Cultures Of Renal Tubular Cells Research Articles

Effect of Aroclor 1248 and Two Pure PCB Congeners upon the Release of Arachidonic Acid from the Intracellular Phospholipids of Rat Renal Tubular Cell Cultures

The exposure of rat tubular cell cultures to different polychlorinated biphenyls (PCBs) stimulated the release of arachidonic acid from the intracellular phospholipids, which were previously labeled with [3H]ara chidonic acid. Studies have been realized with Aroclor 1248 (a commercial PCBs mixture with 48% chlorine by weight) and two pure PCB congeners: 3,3′,4,4′-tetrachlorobyphenyl (a coplanar compound) and 2,2′,4,4′,5,5′-hexachlorobiphenyl (adiortho-substituted nonplanar congener). When Aroclor 1248 was added, a relation between Aroclor concentration and arachidonate release to the culture medium was found. This work concludes that only thediortho-substituted congener that prefers a nonplanar configuration can increase the release of [3H]arachidonic acid from the intracellular stores to the culture medium, while the exposure of cell cultures to the chosen coplanar congener does not alter the release of [3H]arachidonic acid to the culture medium in a significant manner. This means that the toxic effect of PCBs could be due, at least in part, to the coplanarity or noncoplanarity of these compounds.

Isolation and culture of porcine renal tubular cells after cold hypoxia.

Isolation and culture of porcine renal tubular cells after cold hypoxia.

Effect of Aroclor 1248 and Two Pure PCB Congeners upon the Membrane Fluidity of Rat Renal Tubular Cell Cultures

The influence of different polychlorinated biphenyls (PCBs) upon dynamic properties of membranes from rat renal tubular cell cultures has been investigated. Studies have been realized with Aroclor 1248 (a commercial PCB mixture with 48% chlorine by weight), and two pure PCB congeners: 3,3′,4,4′-tetrachlorobyphenyl (a coplanar compound) and 2,2′,4,4′,5,5′-hexachlorobiphenyl (anortho-substituted nonplanar congener). Changes in the membrane fluidity were measured by the fluorescence polarization technique using the probe diphenylhexatriene. The treatment of cells with Aroclor 1248 increased membrane fluidity in a dose-dependent manner. Even though rat renal tubular cell cultures accumulated only a low amount of PCBs when cells were incubated in presence of the toxicant, that small quantity was enough to modify the membrane fluidity. Interestingly, a significant increase of membrane fluidity was found in presence of 2,2′,4,4′,5,5′-hexachlorobiphenyl, but no significant differences were detected in the presence of 3,3′,4,4′-tetrachlorobiphenyl. This means that changes in the membrane fluidity induced by the different PCB congeners are related to the coplanarity or lack of coplanarity of the toxic compounds.

L-ascorbic acid regulates growth and metabolism of renal cells: improvements in cell culture.

The addition of L-ascorbic acid 2-phosphate (AscP) to primary cultures of rabbit renal proximal tubular cells (RPTC) grown under improved culture conditions resulted in an extended growth phase and increased cellular density (1.3-fold increase in monolayer DNA and protein contents). AscP reduced glycolysis, increased net lactate consumption by 38%, and stimulated net glucose production by 47%. Basal O2 consumption increased by 39% in RPTC grown in the presence of AscP and was equivalent to that in freshly isolated proximal tubules. AscP increased ouabain-sensitive O2 consumption (81%) and Na(+)-K(+)-ATPase activity (2.5-fold), which suggested increased active Na+ transport. Addition of AscP increased Na(+)-dependent glucose uptake by 43% and brush-border enzyme marker activities by 46%. It is concluded that supplementation of media with AscP further improves RPTC culture conditions by promotion of cellular growth and stimulation of in vivo-like respiration, lactate utilization, and net glucose synthesis. These changes are accompanied by an increase in brush-border enzyme activities and stimulation of active Na+ transport and Na(+)-dependent glucose transport, which demonstrate an improved expression of brush-border membrane functions in RPTC.

Hypoxia and proliferation are primarily responsible for induction of lactate dehydrogenase activity in cultured cells.

In vitro to in vivo extrapolations require cellular models that closely mimic cells in vivo. Typically, cultured cells revert to glycolysis, have increased lactate dehydrogenase (LDH) activity, have decreased oxidative metabolism, and dedifferentiate. This study examined the role of hypoxia and proliferation in the regulation of LDH activity, and the temporal relationships among induction of glycolysis, LDH activity, and proliferation in primary cultures of rabbit renal proximal tubular cells (RPTC). LDH activity in RPTC grown under standard conditions (STILL) did not increase during the first 3 d of culture, but increased 18-fold by d 7. LDH activity in RPTC grown under conditions of increased oxygen supply (SHAKE) did not increase during the first 3 d of culture, but increased 5.5-fold by d 7. Hypoxia and proliferation were responsible for 73% and 27%, respectively, of the increase in LDH activity in STILL RPTC. Hypoxia had no effect on RPTC proliferation. Neither medium glucose nor insulin concentrations had any effect on LDH activity in SHAKE RPTC. Supplementation of the culture medium with ribose 5-phosphate or ribose diminished the increase in LDH activity in SHAKE RPTC to 62% and 52% of controls but had no effect on monolayer DNA content. Two-day treatment of confluent SHAKE RPTC with epidermal growth factor (EGF) resulted in 1.6-, 1.4-, and 1.9-fold increases in LDH and G6PDH activities and monolayer DNA content, respectively. The stimulatory effect of EGF on LDH and G6PDH activities, but not monolayer DNA content, was abolished by ribose or ribose 5-phosphate. In contrast, transforming growth factor-beta 1 (TGF-beta 1) treatment stimulated lactate production but had no effect on LDH and G6PDH activities, or proliferation of SHAKE RPTC. These results show that (1) both hypoxia and proliferation are primarily responsible for the induction of LDH activity in cultured cells, (2) LDH activity is not an accurate indicator of glycolysis, (3) induction of LDH activity is secondary and due, in part, to the induction of the pentose phosphate pathway, (4) EGF stimulates glycolysis, LDH activity, and proliferation, and (5) TGF-beta 1 stimulates glycolysis but has no effect on LDH activity or proliferation.

Pexicrine effects of basement membrane components on paracrine signaling by renal tubular cells

Paracrine interactions between tubular epithelium and interstitial cells have been assumed to be mediated largely by soluble cytokines. While the role of extracellular matrix (ECM) and matrix metalloproteinases (MMPs) in modifying cell function is widely appreciated, the role of the renal tubular basement membrane in modulation of tubulointerstitial function has not been studied. To establish whether those components of the ECM which support tubular epithelial cells also influence cell function (that is, a pexicrine effect), we studied their effects on paracrine signaling between epithelium and fibroblasts. Primary cultures of rat renal proximal tubular epithelial cells (PTE) were cultured on laminin (LN), collagen types-IV and -I (COL-IV, COL-I) and fibronectin (FN). PTE attained confluence more rapidly when grown on LN = COL-IV > COL-I = FN = plastic. On all substrates PTE produced the MMPS, gelatinase-A and -B and collagenase with an apparent increase in gelatinase-A and -B production when cultured on LN. MMPs were found to be secreted both apically and basally with basal secretion predominating, except on LN where secretion was primarily from the apical surface. Cultures of rat renal cortical interstitial fibroblasts were established and characterized. Cortical fibroblasts (CF) were found to secrete gelatinase-A and collagenase. Conditioned medium (CM) from PTE cultured on COL-IV stimulated proliferation of CF but proliferation was unaltered by CM from PTE grown on other substrates. By contrast, co-culture of PTE on LN with CF suppressed collagenase and gelatinase activity in both cell types, indicating a bi-directional, paracrine modulation of MMP production. Thus in the tubulointerstitium, the BM components LN and COL-IV not only fulfill a structural role but act as signaling molecules with differential effects which modify the function of the tubular epithelium and its paracrine interaction with adjacent fibroblasts. The initiation of interstitial fibrosis induced by injury to the tubular basement membrane may reside in the perturbation of this interaction.

The mechanisms of taurine mediated protection against cell damage induced by hypoxia and reoxygenation.

Taurine administered during hypoxia markedly reduced the cell damage due to O2 deficiency and reoxygenation. Different mechanisms are responsible for the improved survival of the renal cell cultures. Taurine markedly reduces the osmoregulatory deterioration during hypoxia and reoxygenation. Calcium homeostasis was markedly improved. Ca2+ efflux during hypoxia as well as Ca2+ overload during reoxygenation was significantly reduced by the amino acid. The effect of taurine was partly comparable to the effect induced by Ca2+ channel blockers. One of the effects mainly responsible for cellular protection seems to be the taurine-induced acceleration of cellular growth processes in spite of hypoxia and reoxygenation. The spectrum of cytoprotective effects of taurine predisposes this substance to be a physiological protective agent responsible for cellular homeostasis or enantiostasis.

Renal proximal tubular epithelium from patients with nephropathic cystinosis: Immortalized cell lines as in vitro model systems

The renal proximal tubule is a major site of injury in a variety of congenital/metabolic diseases including nephropathic cystinosis, the most commonly known cause of renal Fanconi's syndrome. In this lysosomal storage disease there are defects in proximal tubule function within the first few months of life. While culture of renal tubular cells from the urine of these patients is possible, development of immortalized cell lines would insure large numbers of homogeneous cells for studies of renal epithelial cell morphology and pathophysiology in this disease. To develop immortalized cells, cystinotic and normal proximal tubular cells in culture were exposed to an immortalizing vector, containing pZiptsU19 with the temperature sensitive SV40 T-antigen allele tsA58U19 and a neomycin resistance gene, and neomycin-resistant tubular cells were selected for propagation. Ten clones from cystinotic patients have been developed and characterized. All clones express T-antigen at permissive temperature (33 degrees C). Immortalized cells have an epithelial morphology and grow to form confluent monolayers; doubling times vary from 31 to 86 hours. Cystinotic clones are keratin, MDR P-glycoprotein, and alpha-95 kD brush-border associated protein positive but Tamm-Horsfall protein negative by immunocytochemistry, as are normal proximal tubule cells immortalized with this vector. This is consistent with a proximal tubule origin of the cystinotic clones. The cystine content of the cystinotic cells is 70 to 160 times that of normal renal proximal tubular cells in culture, with most of the cystine sequestered in cell lysosomes, confirming that these cell lines express the storage defect.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of NM23 in Cell Lines Derived from Patients with Metastatic Renal Cell Carcinoma

Reduced expression of nm23 has been associated with increased metastases and decreased survival in a variety of malignancies. In the present study, the expression of nm23 was examined by Northern and Western blot analyses in a series of cell lines derive from patients with metastatic renal cell carcinoma. Two of twelve (17 percent) informative cell lines derived from 9 patients had loss of heterozygosity at Nm23-H1. Twenty-two renal cancer cell lines derived from primary tumors, 5 cell lines derived from metastatic tumors and 4 short-term cultures of normal proximal renal tubular cells all expressed Nm23 mRNA in varying amounts. On average, the level of expression of Nm23 mRNA in short-term cultures of benign proximal renal tubular cells was found to be similar to the level seen in renal cancer cell lines. Twenty-eight cell lines derived from renal primary tumors and 8 cell lines derived from metastatic tumors expressed both the Nm23-H1 and Nm23-H2 proteins. High or low relative expression of nm23 at the mRNA or protein level did not correlate with survival. The absence of any anomalous pattern of expression of the nm23 genes and the lack of correlation of expression with survival suggests that nm23 does not play a central role in the progression of this tumor type.

Loss of Heterozygosity Occurs Centromeric to RB Without Associated Abnormalities in the Retinoblastoma Gene in Tumors from Patients with Metastatic Renal Cell Carcinoma

Tumor suppressor genes have been found to have loss of function in a number of malignancies. This loss of function is believed to contribute to malignant transformation or metastatic spread. In the present study, expression of the retinoblastoma (RB) tumor suppressor gene was examined in cell lines and tumor tissue obtained from primary renal and metastatic sites in patients with metastatic renal cell carcinoma. Three of fifteen (20 percent) of informative renal carcinoma cell lines had loss of heterozygosity (LOH) in the RB gene (intron 20) detected by polymerase chain reaction analysis. Using restriction fragment length polymorphism (RFLP) analysis, 7 of 22 (32 percent) informative cell lines had LOH centromeric to the RB gene at the D13S1 locus. No LOH (0 of 7) was seen telomeric to the RB gene at the D13S2 locus. None of the 28 cell lines examined had decreased RB mRNA expression compared with short-term cultures of proximal renal tubular cells. Western blotting demonstrated phosphorylated and unphosphorylated forms of RB protein of expected molecular weight in all 41 cell lines (33 primary and 8 metastatic) examined. Twenty-nine primary cell lines and 6 metastatic cell lines all demonstrated normal immunohistochemical staining. Loss of RB immunohistochemical staining in paraffin-embedded tissue was detected in none of the primary tumors (0 of 30) or metastatic tumors (0 of 12). The absence of abnormalities of RB expression detected in these renal cell carcinomas suggests that abnormalities of the RB gene are not central to malignant transformation or progression in this tumor type; however, another tumor suppressor gene centromeric to the RB locus may be important in renal cell carcinogenesis.

Calcium oxalate crystallizing properties of polyanions elaborated by cultured renal proximal tubular cells.

To study the influence of renal polyanions on crystallization of urinary calcium oxalate, we recovered polyanionic macromolecules from media conditioned by primary cultures of renal proximal tubular epithelial cells of rats in serum-free, hormonally defined medium. Cells cultured on microporous supports showed a higher degree of morphological and functional proximal differentiation into a polarized monolayer than those on plastic impervious substrata. Papainization of the polyanions yielded the glycosaminoglycans chondroitin/dermatan sulphate and heparan sulphate. These accounted respectively for 60% and 80% of the crystal nucleation-promoting activities of polyanions recovered from the apical and basal media conditioned by polarized cultures on microporous supports. Similar relative activities were observed among the urinary glycosaminoglycans and polyanions similarly tested. Primary cultures of polarized proximal tubular epithelial cells are useful then as an in vitro model to study the crystallizing activities of polyanionic macromolecules produced by renal cells.

Culture of Human Kidney Proximal Tubular Cells – The Effect of Various Detachment Procedures on Viability and Degree of Cell Detachment

Culture of renal proximal tubular cells has become a widely used tool in research. As tubular epithelial cell cultures form monolayers which have to be detached from the culture flasks prior to experiments, it was the objective of this study to evaluate possible damage suffered by the cells during the procedure of transforming monolayers into single-cell suspensions. Various detachment procedures (scraping, EDTA incubation, trypsin/EDTA incubation, incubation with coUagenase alone and coUagenase and EDTA together, and the use of various coUagenase preparations) were studied in order to obtain viable, non-damaged human cells. The measurement of lactate dehydrogenase activity was used as an indicator in the assessment of cell viability/damage. The effect of the various methods of detachment on the release of lactate dehydrogenase from human cells was compared with that from the renal cell lines, MDCK and LLC-PK=. The released lactate dehydrogenase activity following the detachment was significantly lower in the continuous cell lines as compared to the human cells. Scraping of the human cells caused the highest degree of damage, showing a release of about 90% of the total lactate dehydrogenase, whereas collagenases showed a lactate dehydrogenase release between 8 and 25% depending on the coUagenase preparation used. The use of collagenase in the detachment of cultured human kidney proximal tubular cells is recommended. The coUagenase P preparation from Boehringer Mannheim (200 mg/l00 ml; incubation at 37 °C for 10 min) appears to be particularly suitable.

Growth of cultured rabbit renal tubular cells does not require exogenous glutamine

Following the pioneering work of Eagle et a! [1], glutamine, which is the most abundant amino acid in plasma (0.5 to 0.6 mM) [2] and an important source of energy and nitrogen for cell metabolism and growth [3], is considered to be a mandatory component of cell culture media. In renal epithelial cell cultures, it is added often at a final concentration of 2 to 5 mrvi [4—10]. However, this is not necessarily useful in primary cultures of renal proximal tubular cells from the rabbit, the most widely used species for renal cell cultures. Indeed, glutamine synthetase is present in the renal cortex of this species; in addition, the activity of phosphate-activated glutaminase, the enzyme which initiates glutamine metabolism and yields glutamate and ammonium [3], is low in the rabbit kidney [2]. Moreover, freshly isolated rabbit proximal tubules do not or hardly remove glutamine in net amounts when they are incubated in the presence of glutamine concentrations varying from 1 to 5 mtvi [11]. In an attempt to clarify this point, we cultured rabbit proximal tubular cells on a permeable collagen membrane and investigated whether the addition of glutamine to the culture medium influences cellular growth and glycolysis. The results obtained show that omission of exogenous glutamine does not alter the growth of rabbit renal proximal tubular cells in primary culture, and that the provision of glutamine by the glutamine synthetase reaction is sufficient and mandatory for their growth and DNA synthesis. Addition of glutamine, which stimulated glycolysis in freshly isolated proximal tubules, failed to do so in cultured rabbit renal cells.

Increased intracellular glycerophosphoinositol and arachidonic acid are biochemical markers for lindane toxicity

Lindane stimulates the release of both glycerophosphoinositol and arachidonic acid from phospholipids in rat renal proximal tubular cell cultures. When lindane was added to the culture medium, a correlation between the time-course profiles of glycerophosphoinositol and arachidonate release was found. This suggests a pathway in which phosphatidylinositol is not directly broken down by phospholipase C, but can instead be broken down to glycerophosphoinositol and arachidonic acid by phospholipase A enzymes. Therefore, a mechanism of action of lindane is through its effect on glycerophosphoinositol and arachidonic acid metabolism.

Internalization of Proteus mirabilis by human renal epithelial cells

Proteus mirabilis, a common agent of bacteriuria in humans, causes acute pyelonephritis and bacteremia. Renal epithelium provides a barrier between luminal organisms and the renal interstitium. We have hypothesized that P. mirabilis may be internalized into renal epithelium. To test this hypothesis, we added suspensions of three P. mirabilis strains (10(8) CFU) to confluent monolayers of primary cultures of human renal proximal tubular epithelial cells (HRPTEC) and, after 3 h, found the bacteria internalized within membrane-bound vacuoles by light and electron microscopy. Internalization of bacteria by HRPTEC was corroborated by using the gentamicin protection assay. Cytolysis of HRPTEC by the HpmA hemolysin, however, was a confounding factor in this assay, and therefore a hemolysin-negative hpmA mutant was used in subsequent experiments. The nonhemolytic mutant WPM111 did not disrupt the monolayer and was recovered in numbers that were 10- to 100-fold higher than those of the hemolytic parent BA6163. Cytochalasin D (20 micrograms/ml) inhibited internalization of Salmonella typhimurium but not that of P. mirabilis, suggesting that the latter species enters HRPTEC by a mechanism that is not dependent on actin polymerization. We suggest that HpmA hemolysin-mediated cytotoxicity and internalization of bacteria by HRPTEC may play a role in the development of Proteus pyelonephritis.

Lindane affects phosphoiniositide turnover through a different mechanism of the phosphatidylinositol synthesis inhibition in rat renal proximal tubule cell culture

The ability fo lindane to change the metabolism of inositol phospholipids was investigated using rat renal proximal tubular cell cultures labelled with [ 3H]inositol. Lindane addition to the culture medium caused labelling of inositol trisphosphate, inositol bisphosphate and inositol tetrakishosphate to decrease and that of the inositol monophosphate pool to increase. A depletion of radioactivity in phosphatidylinositols was also observed after lindane addition. Most strikingly, the addition of lindane considerably increased the levels of glycerophospho-inositol in a dose-dependent manner. The effect of lindane follows a different pattern from that of bradykinin, and it is suggested to act by stimulating phospholipase A activity(ies). IP-inositol monophosphate; IP 2-inositol bisphosphate; IP 3-inositoll trisphosphate, IP 4-inositol tetrakisphosphate; GPI-glycerophosphoinositol; PI-phosphatidylinositol; PIP-phosphatidylinositol monophosphate; PIP 2-phosphatidylinositol bisphosphate; TCA-trichloroacetic acid; DMEM-Dulbecco's Modified Eagle's Medium; DMEMab-DMEM supplemented with penicillin (100 IU/ml), streptomycin (100 μg/ml) and fungizone (0.25 μg/ml); BSA- bovine serum albumin

Toxicity of ifosfamide, cyclophosphamide and their metabolites in renal tubular cells in culture.

Ifosfamide (IF) and cyclophosphamide (CP) are highly effective alkylating cytostatic drugs. IF and CP have to be activated through a metabolic step in vivo; numerous metabolites are known. While both IF and its structural isomer CP have severe urotoxic side effects, only IF is also a nephrotoxic drug, causing tubular damage resulting in Fanconi syndrome in some cases. Little information is available regarding the pathogenic mechanism of tubular damage by IF. We used the renal epithelial cell line LLC-PK1, which has many properties of the proximal tubule, in order to investigate the toxicity of IF and CP and of their reactive metabolites 4-hydroxy-IF (4-OH-IF), 4-hydroxy-CP (4-OH-CP), acrolein and chloroacetaldehyde (CAA). Protein content of monolayers, DNA and RNA synthesis were determined by standard techniques (thymidine and uridine incorporation). IF and CP had the lowest toxicities of all compounds tested. Both drugs inhibited thymidine incorporation by about 30% at a concentration of 300 mumol/l after 1 h incubation. 4-OH-IF and 4-OH-CP were significantly more toxic than the parent drugs. Thymidine incorporation, the most sensitive parameter, was reduced by about 70% by 300 mumol/l of either compound. In addition, 4-OH-CP reduced the total protein content of monolayers. 4-OH-IF did not effect protein content and RNA synthesis. Acrolein, the most toxic metabolite tested, reduced all three parameters significantly at concentrations of 50-75 mumol/l after 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of taurine transport by external taurine concentration and medium osmolality in renal tubular cells in culture.

Taurine is actively transported into the renal tubular cell by a Na+ — and Cl−-dependent system specific for this β-amino acid and structural analogs (14). The proximal epithelial transport system regulates the urinary excretion of taurine. Taurine transport is regulated by the dietary intake of taurine in the rat (1), by extracellular taurine concentration in kidney cells in culture (7), and by medium osmolality in MDCK cells (11). We have previously demonstrated that taurine transport in both MDCK and LLC-PK1 cells is increased following incubation of cell monolayers in taurine-free medium and in decreased following incubation of cells in high extracellular taurine (8). The LLC-PK1 is a porcine cell line of proximal tubular origin and the MDCK (Madin Darby Canine Kidney) cell line is from the collecting duct of the dog.

Susceptibility of renal tubular cells to lymphokine-activated killer (LAK) cells: application of culture system using a collagen gel matrix.

Monolayer cultures of renal tubular (hKEC) cells were established. These cells formed empty spheroids after 2-3 weeks of culture in a collagen gel matrix. A subcellular polarity from the apex to basement was induced in these "spheroidal" hKEC cells. The weak expression of laminin at the outer surface was evident on spheroidal but not on monolayered hKEC cells. The regulation of HLA-ABC, DR, and intercellular adhesion molecule-1 (ICAM-1) antigens on hKEC cells in the gel matrix was investigated utilizing digestion of gel matrix by collagenase. Enzymatic digestion of the collagen gel did not significantly affect the surface expression of HLA-ABC and ICAM-1, but reduced HLA-DR expression as shown by flow cytometry. The MHC and ICAM-1 molecules on both spheroid-forming and monolayered hKEC cells were upregulated by adding a supernatant of mixed lymphocyte reaction (MLR) and recombinant human interferon (IFN)-gamma. HLA-DR antigen expression was inconsistently induced on the hKEC cells cultured in collagen gel without MLR supernatant or IFN-gamma. In contrast, no HLA-DR expression was found on monolayered hKEC cells in the absence of MLR supernatant or IFN-gamma. Spheroid-forming hKEC cells, when dispersed by enzymatic digestion, were more susceptible to cytolysis by lymphokine-activated killer (LAK) cells than were the enzymatically dispersed, monolayered cells in the 51Cr-release assay. The LAK cells were seen to migrate into the collagen gel and kill the hKEC cells. Thus, LAK cells may function to favor the acceleration of graft rejection.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro mechanisms and models of gentamicin nephrotoxicity

Nephrotoxicity is the dose limiting feature of gentamicin adminstration. The nephrotoxic potential of gentamicin was investigated in primary cultures of rat renal proximal tubular cells and in the established renal epithelial cell lines, LLC-PK 1 cells and MDCK cells. Polyaspartic acids have been reported to ameliorate the nephrotoxicity of gentamicin in vivo. However, these compounds have been reported to exert other toxic side effects. We investigated the role of magnesium- l-aspartate-hydrochloride in nephroprotection against gentamicin in these models of nephrotoxicity. Gentamicin admistration resulted in a reduction in the DNA and protein content of the primary proximal tubular cells and an impaired calcium uptake into these cells. Magnesium- l-aspartate-hydrochloride significantly reduced the extent of [ 3H]gentamicin binding to these cells. Transepithelial resistance determination in the established renal cell lines revealed that gentamicin disrupted intercellular communications, while magnesium- l-aspartate-hydrochloride abolished this gentamicin-induced alteration. The actions of gentamicin were shown to occur prior to any loss of cell viability as assessed by flow cytometry. It is concluded that these systems provide useful models for the investigation of gentamicin nephrotoxicity and that magnesium- l-aspartate-hydrochloride may be useful in ameliorating gentamicin nephrotoxicity in clinical conditions.