histological study on the effect of tobramycin dosage...

Pak J Med Sci 2007 Vol. 23 No. 1 www.pjms.com.pk 71

HISTOLOGICAL STUDY ON THE EFFECT OFTOBRAMYCIN DOSAGE REGIMENS ON RENAL

PROXIMAL TUBULAR CELLS IN THE RATSMohamed Akram Al-Motabagani1

ABSTRACTObjective: The present study was undertaken to show the potential nephrotoxicity of tobramycin,given in two different dosage regimens, on the proximal convoluted tubules by using the lightand transmission electron microscopic techniques.Materials and Methods: Thirty-five rats were divided into three groups: Group I served ascontrol, Group II received tobramycin at 4mg/Kg of body weight intraperitoneally every 8 hoursfor ten days, and Group III received once-daily dosing of intraperitoneal injection of tobramycinat 12mg/Kg of body weight for ten days. The rats were sacrificed 3 days after the last injection.Small pieces of the right kidneys of all the animals were processed for light and electronmicroscopic examination.Results: The study showed that tobramycin resulted in certain structural and ultrastructural changesin the proximal convoluted tubules. These changes included vacuolar degeneration in theepithelial cells, increased number of lysosomes with variably sized myeloid bodies, mitochondrialoedema, and loss of apical microvilli. These changes were clearly evident following multiple-dailydosing and were less obvious following once-daily dosing. Furthermore, regenerating tubularepithelial cells were evident following once-daily dosing administration.Conclusion: The experimental tobramycin toxicity can be reduced by administering equivalentamounts of the antibiotic in a once-daily dosing as opposed to multiple-daily injections. It ishoped that this study will contribute in the selection of a more appropriate dosing regimen fortobramycin in human beings.

KEY WORDS: Tobramycin, Dosage regimens, Proximal convoluted tubules, Myeloid bodies,Regenerating cells.

Pak J Med Sci January - March 2007 Vol. 23 No. 1 71-77

1. Dr. M. A. Al-MotabaganiChairman and Head,Department of Anatomy,College of Medicine, King Faisal University,P.O. Box: 2114, Dammam -31451,Saudi Arabia.

Correspondences:

Dr. Mohamed Akram Al-Motabagani,E-Mail: [email protected]

* Revision Received: June 21, 2006

* Revision Accepted: July 20, 2006

INTRODUCTION

Tobramycin is a member of theaminoglycosides antibiotics. The distinguishingcharacteristics and clinical uses ofaminoglycosides have been described previ-ously.1 These potent antibiotics are commonlyused to treat serious infections caused by manygram-negative bacilli and some gram-positive

organisms. Aminoglycosides, being polar cat-ions, are poorly absorbed from the gastrointes-tinal tract and hence are administered parenter-ally. Although they are widely used in clinicalpractice, serious toxicity is a major limitationto the usefulness of the aminoglycosides. Nu-merous toxicological studies have demon-strated the associations of nephrotoxicity, oto-toxicity (vestibular and auditory), neuromus-cular blockade and hypersensitivity reactionswith the use of these drugs.2-9

Because aminoglycosides display bacteri-cidal, concentration-dependent killing action,there has been a long-standing debate regard-ing whether aminoglycosides should be admin-istered on a once-daily dosing or multiple dailydosing regimens to achieve optimal efficacy andsafety.4,8,10,11 Traditionally, the aminoglycosides

72 Pak J Med Sci 2007 Vol. 23 No. 1 www.pjms.com.pk

Mohamed Akram Al-Motabagani

have been given two to four times daily. How-ever, recent clinical studies have suggested thatless frequent, once-daily dosing, administrationof aminoglycosides may achieve equal or bet-ter efficacy with less toxicity in the adults,2,4,8

children10 and neonates.12 In these clinical stud-ies nephrotoxicity was assessed by serumconcentrations analyses and immunoassay. Inaddition numerous studies are availabledescribing nephrotoxicity in terms of measure-ments of serum creatinine and inorganic phos-phate levels, levels of alkaline and acid phos-phatase in kidney homogenates, detection ofurinary enzymes, and sphingomyelinase andcathepsin B activities.3-12 On the other handonly few studies are available describing themorphological alterations in the renal histol-ogy after aminoglycosides administration,13-16

but did not relate the changes in the renal his-tology to the dosage regimens. In view of thisthe present work was undertaken to comparethe potential nephrotoxicity of tobramycin,given in two different dosage regimen, on theproximal convoluted tubules by using the lightand transmission electron microscopy.

MATERIALS AND METHODS

Thirty-five adult male Wistar albino ratswere used in the present study. The weight ofthe rats varied from 250 to 300 grams, and theirages ranged from 9 to 11 weeks. They werefed a standard rat and mouse diet and waterwas supplied ad libitum. They were divided intothree main groups. Group I (15 rats) served ascontrol; five of the control rats were untreated,and the remaining ten rats were dividedequally into two subgroups which receivedintraperitoneal injections of normal saline(NaCl, 0.9%) in the same dosage regimens asin groups II and III, respectively. Groups II andIII received the same dose of intraperitonealinjections of tobramycin (Tobramycin sulfateinjections, Lilly Co.) In group II (10 rats) thetobramycin was given in multiple-dailydosing in the form of a single dose (4mg/Kg ofbody weight) every 8 hours for ten days. GroupIII (10 rats) received once-daily dosing oftobramycin at 12mg/Kg of body weight for tendays. These dosage regimens were similar to

those employed by previous investigators onpatients undergoing treatment withtobramycin.1,11,17

The animals of all groups were sacrificed 3days after the last injection. Prior to sacrificethe animals were anesthetized and the abdomi-nal cavity was opened by a midline incision.The renal cortex of the right kidney of eachanimal was dissected, and small blocks of tis-sue were collected and processed for light andelectron microscopy. Paraffin sections werestained with haematoxylin and eosin (H&E).Ultrathin sections were contrasted withuranyl acetate and lead citrate and examinedin a Jeol JEM 100 SX transmission electronmicroscope operated at 80kv.

RESULTS

Group-I (control): The results obtained from thecontrol group (untreated and saline-treatedrats) were similar. Fig-1A, 1B and 1C are rep-resentatives of such results. Examinations ofthe specimens taken from the control grouprevealed the normal structure of the proximalconvoluted tubules by both light and electronmicroscopy. Profiles of the proximal convo-luted tubules made up the bulk of the renalcortex. The proximal convoluted tubules werelined by of a single layer of large cuboidal epi-thelial cells resting on a well-defined basallamina. The nuclei were spherical and centraland the cytoplasm was granular and eosino-philic (Fig-1A). Ultrastructurally, normal cel-lular components such as mitochondria, lyso-somes, Golgi apparatus, and smooth and roughendoplasmic reticulum were present in theproximal convoluted tubules cells (Fig-1B).These cells had all the cytological features nec-essary for fluid and ion exchange. The apicalsurface had a well-developed luminal brushborder consisting of numerous closely packedmicrovilli covered by a coat of glycocalyx. Api-cal canaliculi originated between the bases ofthe microvilli and extended into the apicalcytoplasm giving rise to small pinocytoticvesicles. The vesicles coalesced to form largervacuoles (Fig-1B). Cellular processes extendedfrom the basolateral surfaces of each cell tointerdigitate with similar processes of adjacent


cells forming an extensive intricate system ofinterdigitating processes. Elongated mitochon-dria resided in the basal processes and wereoriented with their long axes perpendicular tothe basal lamina (Fig-1C).Group II (Animals treated with multiple-dailydosing of tobramycin; once every 8 hours for twodays): The results obtained from the rats ofgroup II were similar. Fig-2A, 2B, 2C and 2Dare representatives of such results. In two-thirds of the proximal convoluted tubules pro-files in a visual field, a brown material ap-peared in the form of fine discrete granules oraggregated into more prominent paranuclearmasses (Fig-2A). Extensive vacuolation of thecytoplasm occurred, often disrupting the cellbody so that only a cellular remnant remainsin contact with the basement membrane (Fig-2B).Ultrastructurally, the brush border of theproximal convoluted tubules showed areas ofcomplete loss and the mitochondria appearedswollen. The cytoplasm of the proximal con-voluted tubule cells contained aggregates ofconcentrically whorled membranes (Fig-2C).The proximal convoluted tubule cells containedincreased numbers of large, irregular, lysos-omes with numerous myeloid bodies (Fig-2D).A few proximal convoluted tubules with mini-mally altered epithelium were found side byside with those having severely disrupted orsloughed epithelium. In the severely disruptedproximal convoluted tubules, there were rem-nants of proximal convoluted tubule cells con-sisting of cytoplasmic collections of disorga-nized, severely damaged organelles.Group III (Animals treated with once-dailydosing of tobramycin for six days): The resultsobtained from the rats of group III were simi-lar. Fig-3A, 3B and 3C are representatives ofsuch results. In one-third of the proximal con-voluted tubules profiles in a visual field, someof the proximal convoluted tubule cells showedvacuolation of the cytoplasm and destructionof the brush border (Fig-3A). Many proximalconvoluted tubules segments were lined by re-generative epithelial cells. These cells appearedlow cuboidal in shape with large vesicularnuclei and basophilic granular cytoplasm.Mitotic figures were numerous (Fig-3A).

Figs-1A-1C: A light photomicrograph and transmissionelectron micrographs of sections of control kidneys of

adult albino rats (group I)

Fig-1A: Showing the normal structure of the proximal(PT) and distal (DT) convoluted tubules and a renal

corpuscle (RC). (H&E, X400).

Fig-1B: This electron micrograph spans the upper por-tion of the proximal convoluted tubule cells with theircharacteristic long microvillous border (MV), intercellu-lar junctional complexes (J), pinocytotic apical vesicles(V), lysosomes (L), mitochondria (M), and a portion of

the nucleus (N).(X7000).

Fig-1C: This electron micrograph spans the basal portionof the two adjacent proximal convoluted tubule cells. Thenuclei (N) are spherical, euchromatic and located nearthe base of the cells. The basal part of the cells showsbasal striations resulting from the enfolding of the basalcell membrane (BI) and the closely associated elongated

mitochondria (M).( X7000).

Tobramycin Dosage on Renal Proximal Tubular Cells


By electron microscopy, most of the proxi-mal tubules appeared same as control exceptfor the loss of definite brush border (Fig-3B).However in a few tubules a patchy nature of

Figs-2A-2E: Light and transmission electron micrographsof sections of proximal convoluted tubule cells ten daysafter tobramycin administration with multiple-dailyinjections, once every 8 hours for two days (group II).

Fig-2A: Showing intracellular brown mass (BM) in theproximal convoluted tubule cells. (H&E, X400).

Fig-2B: Showing extensive tubular vacuolation (TV)disrupting the cell body with remnants in contact with

the basement membrane (arrows). (H&E, X400).

Fig-2C: Showing destruction and loss of brush border(DBB), unicentric myeloid body (MB), swollen

mitochondria (M) and granular nuclei (N). (X 7,000).

Fig-2D: Showing large secondary lysosomes (L)containing numerous myeloid bodies (MB). The

nucleus (N) is irregular in shape. (X 7,000)

Figs-3A-3C: A light photomicrograph and transmissionelectron micrographs of sections of kidneys ten daysafter tobramycin administration with multiple-daily

injection for six days (group III).

Fig-3A: Showing regenerating tubules (T3) with regu-larly arranged large vesicular nuclei and basophilic cyto-plasm and mitotic figures. Vacuolation (V) of cytoplasm

is apparent in some PCT cells (H&E, X 400).

Fig-3B: Showing two adjacent proximal convolutedtubule cells. The nucleus (N) is spherical and euchromatic.The apical surface of the tubule cells has few shortmicrovilli (MV) but lacks a brush border. The basal part

of the cells shows basal striations resulting from theenfolding of the basal cell membrane and the closely

associated mitochondria (M). (X7000).

Fig-3C: Showing clear cytoplasm of one proximal tubulecell (C) with simplification of basal infoldings (BI) andloss of most of its organelles. The adjacent cell contains a

few myeloid bodies (MB). (X 7,000)



cell injury was observed. Within the sameproximal convoluted tubule, the extent of cellchanges was highly variable. The cytoplasmof some cells was clear and completely disar-rayed and contained few organelles, with sim-plifications of basal membrane infoldings.Whereas other cells in the same tubule ap-peared with minor changes and a few myeloidbodies (Fig-3C).

The results of the morphological changesobserved with multiple-daily and once-dailydosing regimens as compared to control aresummarized in Table-I.

DISCUSSION

Aminoglycosides bactericidal activity isdirectly related to the concentration achievedbecause they have a concentration-dependentkilling and a concentration-dependentpostantibiotic effect.1 This enhanced activity athigher concentration, combined with the toxicside-effects, has resulted in some uncertaintywith regard to the precise dosage regimen. Thequestion of multiple-daily dosing versus

once-daily dosing regimens remains in dispute.Traditionally, the total daily dose ofaminoglycosides is administered as two or threeequally divided doses.1 On the other handnumerous studies in a variety of clinical set-tings employing every commonly usedaminoglycoside have favoured once-daily dos-ing regimen.4,8,10-12,17 In an attempt to contrib-ute some resolution to the above dispute, thepresent study was undertaken with theobjective of determining the effect of frequencyof dosing of tobramycin on the morphology ofthe proximal convoluted tubules by light andelectron microscopy. To achieve this objectivethe experimental groups of animals receivedequivalent total doses of tobramycin withdifferent frequency of dosing, namely once-and multiple-daily dosing for a total durationof ten days.

Examination of the renal cortex of the con-trol group (untreated and saline-treated rats)revealed normal structural and Ultrastructuralappearance. However the kidneys of theexperimental (tobramycin-treated groups)

Table-I: The summary of the relative microscopic changes in the proximal convolutedtubules of the tobramycin-treated groups (II and III) as compared to control (group I)

Relative Control Multiple-daily injections Once-daily injectionsmicroscopic changes (group I) of tobramycin (group II) of tobramycin (group III)

Intracellular brown mass Nil +++ + (light microscopy)Myeloid bodies in large Nil +++ + secondary lysosomes (electron microscopy)Loss of brush border Nil +++ +Mitochondrial swelling Nil +++ +Tubular vacuolation and Nil +++ + disruptionTubular degeneration Nil +++ +Loss of basal striations Nil +++ +Tubular regeneration +++ + +++BI: Basal infoldings N: NucleusBM: Brown masses PT: Proximal convoluted tubule(s)C: Cell with clear cytoplasm. RC: Renal corpuscleDBB: Destructed brush border RBC: Red blood corpusclesDT: Distal convoluted tubule(s) T1: Tubule with minimally altered epitheliumJ: Junctional complexes T2: Tubule with severely disrupted epitheliumL: Lysosomes T3: Regenerating tubulesM: Mitochondria TV: Tubular vacuolationMB: Myeloid body V: VacuolationMV: Microvilli


revealed potential signs of nephrotoxicity withmost of the damage confined to the proximalconvoluted tubules. This finding is in confor-mity with a number of similar observations inthe literature.16,18 The simplest explanation forthis phenomenon is that the proximal convo-luted tubules are the largest, highly specializedsegments of the nephrons and collectively makeup most of the renal cortex. In addition theyare the first regions of the nephrons coming incontact with drugs after being filtered by theglomeruli. In addition, accumulation ofaminoglycosides in the proximal convolutedtubules cells has been attributed to theaminoglycosides-concentrating capacity ofthese cells.18

Nephrotoxicity due to aminoglycosides hasbeen ascribed to endocytosis and sequestrationof the aminoglycosides to lysosomes, formationof myeloid bodies, and phospholipidosis. Rup-ture of the lysosomal membrane leads to releaseof acid hydrolases with subsequent necrotic celldeath.19,20 However, an alternative mechanismmediating aminoglycosides cellular toxicity hasalso been identified in the cell-surface, namelyG protein-coupled Ca2+ (polyvalent cation)–sensing receptor (CaR). It has been suggestedthat the presence of the CaR in PCT, in con-junction with the ability of the aminoglycosidesto activate the receptor might be involved inthe process of cellular toxicity induced by theaminoglycosides.16

The current study has shown thattobramycin administration resulted in certainmorphologic changes in proximal convolutedtubules. These changes included vacuolar de-generation in the epithelial cells, increasednumber of lysosomes with variably sized my-eloid bodies, mitochondrial oedema, and lossof apical microvilli (Table-I). It is interesting tonote that these changes were clearly evidentfollowing multiple-daily injections whereasfollowing once-daily administration the mor-phological changes were less obvious. This maybe regarded as indicative of the marked effectof frequency of dosing on tobramycin-inducedmorphological changes because tobramycinwas introduced at equivalent total dosage. This

mean that the tobramycin-induced tubulardamage increases as the interval of adminis-tration is reduced from 24 to 8 hours. Fromthese findings it may be concluded thattobramycin-induced toxicity is directly propor-tional to the frequency of dosing. It is possiblethat the intracellular accumulation oftobramycin18 resulting from activation of CaRreceptors16 and binding of the tobramycin tothe acidic phospholipids21 in the brush bordermembrane of proximal convoluted tubule cellswith subsequent receptor-mediated endocyto-sis22 were markedly enhanced following mul-tiple-daily as compared to once-daily injectionsof equivalent amounts of the antibiotictobramycin. It is also possible that the renalclearance and elimination of tobramycin washigher following once-daily dosing.1 Moreover,it has been known that an increase in fluid in-flux results in cellular changes such as vacu-olar degeneration, mitochondrial swelling andloss of microvilli, and that these changes arereversible.23 Thus it may be inferred that thefluid influx in the proximal tubular epithelialcells was markedly enhanced following mul-tiple-daily as compared to once-daily dosingregimens.

In the present work, the regeneration oftubular epithelium was evident following ad-ministration of the tobramycin at once-dailyinjections; many mitotic figures were observedin the regenerating tubules. The regeneratingepithelial cells had large vesicular nuclei andbasophilic cytoplasm. Electron microscopyrevealed that these cells were squamoid inshape with few apical microvilli. The nucleuswas flat with large prominent nucleolus, andthe cytoplasm contained many mitochondria.Similar patterns of tubular regeneration havebeen described previously.24,25 The appearanceof the regenerating cells following administra-tion of aminoglycosides was attributed to themorphologic and metabolic immaturity of thesecells which may reflect a degree of protectionfrom the toxic effects of tobramycin.26 Theregenerating cells probably arise from therelatively unaffected residual epithelial cellsthat can be found in areas of tubular damage.27

It has also been reported that damage to



epithelial cells in the proximal convoluted tu-bules may in some manner act as a stimulus toregeneration by release of cellular substances.28

Since renal injury was mild followingtobramycin administration at once-daily dos-ing regimen, coupled with the appearance ofregenerating epithelial cells, it may be con-cluded that the experimental tobramycin tox-icity can be reduced by administering equiva-lent amounts of the antibiotic in a once-dailydosing as opposed to multiple-daily dosing in-jections, which is important for the discussionof the most appropriate dosing regimen foraminoglycosides in humans.

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