condition, 98–99% of the calcium which passes into the tubule is reabsorbed. Approximately 60–70% of the filtered calcium is reabsorbed in the proximal convoluted tubule, 20% in the loop of Henle, 10% by the distal convoluted tubule, and 5% by the collecting duct9. Any calcium that is not reabsorbed then passes into what is now urine and into the bladder.

Copper(Cu) is important for all functions of many cellular enzymes10. The copper transporters ATP7A and ATP7B are expressed at distinct regions of the kidney11. They are both expressed in the glomeruli in minor amounts. ATP7A is localized in the proximal and distal tubules, while ATP7B is expressed in the loop of Henle. The two copper transporters work together to maintain copper balance within this tissue. The ability of ATP7A to traffic to basolateral membrane (an event associated with copper export) in response to copper elevation strongly suggests that ATP7A plays the major role in exporting copper from renal cells for reabsorption into the blood and in protecting renal cells against copper overload12. In another research, ATP7B expression in the loops of Henle suggests that once in the filtrate, ATP7B is responsible for copper reabsorption11. Previous data suggest that copper may be reabsorbed by the same pathway as that involved in iron reabsorption13, which occurs between the proximal and distal convoluted tubules, interpreted as the loops of Henle.

Zinc (Zn) exists as a divalent cation (Zn2+). The kidney epithelium is the site of urinary zinc excretion and reabsorption. Zinc reabsorption occurs in all the downstream segments of the nephron; proximal tubule, Loop of Henle, and terminal nephron segments14. Most of the filtered Zn is reabsorbed along kidney proximal tubule15. The kidney is considered the primary eliminator of exogenous drugs and toxins; impairment of excretion predisposes it to various forms of injury. Development of new biomarkers is needed for the specific diagnosis of nephrotoxicity at earlier stage16. These biomarkers include brush bodrder enzyme such as Leucineaminopeptidase (LAP)17, Lysosomal enzyme such as N-acetyl-β-D-glucosaminidase (NAG)18, and cytosolic enzyme such as α- glutathion S-transferase (α-GST)18. Thus, detection of enzymes in the urine potentially provides valuable information not only related to the site of tubular injury (proximal & distal tubule) but also to the severity of injury16.

Renal tubular cells, in particular proximal tubule cells, are vulnerable to the toxic effects of drugs because their role in concentrating and reabsorbing glomerular filtrate exposes them to high levels of circulating toxins19. Although most nephrotoxicity occurs in the proximal part of the nephron, some chemicals damage distal tubules. The function of these structures facilitates their vulnerability to toxicants20.

Drug addiction has been associated with various forms of renal diseases which may be related to direct effect of the drugs themselves while others are caused by complications related to drug abuse21.

The present work aims to investigate the effect of tramadol addiction on urinary excretion of some metals (e.g. copper, zinc and calcium) in relation to structural integrity of proximal tubules which were evaluated by measuring the urinary excretion of proximal tubular specific enzymes e.g. LAP and NAG. In addition, the study was extended to evaluate the effect of tramadol addiction coabused with cannabis.