Talat Yasmeent  ( Department of Anatomy, Sindh Medical College, )
Ghulam Sarwar Qureshi  ( Department of Anatomy, Sindh Medical College, )
Sughra Perveen  ( Department of General Surgery3, Jinnah Postgraduate Medical Centre, Karachi. )

Objective:To see the toxic effects of NSAID on kidney tissue of albino rats. Methods:For this experimental study, 16 albino rats were taken. They were divided into two groups; Aand B.The animals in group-Awere given Normal Saline. Group-B received Diclofenac Sodium 2 mg/kg/day by feeding tube for 14 days. On day-15 all animals were sacrificed. Kidneys were removed, fixed, embedded in paraffin,section cut at 4 µm thick and stained with H&E, PAS, and silver methenamine. Renal histology was done underlight microscope to see the renal tubular diameter, count and cellular detail.Results:The result of present study revealed that diclofenac sodium in single daily dose of 2 mg/kg for a periodof two weeks effectively produced destruction of proximal and distal convoluted tubules in adult albino rats showing the dilatation of tubule and flattening of tubular epithelium, disruption of brush border in proximal tubuleand thickening of basement membrane around proximal and distal tubular epithelium.Conclusion:Diclofenac sodium induced nephrotoxicity causes the destruction of proximal and distal convoluted tubules showing the dilatation of tubule and flattening of its epithelium in albino rats (JPMA57:349:2007).

Diclofenac sodium, an analgesic, anti-inflammatoryand antipyretic compound, now adays, is one of the commonest used non-steroidal anti-inflammatory drugs(NSAID).1It is used in a variety of painful conditions like osteoarthritis, rheumatoid arthritis, ankylosing spondylitis,renal colic, in dentistry and preoperatively 2 to reducepostoperative pain. Under such conditions if the use isprolonged, these drugs sometimes produce moderate tomarked degree of nephrotoxicity. Kidney is an importanttarget site for untoward effect of diclofenac sodium inhumans as well as in animals.3 The usage of this drug by large number of patients indicates its efficacy; however it also indicates that a largepopulation is at risk.4 Present study is therefore designed to study the grossand microscopic changes in the kidney following administration of therapeutic dose of diclofenac sodium inalbino rats.

The animals used in this experimental study were albino rats of 12 weeks age, weighing between 180~200grams. They were originally obtained from Charles River Laboratories, Brooklyn, Massachusetts, USA, crossbredand kept at the Animal House of Basic Medical Sciences Institute, JPMC, Karachi. The study period extended fromJune 2002 to November 2002.Sixteen animals were used in this study; they were divided into two groups; Aand B. The animals in eachgroup were kept in a separate cage and labeled. Each animalwas weighed prior to the treatment. The animals in group-Aserved as control and received Normal Saline, 10 ml/kgbody weight orally daily for two weeks. The animals ingroup-B received diclofenac sodium at a dose of 2mg/kg/day5, dissolved in distilled water and administeredorally by a feeding tube once daily for two weeks. On day-15 the animals were sacrificed by deep ether anesthesia.They were dissected; their kidneys were removed and weighed with the help of Sartorious balance. Each kidneywas bisected into two halves; one half fixed in 10% the formalin and other half in alcoholic formalin.The tissue was then processed for paraffinembedding, sectioned on a rotary microtome, 4 µm thick longitudinal sections were cut, and were mounted ongelatinized slides. The tissue sections fixed in 10% formal inwere stained with H&E to access the general architecture ofrenal parenchyma and with silver methenamine forbasement membrane. The sections of tissue fixed inalcoholic formalin were stained with PAS.6 The morphological changes in renal parenchyma were observed.Micrometry was done and the data was subjected tostatistical analysis.The parameters were proximal and distal tubularcount, their diameter and number of cells per unit area.Tubular count was made under 8 ocular and 40 objective with counting reticule in randomly selected five fields in thecortex of kidney, while tubular diameter was recorded withthe help of ocular micrometer. Nuclear count, cytoplasmicand nuclear details were made under low power, high powerand oil immersion objectives.

Regarding the general behaviour, the animals ingroup- Aremained active, quick to respond, and their food intake was normal. In group- B the animals looked ill and weak from day- 4 of experimental period. They were lethargic, response to stimuli sluggish, and were reluctant to take food.The H&E stained sections in group-A showed thehistological structure in the cortical and medullary portionto be absolutely normal without any change in either glomeruli or tubules. No sign of any degenerative change was observed in the cytoplasm of renal tubular epithelialcells. The interstitium of the renal cortical and medullaryarea was sparse and contained small capillaries filled with RBCs. The brush border on the apical surface of proximaltubular epithelial cells stained magenta in colour and almostfilled the tubule. The glycogen content of the cytoplasm ofproximal tubular cells was quite normal. The basement membrane of proximal and distal tubules also stained magenta, which was distinct and regular.Silver methenamine stained sections revealed basement membrane of glomeruli, Bowman's capsule, andproximal and distal tubules, which was faint in outline, andun-measurable by light microscopy.The mean number of proximal convoluted tubulesper unit area in group-Awas 23.750±0.559, which when compared with group-B, a highly significant increase(P<0.001) was noticed.The H&E stained sections in group-B revealed the proximal tubules in juxtaglomerular region to be dilated,circular oval or elliptical in section and filled with cellulardebris. The lining epithelial cells of the proximal convoluted tubules were low columnar, many of these cells showed degenerative changes. The nuclei of the intact cells wereeither central or towards the apical portion of cells. The nuclei of some of the cells appeared condensed indicating pyknosisleading to cell death. Many of the cells in proximal tubulesshowed vacuolation obscuring all cytoplasmic details.The distal convoluted tubules also appeared dilatedand circular oval or elliptical in outline. Some of themcontained cellular debris in their lumen. The liningepithelial cells were cuboidal, some of these cells showed degenerative changes. The nuclei of the intact cells wereeither central or towards the luminal aspect but some nucleiof the cells appeared condensed.The interstitium of renal cortical area was sparsewith few inflammatory cells but no marked oedema. Many dilated and congested blood vessels were frequently observed.The medulla showed infiltration of mononuclearcells as well as marked congestion of blood vessels. In PASstained sections the brush border at the luminal surface appeared scanty and indistinct and at some places it was completely absent. The intracellular glycogen content of the proximal as well as distal tubules was moderately depleted.However, the basement membrane of proximal and distaltubules was regular and intact.In silver methenamine stained sections the basementmembrane was visible as intensely stained black line around proximal and distal tubules which was quite thickened in

Table 1. Comparison of Proximal Tubular Count, Diameter, and number of Cells between Control and Diclofenac Sodium treated animals.

Table 2. Comparison of Distal Tubular Count, Diameter, and number of Cells between Control and Diclofenac Sodium treated animals.

some tubules but still not measurable by light microscope.

The main effect of diclofenac is like all other NSAIDS,to prevent the synthesis of prostaglandin by inhibiting the enzyme cyclooxygenase in the cells of the body. The kidney is extremely active in the synthesis and metabolism of prostaglandin. These compounds participate in several processes in renal physiology including auto-regulation ofrenal blood flow and glomerular filtration, modulation of reninrelease, tubular ion transport and water metabolism.7It is notsurprising that diminished prostaglandin synthesis may be aninitiating event in the patho-physiologic process of diclofenacsodium induced renal dysfunction.8

After treatment with diclofenac sodium in group-B,the general behaviour of the animals changed to ill,sluggish, and food intake decreased, which may be attributed to loss of appetite due to side effects of diclofenacsodium on GIT. Our findings are in conformity with Beun etal 9 who also observed anorexia in patients receiving diclofenac sodium for arthritis.Asignificant decrease in number of tubules per unitarea of the kidneys was noticed in group-B animals, which may be attributed to damage to the tubular epithelial cells byischaemia produced by inhibition of prostaglandin in renalarterioles causing their constriction. These results are inagreement with Gray et al10and Clive and Stoff4who observedvacuolar degeneration of proximal tubule and focal tubular

atrophy in NSAID (indomethacin) with renal failure.Ahighly significant increase in the diameter of proximal tubules in group-B animals as compared to group-Awas noted, which may be attributed to degeneration ofcells in proximal tubules resulting in apparent increase intheir diameter. Our findings are in conformity with Scott etal11who observed renal tubular cells in urine after ingestion of salicylates and concluded that NSAID (diclofenacsodium) causes transient shedding of renal cells. The damage to renal tubules may be attributed to decrease inblood supply to kidney tissues as the renal vascular tone isdetermined by autonomous intrinsic activity of the renalarterioles and continuous production of renal prostaglandinwhich was inhibited by this drug and caused unopposed constriction of arterioles resulting in ischaemia of tubulesand epithelial cell death.The interstitial nephritis was noted in the cortex andmedulla of the kidneys in group-B animals. This may be attributed to decrease in cyclooxygenase by NSAID, lead to shunting of arachidonic acid precursor into lipooxygenasepathway, favouring the production of inflammation inducing metabolites of eicosapentaenoic acid which functions as lymphokinase, leading to recruitment of T-lymphocytes and perpetuation of the inflammatory process.Finally, it may be concluded that diclofenac sodium produces changes in kidney, which may be attributed toischaemia induced by inhibition of prostaglandin synthesisresulting in tubular necrosis