Growth Characteristics of Cultured Human Proximal Tubule Cells Exposed to Aminoglycoside Antibiotics*

MARYANN SENS, M .D ., P h .D ., DEBRA J. HAZEN-MARTIN, P h .D .,

JOHN G. BLACKBURN, P h .D ., GORDON R. HENNIGAR, M .D .,

and DONALD A. SENS, Ph.D .

Departments o f Pathology and Physiology Medical University o f South Carolina,

Charleston, SC 29425

ABSTRACT

It is well known that the nephrotoxic lesions that occur during amino­glycoside-induced nephrotoxicity are both dose- and tim e-dependent. It was the purpose of this study to determ ine if a cell culture model based on the hum an proximal tubule would exhibit similar dose- and tim e-depen- dent relationships when exposed to aminoglycosides of various nephrotoxic potential. For this determination, the hum an proximal tubule (HPT) cells w ere exposed to increasing concentrations of streptomycin, kanamycin, gentamicin, and neomycin and m onitored for cell growth and toxicity over an 18-day period of exposure. Both actively-dividing and resting cells w ere assessed with regard to aminoglycoside exposure. At high levels of aminoglycoside exposure, linear regression analysis disclosed that the rank order of toxicity of the aminoglycosides to be: neomycin > kanamycin > gentamicin > streptomycin. Both actively-dividing and resting cultures of HPT cells displayed both dose- and tim e-dependency with regard to toxic­ity and the ability of the cells to regenerate in the continued presence of aminoglycoside exposure. This pattern of dose- and tim e-dependency was unique for each aminoglycoside and varied depending on the replicative state of the cells.

W ith the exception of neomycin, clear evidence was obtained that toxic­ity and cell regeneration were occurring simultaneously throughout the tim e course of aminoglycoside exposure; the equilibrium betw een the two processes determ ining overall cell toxicity or regeneration. In addition, the HPT cells exposed to gentamicin displayed a unique pattern of toxicity and cell regeneration when compared to the o ther aminoglycosides tested, with gentamicin having an increased ability to stimulate cell proliferation. W hile the results obtained are in excellent agreem ent with that known from the clinical experience with the aminoglycosides, the dose- and tim e-dependency of the responses will require careful attention to growth state during em ploym ent in experimental protocols.

ANNALS O F CLINICAL AND LABORATORY SCIEN CE, Vol. 19, No. 4Copyright © 1989, Institute for Clinical Science, Inc.

Send reprint requests to: Mary Ann Sens, M .D ., m ent of Pathology, Medical University of South Car-Ph.D ., Division of Cellular Biopathology, Depart- olina, 171 Ashley Avenue, Charleston, SC 29425.

2660091-7370/89/0700-0266 $02.00 © Institute for Clinical Science, Inc.

AMINOGLYCOSIDE EFFECTS ON CELL GROWTH 267

Introduction

The am inoglycoside an tib io tics are used ex tensively in th e tre a tm e n t of gram-negative infections, and their clini­cal use is associated with dose-limiting nephrotoxicity .2’1018,27 The renal patho­genicity associated with the aminoglyco­sides can be attribu ted to their selective accu m u la tio n in th e ren a l proxim al tubule of hum ans2,7,24,27 and experim en­tal anim als.9,21 That pathogenicity is due to accumulation is suggested since it is known that the aminoglycosides are not m e ta b o liz e d by th e k id n ey b u t are excreted unchanged in the urine (paren­teral administration) or in the feces (oral administration ) . 2,20'21 The accumulation of the aminoglycosides within the proxi­mal tubule results in the early ultrastruc- tural appearance of cytoplasmic myeloid bodies19 along with alterations in tubular function21 and release of tubular proteins into the u r in e . 1,24 O ne of the striking features of aminoglycoside nephrotoxi­city is that the morphologic lesions are both dose- and tim e-dependent18,25 and run a course best characterized as one of proximal tubular damage with potential recovery.

Histologic examination of the kidneys of rats following aminoglycoside expo­sure reveals proximal tubule cell damage even at low m ultip les of the hum an dose . 15,16,19 This tubular degeneration is focal in nature and usually progresses to tu b u la r n ec ro sis , w hich may e ith e r resolve by com plete regeneration of the necrotic proxim al tubu le or resu lt in scarring and interstitial fibrosis. 15,16 Con­current recovery from the nephrotoxicity induced by large doses of aminoglyco­sides in rats has been docum ented with active proximal tubule cell necrosis and regeneration occurring simultaneously in adjacent nephrons . 5,13,16,22 D espite the localization of pathologic damage to the

proximal tubule , the initial injury and m echanism (s) of ce llu la r toxicity and regeneration rem ain unknown.

W hile num erous cellular alterations have been noted to occur with aminogly­coside exposure , 15,18 no agreem ent has been reached on which are prim ary or secondary events in nephrotoxicity. This difficulty arises prim arily owing to the complexity of the in vivo environm ent w h e re fa c to rs su c h as g lo m e ru la r involvement, tubular obstruction, vascu­lar compromise, electrolyte imbalances, and infections render the definition of p rim ary v e rsu s seco n d ary ev en ts in n e p h ro to x ic ity v e ry d ifficu lt. In an a t te m p t to l im it th e in te r fe re n c e s present in the in vivo setting, this labo­ra to ry has d e v e lo p e d a ce ll c u ltu re model derived from the human proximal tu b u le 3,6 w hich, w hen exposed to the aminoglycoside antibiotic, streptomycin, dem onstrated growth responses and cel­lu la r changes sim ilar to those d o cu ­m ented to occur in vivo.26 In this report, the characterization of this model system is ex tended to d e te rm in e the growth response of the cells to several aminogly­coside antibiotics chosen to represent a spectrum of known nephrotoxicities. It will be shown that the effects on cell growth are dose- and tim e-dependent and that cellular toxicity correlates to the nephrotoxicities known from the in vivo experience w ith these antibiotics. Of particular in terest was the finding that gentamicin dem onstrated the ability to stimulate proximal tubule cell growth.

Materials and Methods

T is s u e C u l t u r e

Sources of reagen ts and the p roce­dures for the initiation and maintenance of stock c u ltu re s of hum an proxim al tubule (HPT) cells have been previously d e sc r ib e d . 6,26 Briefly, the H P T cells were grown in a serum -free formulation

268 SENS, HAZEN-MARTIN, BLACKBURN, HENNIGAR, AND SENS

consisting of a 1:1 mixture of Dulbecco’s M o d if ie d E a g le s (D M E ) a n d F -12 medium supplem ented with selenium (5 ng per ml), insulin (5 fig per ml), trans­ferrin (5 (Jig per ml), hydrocortisone (36 ng p e r ml), triiodothyronine (4 pg per ml), and epiderm al growth factor (1 0 ng per ml). The cells were fed fresh growth medium every three days. For the mea­surem ent of cell growth and ultrastruc­ture , cultures of HPT cells at passage 3 - 4 were subcultured into 12-well plates w ith each w ell con ta in ing two ml of growth m edium . C onfluent or resting culture experim ents were performed fol­lowing a 1:1 subculture of the cells. In th e s e s tu d i e s , th e c e l l n u m b e r s rem ained constan t in the control cu l­tu re s (no a m in o g ly co sid e ad d itio n ) th ro u g h o u t th e e x p e r im e n ta l tim e course. Subconfluent studies were per­formed following a 1:3 subculture such that the control wells dem onstrated an exponential growth phase during the ini­tial four days of aminoglycoside expo­sure. Twenty-four hours following the ap p ro p ria te su b cu ltu re , the cu ltu res were fed fresh growth medium contain­ing a given concentration of aminoglyco­side. The aminoglycosides assessed were streptom ycin, kanamycin, gentam icin, and neom ycin, and all concentrations w ere tested in triplicate. The effect of each aminoglycoside concentration on cell growth was tested over an 18-day tim e course. The concentrations of each am inog lycoside te s te d w ere: 6,500; 5,000; 3,500; 2,500; 1,250; 750; 500; 250; 100; 50; 25; and, 5 (xg per ml. The cells were fed fresh growth medium contain­ing th e ap p ro p ria te am inoglycosides every three days.

Throughout the experimental course, th e ce lls w e re m o n ito re d u s in g an inverted microscope.* Every third day, cell coun ts w ere o b ta ined using the

inverted microscope interfaced via video ca m era f to a co m p u te r-lin k ed im age analysis system. $ Random fields of cells were selected and the cells in the fields counted using the mouse attachm ent and digitizing tablet. For each concentration, a m inim um of 2 0 fields w ere selected and counted in each well at each tim e p o in t. All c o n c en tra tio n s w ere p e r ­form ed in triplicate. The criterion for acceptance of individual tim e points was the finding of no statistical difference in cell num bers within the triplicate sam­ples at each concentration. All statistical analyses w ere perform ed utilizing the V ideoplan software and included the S tudent’s two-sided t-test as well as basic statistical m ethods such as mean, stan­dard deviation, and standard variance. Trypan blue staining was utilized in p re ­liminary determ inations and at selected points of each tim e course to judge cell viability. Cells that were floating free in the growth m edium after concentration by centrifugation were dem onstrated to be over 99 percent non-viable. The HPT cells a tta c h e d to th e c u ltu re vesse l growth surface w ere shown to exclude trypan blue regardless of aminoglycoside concentration or time of exposure.

The results presented are representa­tive of one isolate of hum an proxim al tubule cells. Two other independent iso­la tes w ere te s te d , and b o th y ie ld e d eq u iv a len t resu lts to th a t p re se n te d herein.

Results

C o n f l u e n t HPT C e l l s E x p o s e d t o A m in o g l y c o s id e A n t ib io t ic s

Confluent cultures of HPT cells were m onitored for cell growth and survival when exposed continuously over an 18- day period to a wide concentration range

* Zeiss IM35.t Hitachi GP5A4. t Zeiss Videoplan.

AMINOGLYCOSIDE EFFECTS ON CELL GROWTH 269

Dose Response of Human Proximal Tubule Cells to Aminoglycosides

TABLE I

P e r c e n t V i a b i l i t y *C o n c e n tr a t io n

(m g/m l)S t r e p t o ­

m ycinKana­m ycin

G en ta ­m ic in

Neo­m ycin

2.5 87.1 8 4 .1 96.5 37.93.5 90.4 80.1 90.4 32.25.0 90.9 74.3 78.9 28.26.5 68.9 62.4 54.5 25.9

TD 50+ 12.2 9.0 7.3 2.2

♦Percent viability expressed as percent of control viability.

+TD 50 = dose producing 50 percent lethality.Values were calculated using linear regression analyses employing Systat software on IBM PS-2.The regression was linear at a = 0.005 with ANOVA.

of the aminoglycoside antibiotics strep­tom ycin, kanam ycin, gentam icin , and neomycin. The period of exposure and large concentration range (5 jjLg per ml to6,500 (JLg per ml) were chosen to afford the maximal opportunity to detect: (1 ) the rank o rder of toxicity of the four aminoglycosides; (2 ) the dose- and time- dependency of each aminoglycoside; (3) the ability of the cells to recover and regain proliferative capacity in the con­tinued presence of antibiotic; and, (4) p o te n tia l d iffe ren ces b e tw e e n th ese closely related antibiotics.

The rank o rder of toxicity for HPT cells exposed to streptom ycin, kanamy­c in , g en tam ic in , and neom ycin was d e te rm in e d follow ing seven days of exposure to 2.5, 3.5, 5.0, and 6.5 mg per ml of antibiotic. As shown in table I, the rank order of toxicity for the HPT cells, as determ ined through linear regression analysis, was neomycin > kanamycin > gentamicin > streptomycin. The highest concentration range of aminoglycoside exposure was chosen for analysis since, as will be subsequently dem onstrated, exposure of HPT cells to lower concen­trations of these antibiotics resu lted in selected instances of cell renew al and resistance th a t w ere b o th dose- and tim e-dependent. As such, inclusion of

these lower concentrations of exposure into the calculations, while resulting in consistent T. D .50 values and rank order of toxicity, produced a significant devia­tion from linearity. This problem was avoided when employing the higher con­cen tra tio n s and sh o rt tim e p e rio d of exposure.

The response of the confluent HPT cells to the aminoglycoside antibiotics was both dose- and tim e-dependent. As depicted in figure 1A, exposure of HPT cells to neomycin, the most nephrotoxic of the aminoglycosides, at a concentra­tion of five (jig p e r ml resu lted in the death and detachm ent from the culture growth surface of 2 0 percent of the cells. This occurred w ithin the initial 10 days of exposure and, thereafter, no further d e c re a se s in c e ll n u m b e r o c c u rre d despite continued presence of the antibi­otic. In creasin g th e co n cen tra tio n of neomycin resulted in a continued reduc­tion in the percen tage of viable H PT cells within the initial 1 0 days of expo­sure; however, this reduction was clearly not in proportion to the increase in neo­mycin concentration. W hereas a five |xg p e r m l c o n c e n tra t io n o f n e o m y c in resulted in 20 percent cell death, 25 (JLg

per ml resulted in only an additional 13 percent reduction, and exposure to 250 jjLg per ml in only an additional 20 per­cent reduction. This slow increase in cel­lular toxicity with increasing neomycin concentration was ev iden t during the initial 1 0 days of exposure for all the con­centrations tested. Additionally, regard­less of concentration, the toxicity of neo­mycin occurred mainly within the initial 1 0 days of exposure and, thereafter, sta­bilized at a constant value despite con­tinued exposure to the drug. Daily light microscopic examination of the cultures re v e a le d few d e ta c h e d ce lls in th e growth medium after 1 0 days of neomy­cin exposure, an observation suggesting that the rem aining cells were resistant to c o n tin u e d n e o m y c in ex p o su re and ,

270 SENS, HAZEN-MARTIN, BLACKBURN, HENNIGAR, AND SENS

ow ing to th e c o n s ta n t a tta c h e d cell n u m b er, no t u n dergo ing ap p rec iab le proliferation.

An identical analysis perform ed utiliz­ing streptom ycin, the least nephrotoxic of th e am inoglycosides, disclosed this antibiotic to be far less toxic to the HPT cells (figure IB 26). Exposure of the HPT cells to concentrations of streptom ycin be tw een five and 50 ¡xg p e r ml w ere w ith o u t e ffec t on th e H P T ce lls as ju d g e d bo th by cell coun ts and ligh t microscopy. Concentrations of s trep to ­mycin betw een 100 and 500 (jug per ml elicited a small, bu t repeatable, gradual decrease in the num ber of attached HPT cells during the initial 1 0 days of expo­sure, an event followed by renew ed cell proliferation to control values in the con­tin u ed p resence of the drug. F u r th e r increasing the streptomycin concentra­tion (750 to 3,500 |JLg per ml) resulted in im m ediate cell toxicity within the initial th ree days of exposure followed by rapid prolifertion of the cells to near-control leve ls of g row th . L igh t m icroscop ic examination of the cultures revealed the presence of detached, non-viable cells in the growth medium throughout the time course, suggesting that toxicity and p ro­liferation were occurring as simultaneous

FIGURE 1. Time course of confluen t hum an proximal tubule cells exposed to various concentra­tions of streptom ycin, neomycin, and kanamycin. The data points are expressed as the percent of con­trol human proximal tubule cells, plated at the same time at the same density, and maintained in antibi­otic-free media. They are derived from the mean of 20 cell count measurements, repeated in triplicate. The individual standard error of the mean (SEM) for each point was less than five percent of the mean (error bars not shown for graphical simplicity).

F ig u r e 1A. Confluent human proximal tubule cells exposed to neomycin. O = 5 (xg per ml; V = 25 (Jtg per ml; □ = 250 |xg per ml; A = 2500 p.g per ml; • = 6500 (jLg per ml.

F ig u r e IB . Confluent human proximal tubule cells exposed to streptomycin. O = 50 (xg per ml; V = 250 (xg per ml; □ = 750 |xg per ml; A = 2500 (xg per ml; 0 = 6500 |xg per ml.

F ig u r e 1C. Confluent human proximal tubule cells exposed to kanamycin. O = 50 ¡xg per ml; V = 500 |xg per ml; □ = 1250 ¡J.g per ml; A = 3500 |xg per ml; 0 = 6500 |j,g per ml.

AMINOGLYCOSIDE EFFECTS ON C ELL GROWTH 271

events. The HPT cells exposed to con­centrations of streptom ycin greater than3,500 fxg per ml displayed similar pat­terns to that previously described, but dem onstrated only a marginal ability to proliferate during continued streptom y­cin exposure and regain confluent cell density.

Kanamycin, an aminoglycoside antibi­otic with clinical nephrotoxicity in term e­diate to that of neomycin and streptom y­cin, was also assessed w ith regard to HPT cell viability and growth (figure lc). Exposure of the HPT cells to kanamycin at concentrations betw een five and 1 0 0 (jug per ml resulted in a small, bu t repea­table, decrease in viable cells within the first seven to 10 days of exposure. This was followed by renew ed proliferation of the cells to control values during the rem aining days of the tim e course and occurred in the continuing presence of this antibiotic. Light microscopic exami­nation during the period of cell renewal d isc losed the p resen ce of significant quantities of detached cells, indicating th a t p ro life ra tio n and tox icity w ere occurring simultaneously. Increasing the concentration of kanamycin (250 to 750 (J ig p e r ml) re su lte d in fu rth e r small decreases in viability during the initial 1 0 days of exposure and in the subse­quen t ability of the cell population to undergo regeneration to control values. At a kanamycin concentration of 1,250 |xg pe r ml and above, findings similar to th e p re v io u s ones d e m o n s tra te d w ith fu rth e r gradual decreases in cell viability as the kanamycin concentration increased.

An identical assessment of the toxicity of gentam icin, an aminoglycoside with sim ilar clinical nephrotoxicity to kana­m ycin, on the viability and grow th of H PT cells y ielded contrasting results when compared to the other aminogly­cosides. E xposure o f co n flu en t H PT cells to five |xg per ml of gentamicin dis­closed a small increase in the cell growth

w ithin th ree days of exposure (figure 2A). This was followed by a decrease in attached cells within the next four days of exposure and then renew ed cell prolif­eration over the rem aining seven days of exposure that exceeded that of HPT cells unexposed to gentamicin. This pattern of g ro w th s t im u la t io n , to x ic ity , an d regrowth (exceeding control values) was also dem onstrated for gentam icin con­centrations betw een 25 and 100 (xg per m l, w ith th e only d ifference b e ing a gradual decrease in the m agnitude of the growth phases.

Increasing the gentamicin concentra­tion to 250 jjug per ml (figure 2B) resulted in a small, gradual decrease in cell viabil­ity during the first 1 0 days of exposure and a gradual regrowth of the cell popu­lation to near control levels. F u r th e r increasing the gentamicin concentration to 500 jxg per ml resulted in a decrease in cell viability of approximately 2 0 per­cent within the initial 1 0 days of expo­sure; however, this was followed by a strik ing increase in cell p ro life ration which surpassed control levels (figure 2B). Furtherm ore, exposure of the HPT cells to h igher dosages of gentam icin (750 and 1,250 jjug per ml) resulted in no appreciable reduction in cell num ber, b u t ra ther a clear, striking increase in cell proliferation which exceeded control values by over 20 percen t (figure 2B). Light microscopic examination of these cu ltu res rev ea led very few d e tach ed cells at any point during the tim e course.

Additionally, this increase in cell pro­liferation as a result of gentamicin (750 (xg per ml for 15 days) presented light microscopically as areas of the monolayer containing tightly packed cells with some evidence of focal m ultilayer formation (figures 3A and 3B). O f additional in ter­est is the observation that the control HPT cells (figure 3B) appear with a flat­te n e d m orphology, a m orpho log ica l characteristic routinely noted after these ce lls h av e b e e n m a in ta in e d fo r an

PERC

ENT

OF

CON

TRO

L

272 SENS, HAZEN-MARTIN, BLACKBURN, HENNIGAR, AND SENS

extended tim e period in a non-prolifera- tive state. This is in m arked contrast to the cells exposed to gentamicin (figure 3A), which possess a m ore epithelioid morphology which is routinely noted for d iv id in g o r re c e n tly c o n flu e n t H PT cells. F u rth e r increases in gentam icin concentration (2,500 and 3,500 |xg per ml) resu lted in initial cell toxicity, fol­lowed by appreciable regrow th of the monolayer, and subsequent total cell tox­icity by the end of the tim e course (fig­ure 2C). Concentrations of gentamicin in excess of 3,500 jJLg p e r ml resu lted in almost total cell death without evidence of any recovery period (figure 2C).

S u b c o n f l u e n t HPT C e l l s E x p o s e d t o A m in o g l y c o s id e A n t ib io t ic s

In order to com pare possible differ­e n c e s in a m in o g ly c o s id e to x ic ity betw een resting versus actively-dividing HPT cell cultures, the previous protocol was repeated using HPT cells plated at subconfluent density. To achieve active cell growth, the HPT cells w ere subcul­tu red at a ratio of 1:3, w hich allowed approxim ately four days of active divi­sion before the cells attained confluency.

F i g u r e 2. Time course of confluen t hum an proximal tubule cells exposed to various concentra­tions of gentamicin. The data points are expressed as the percent of control human proximal tubule cells, plated at the same tim e at the same density, and m ain tained in an tib io tic -free m edia. They are derived from the mean of 20 cell count m easure­ments, repeated in triplicate. The individual SEM for each point was less than five percent of the mean (error bars not shown for graphical simplicity).

F i g u r e 2A. Confluent human proximal tubule cells exposed to low dosages of gentamicin. 0 = 5 H -g per ml; V = 25 ( J ig per ml; □ = 50 (xg per ml; A = 100 (xg per ml.

F i g u r e 2B. Confluent human proximal tubule cells exposed to moderate dosages of gentamicin. O = 250 (jLg per ml; V = 500 (xg per ml; □ = 750 |xg per ml; A = 1250 |xg per ml.

F i g u r e 2C. Confluent human proximal tubule cells exposed to high dosages of gentamicin. O = 2500 (xg per ml; V = 3500 |xg per ml; □ = 5000 |JLg per ml; A = 6500 (Jig per ml.

AMINOGLYCOSIDE EFFECTS ON CELL GROWTH 273

F i g u r e s 3A and 3B. Confluent human proximal tubule cells exposed to: A = 750 |xg per ml of genta- micin for 15 days; B = control cells at 15 days which were not exposed to gentamicin. Cells were fixed and stained with toluidine blue and photographed using a Zeiss IM 35 microscope equipped with Hoff­man optics. Magnification = 250 x .

A comparison of the toxicity of neomycin betw een actively-dividing (figure 4A) and stationary cultures (figure 1A) clearly d em o n stra ted tha t neom ycin was far m ore toxic to H PT cells undergo ing active cell division. W hereas exposure to neomycin at five |xg per ml elicited a 20 percent reduction for HPT cells at con­f lu e n t density , th is reduc tion was in excess of 50 percent for actively-dividing cells. This increased toxicity of neomycin for actively-dividing HPT cells was true for all the concentrations of neomycin

tested. In addition, the toxicity of neo­mycin for rapidly dividing H PT cells occurred throughout the tim e course in contrast to resting cultures, which exhib­ited initial toxicity followed by a plateau phase.

As reported previously,26 a decrease in toxicity was observed for actively-divid­ing HPT cells exposed to streptom ycin (figures IB and 4B). This observation was most apparent for streptomycin concen­trations early in the time course within the initial seven days of exposure. There­after, the HPT cells exposed to strepto­mycin displayed a decreased ability to regenerate to control values. An identi­cal analysis of the HPT cells exposed to kanamycin dem onstrated, during the ini­tial three days of exposure, a very m ar­ginal trend for an increase in toxicity for actively-dividing cells compared to sta­tionary cells (figures 1C and 4C). How­ever, following this initial increase in tox­ic ity , th e s u b c o n f lu e n t H P T c e lls exposed to kanamycin dem onstrated no additional loss of viable cells and, at most dosages , d e m o n s tra te d a t re n d for ren ew ed cell p ro life ra tion to contro l levels. This is in contrast to stationary cultures, w here continued exposure to kanamycin resulted in further decreases in attached, viable cells. Light m icro­scopic examination of the actively-divid- ing cultures exposed to kanamycin dem ­o n s tr a te d a p p re c ia b le n u m b e rs of detached, non-viable cells in the growth m edium th roughout the tim e course, providing evidence that both cell death and proliferation were occurring simulta­neously.

Exposure of the actively-dividing HPT cells to gentam icin disclosed a unique pattern of cell response when compared both to that dem onstrated with the other aminoglycosides and to that found for confluent HPT cells exposed to gentam i­cin. Actively-dividing cells exposed to five |xg p e r ml of gentam icin dem on-

274 SENS, HAZEN-MARTIN, BLACKBURN, HENNIGAR, AND SENS

strated a large increase in cell prolifera­tion during the first seven days of expo­sure, an event which was followed by m arked cell toxicity (figure 5A). This pat­tern of prom inent cell proliferation fol­lowed by m arked toxicity was dem on­strated for all gentamicin concentrations up to and including 500 |xg per ml (fig­ures 5A and 5B). Light microscopy of the cells undergo ing m arked proliferation above control levels revealed focal areas of th e m ono layer c o n ta in in g tig h tly packed cells and evidence of multilayer formation in a m anner identical to that described ea rlie r for confluen t cells. Cells exposed to gentamicin at 1,250 |xg per ml displayed minimal cell toxicity, fo llow ed by m arg inal recovery , and finally m arked toxicity (figure 5B). Con­centrations of gentam icin in excess of 1,250 per ml were toxic to the HPT cells w ithout evidence of cell recovery (figure 5B). Concentrations of gentam i­cin in excess of 1,250 |xg p e r ml were toxic to the HPT cells w ithout evidence of cell recovery (figure 5C). Light micro­scopic examination revealed that even during periods of significant cell recov­e ry , th e r e w e re s t i l l a p p re c ia b le

F igu re 4. Time course of subconfluent human proximal tubule cells exposed to various concentra­tions of streptom ycin, neomycin, and kanamycin. The data points are expressed as the percent of con­trol human proximal tubule cells, plated at the same time at the same density, and maintained in antibi- otic-free media. They are derived from the mean of 20 cell count measurements, repeated in triplicate. The individual SEM for each point was less than five percent of the mean (error bars not shown for graphi­cal simplicity).

F ig u re 4A. S ubcon fluen t hum an proxim al tubule cells exposed to neomycin. O = 5 (Xg per ml; V = 250 ( x g per ml; □ = 2500 | x g per ml; A = 6500 fig per ml.

F igure 4B. S ubcon fluen t hum an proxim al tubule cells exposed to streptomycin. O = 50 (xg per ml; V = 250 |xg per ml; □ = 1250 (xg per ml; A = 2500 (ig per ml; • = 6500 |xg per ml.

F igure 4C. S ubcon fluen t hum an proxim al tubule cells exposed to kanamycin. O = 50 (xg per ml; V = 500 (xg per ml; □ = 1250 |xg per ml; A = 5000 |xg per ml.

AMINOGLYCOSIDE EFFECTS ON C ELL GROWTH 275

num bers of detached, non-viable cells in the growth medium. W hen compared to co n flu en t cells, the actively-dividing cultures appeared to possess a greater capacity for proliferation in the presence of gentamicin at early stages of exposure, bu t also a m arked increase in toxicity at later times of continued exposure to the drug.

Discussion

The artificial environm ent provided by cell culture, through its elimination of the many complex factors present in the in vivo environm ent, could be a valuable adjunct in research efforts probing the cellular basis of aminoglycoside-induced nephrotoxicity. This concept was tested in the p resent study by determ ining the dose- and tim e-dependency of aminogly­coside exposure on the growth and via­bility of cultured human proximal tubule (HPT) cells. The HPT cell culture sys­tem, which proliferates in a serum-free growth media, has been dem onstrated to retain consistent properties from isolate to isolate through a combination of m or­phological, u ltrastructu ra l, electrical, and enzyme histochemical techniques . 3,6 W hile clearly hom ogeneous in d e te r-

F i g u r e 5. Time course of subconfluent human proximal tubule cells exposed to various concentra­tions of gentamicin. The data points are expressed as the percent of control human proximal tubule cells, p lated at the same tim e at the same density, and m ain ta ined in an tib io tic-free m edia. They are derived from the man of 20 cell count m easure­ments, repeated in triplicate. The individual S E M for each point was less than five percent of the mean (error bars not shown for graphical simplicity).

F i g u r e 5A. S ubcon fluen t hum an proxim al tubule cells exposed to low dosages of gentamicin. O = 5 (xg per ml; V = 25 (xg per ml; □ = 50 (xg per ml.

F ig u r e 5 B . S u b c o n f lu e n t h u m a n p ro x im a l tu b u le cells ex p o sed to m o d e ra te dosages o f g e n ta ­m icin . ▲ = 100 (xg p e r m l; O = 250 jxg p e r m l; V = 500 (Xg p e r m l; □ = 750 (xg p e r ml; A = 1250 (xg p e r m l.

F i g u r e 5C. S u b c o n f l u e n t h u m a n p r o x i m a l t u b u l e c e l l s e x p o s e d t o h i g h d o s a g e s o f g e n t a m i c i n .O = 2500 ( X g p e r m l ; □ = 5000 ( x g p e r m l ; A = 6500 ( x g p e r m l .

276 SENS, HAZEN-MARTIN, BLACKBURN, HENNIGAR, AND SENS

m ined structure and function, the cul­ture itself is likely composed of all repre­sen tative segm ents (S1; S2, S3) of the proximal tubule. As such, it is possible th a t cells from ce rta in segm ents are m ore susceptible to a given antibiotic than those of o ther segments.

A previous report from this laboratory has detailed that these cultures of human proximal tubule cells, when exposed to s trep tom ycin , d isp layed m any of the characteristics associated w ith in vivo am in o g ly co sid e-in d u ced n e p h ro to x i­city. 26 These alterations included the for­mation of cytoplasmic m yeloid bodies, both cell toxicity and regeneration, and differing toxicity betw een stationary and ac tiv e ly -d iv id in g ce lls . T he p re se n t study was designed to extend the p re ­vious findings to other aminoglycosides of varying nephrotoxic potential, espe­cially as it applies to the dose- and time- dependency of cell toxicity and regener­a tio n . E m p lo y in g l in e a r re g re ss io n analysis of the HPT cells exposed to high concen tra tions o f am inoglycosides, a rank order of toxicity (neomycin > kana- mycin > gentam icin > streptom ycin) was determ ined which agreed well with th a t know n from in v ivo s tu d ie s in h u m an s and an im al m o d e ls . 2,18,20,21 Additionally, the total dose necessary to produce 50 percent lethality for the HPT cells exposed to neomycin and gentami­cin was in close ag reem en t w ith that found by W illiam s and co -w orkers28 em ploying sim ilar cu ltu res of hum an proximal tubule cells. Thus, the HPT cells respond to aminoglycoside expo­sure with a rank order of toxicity similar to that known from in vivo experience with the antibiotics.

The response of the H PT cells to the aminoglycoside antibiotics was clearly dose- and tim e-d e p e n d en t. This is a w ell-docum ented finding w ith in vivo am inoglycoside nephrotoxicity , w here the m orphologic lesions produced are both dose- and tim e-dependent18,25 and

run a course characterized best as one of proximal tubular damage with potential recovery. In the HPT cell culture model, the growth response during exposure to streptom ycin, kanamycin, and gentam i­cin a t low and m o d era te doses was clearly one of simultaneous toxicity and reg en e ra tio n . This was convincingly dem onstrated for each aminoglycoside at several doses in areas of the time courses where cell growth was increasing while light m icroscopic observation dem on­stra ted appreciable cells detached and floating free in the growth medium. The only exception to this was with neomy­cin , w h e re a p p re c ia b le tox icity was present even at the lowest dosage tested and visual observation did suggest some, bu t not appreciable, cell regeneration. This simultaneous occurrence of prolifer­ation and toxicity correlates well to the in vivo circumstance where nephrotoxi­city induced by large doses of aminogly­cosides in rats dem onstrated active prox- im a l t u b u l e c e l l n e c r o s i s a n d regeneration as simultaneous events in ad jacen t n e p h ro n s . 5,13,16,22 Thus, the HPT cell culture model effectively m ir­rors the dose- and tim e-dependency of in vivo aminoglycoside-induced nephrotox­icity.

The ability of proximal tubule cells in vivo to undergo regeneration in the con­tinued presence of gentamicin exposure8 has led to the hypothesis that regenerat­ing rena l tu b u la r ep ith e liu m may be more resistant to the nephrotoxic insults of am inoglycosides than m ore m ature tubular epithelium . 12,16,22 In the present studies, both neomycin and streptom y­cin elicited increased toxicity on HPT cells that w ere undergoing active divi­sion when compared to cells which were confluent and not undergoing apprecia­ble cell division. However, this finding of increased toxicity for dividing cells was not noted for HPT cells exposed to kanam ycin or gen tam icin . For these am inoglycosides, the com petence for

SENS, HAZEN-MARTIN, BLACKBURN, HENNIGAR, AND SENS 277

cell renew al of the H PT cells was far m ore apparent for actively-dividing cells than for those in the resting stage of cell d iv ision . T his was v e ry s tr ik in g for actively-dividing H PT cells exposed to gentam icin, w here all bu t the highest levels of exposure y ielded significant increases in cell growth above control levels during the early periods of the tim e course. However, once cell prolif­eration reached control levels or above, gen tam ic in was th e n found to e lic it appreciable cell toxicity. The finding that a c tiv e ly -d iv id in g H P T c e lls , w h en exposed to gentam icin , dem onstra ted increased capability for cell growth cor­relates tem porally to the regeneration of proximal tubular elem ents during genta- micin-induced nephrotoxicity as noted in animals and man.

The present results also suggest that each individual aminoglycoside, as well as having a definable toxicity, may also have a definable capacity to stim ulate cell regeneration. This is suggested by the findings that exposure of HPT cells to neom ycin disclosed very little evi­dence for cell regeneration, while similar exposure to gentamicin elicited clear evi­dence for enhanced H PT cell growth. F u r th e rm o re , b o th kanam ycin and streptom ycin exhibited evidence for pro­moting cell regeneration in excess of that noted for neomycin, bu t at a much lower m a g n itu d e th an g e n ta m ic in . T h e se results suggest the possibility that the overall toxicity of a given aminoglycoside might be the average of both the toxic potential and the corresponding poten­tial to alter cellular events in a m anner leading to a signal for cell renewal. It will be of in te re s t to expand th e p re se n t studies to include o ther clinically-useful am inoglycosides such as tobram ycin , amikacin, and netilmicin to determ ine if they have an intrinsic potential to stim u­late proximal tubule cell growth.

The dose- and tim e-dependency of the HPT cells to aminoglycoside exposure is

consistent with these cells presenting as a valid in v itro m odel system for the s tu d y o f a m in o g ly c o s id e - in d u c e d nephrotoxicity. Furtherm ore, the results em ploying gentam icin also suggested th a t th e H P T c e ll c u l tu r e m o d e l responds to aminoglycoside exposure at concentrations equivalent to that know to occur in th e clin ical se tting . I t is known that a serum concentration of 1 2 |xg pe r ml is the limit suggested during clinical treatm ent in the hospital setting. As such, subconfluent HPT cells w ere dem onstrated to respond to five p-g per ml of gentam icin with over 40 percen t cell death following 1 0 days of exposure. W hile n o n -d iv id in g H PT cells w ere d em onstra ted to be m ore resis tan t to g e n ta m ic in e x p o s u re , to x ic ity s ti l l occurred w ithin a range that m ight be present within the proximal tubule. This is due to the fact that the serum level represents only a m inimal estim ate of the amount localized within the proximal tubule.

It is known that the aminoglycosides concen tra te w ithin the kidney cortex, and in aminoglycoside treated rats, corti­cal gentamicin concentrations have been shown to range from 900 to 2,230 |xg per ml w et w eight . 18,23 Assuming that the lysosomal volume represents five to 1 0 percent of the cell volume and given that most of the gentamicin is localized to the lysosom e , 13 it can be ca lcu la ted th a t in tra ly sosom al co n c en tra tio n s cou ld reach 18 to 80 m M . 5 Thus, even at the increased dosages needed to effect con­fluent HPT cells, it would appear that this cell culture model responds appro­priately in term s of the aminoglycoside concentration needed to elicit cellular alterations.

W hile it is encouraging that this cell culture model appears valid for the study of the cellular basis of aminoglycoside- induced nephrotoxicity, the dose- and tim e-dependency of both toxicity and c e l l r e g e n e r a t i o n w ill r e n d e r i t

278 AMINOGLYCOSIDE EFFECTS ON CELL GROWTH

extremely difficult to employ. The ratio­nale for this statem ent is that during an individual tim e course and dose, the HPT cells can undergo both toxicity and regeneration , usually as sim ultaneous events. This indicates that at any given time point, depending on the extent of the two processes, one could define cel­lu la r a lte ra tio n s o c c u rr in g ow ing to regeneration of the cells and not neces­sarily owing to toxicity of the aminogly­c o sid es , o r v ice v e rsa . W h ile b o th h u m a n 4,11 a n d a n im a l14,17 c u l tu r e s derived from the proximal tubule have been used to study the alterations of cel­lular metabolism that occur owing to the am inoglycosides, the resu lts have not been addressed as regards the growth state of the cells. O ne particular diffi­cu lty arises w ith the use of defin ing appropriate cells for controls. If amino­glycoside exposure elicits both cell toxic­ity and renew al, th en confluen t cells a fte r various days of am inoglycoside exposure in culture are representative of new ly rep lica ted cells, w hile contro l cells would be quiescent during this time frame. W hile the use of proximal tubule- derived cell cultures will allow studies to be perform ed free of in vivo environ­mental influences, results will have to be ca re fu lly c o n tro lle d as re g a rd s th e growth state of the cultures.

Acknowledgments

This research was su p p o rted bv N IH grant DK35311.

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