Cultures Of Human Proximal Tubule Cells Research Articles

Transient induction of metallothionein isoform 3 (MT-3), c- fos, c- jun and c- myc in human proximal tubule cells exposed to cadmium

Cadmium (Cd +2) has been shown to transiently increase the expression of mRNA for the third isoform of the metallothionein (MT-3) gene family in cultured human proximal tubule (HPT) cells. The goal of the present study was to further define the expression of MT-3 in mortal (HPT) and immortal (HK-2) cultures of HPT cells when exposed to lethal and sub-lethal concentrations of Cd +2 under both acute and chronic time periods of exposure. Expression of MT-3 mRNA and protein was determined in cultured HPT cells and HK-2 cells using reverse-transcription-polymerase chain reaction (RT-PCR) and immuno-blotting, and expression of c- fos, c- jun and c- myc mRNA by RT-PCR. The results confirmed that exposure of the HPT cells to Cd +2 induced a transient increase in MT-3 mRNA and extended the induction to include a subsequent transient increase in the level of the MT-3 protein. The induction of MT-3 was rapid and returned to control values within 48 h of exposure despite the continued presence of lethal and sublethal concentrations of Cd +2. It was also demonstrated that the pattern of expression of MT-3 mRNA was similar to that of the early response genes, c- fos, c- jun and c- myc. It was shown that the HK-2 cells did not express MT-3 when exposed to Cd +2, but had similar expression of the c- fos, c- jun and c- myc genes. The results demonstrate that MT-3 expression is metal responsive in HPT cells.

In vitro characterization of renal reabsorption and secretion of folate using primary cultures of human kidney cells

Proximal tubule cells in the kidney play an important role in the homeostasis of folate. Mechanistic studies on folate reabsorption and secretion across the proximal tubular epithelium have been limited. The present studies were aimed at assessing the suitability of human proximal tubule (HPT) cells cultured on membrane inserts as an in vitro model of renal folate processing by examining aspects of folate transport from the apical to basolateral (A-B, reabsorptive) and from the basolateral to apical (B-A, secretory) directions. The data demonstrate that apical membrane binding and cellular uptake of folate in HPT cells were mediated via specific high affinity mechanisms. Apical binding and cellular uptake of folate occurred in a greater proportion from the A-B direction than from the B-A direction, suggesting that folate reabsorption may be the dominant process in HPT cells. However, cellular uptake did occur readily from the B-A direction, in amounts greater than could be explained by leakage through the cell barrier to the apical media, followed by uptake from the A-B direction. These data suggest the presence of specific folate secretory processes in HPT cells. Folate was transferred across the cell layer into the opposite media compartments from both directions, but apparently by nonspecific processes. The present studies suggest that primary cultures of HPT cells grown on microporous membrane inserts may serve as a valuable in vitro model to study the bidirectional transport of folate by kidney cells.

Acute Toxicity of Propylene Glycol: An Assessment Using Cultured Proximal Tubule Cells of Human Origin

Acute Toxicity of Propylene Glycol: An Assessment Using Cultured Proximal Tubule Cells of Human Origin. Morshed, K. M., Jain, S. K., And McMartin. K. E. (1994). Fundam. Appl. Toxicol. 23, 38-43.The present study assesses the acute toxicity of propylene glycol (propane-1,2-diol, PD) using cultured human proximal tubule (HPT) cells as the model. Confluent monolayers of HPT cells were treated with 0-263 mM (0-2%) isotonic solutions of racemic, sinister, and rectus PD (rac-, S-, and R-PD, synonym: DL-, L-, and D-PD, respectively). Release of lactate dehydrogenase (LDH) and of preloaded 51Cr as markers of in vitro toxicity, were increased by PD in a time- and concentration related manner, suggesting significant PD induced damage of the HPT cell membrane. These toxic effects reached an apparent maximum within 2 hr. Further studies were performed to determine whether adverse effects of PD may occur prior to development of membrane damage. Because significant membrane damage was not apparent during the first 15 min of PD exposure, cellular release of lactate and accumulation of glucose were studied after preincubating cells in PD (treated) or pH 7.4 buffer (control) for 10 min. PD significantly increased the subsequent release of lactate by concentration- and enantiomer-dependent mechanisms with R-PD being the most potent agent. PD also inhibited the Na+-independent, carrier-mediated glucose accumulation without significant effects on the Na+-dependent pathway. These data suggest a rapid onset of cellular toxicity even when the plasma membrane integrity and viability remained apparently normal. The present studies show significant toxic effects of PD and suggest that a primary culture of HPT cells may be useful in evaluating the toxicity of xenobiotics.

Variation in the electrical properties of cultured human proximal tubule cells

Monolayers of human proximal tubule (HPT) cells, when grown on permeable supports and mounted in Ussing chambers, spontaneously display a transepithelial potential difference (PD), short-circuit current (Isc), and transepithelial specific resistance (RT). These electrical parameters were used to determine the degree of heterogeneity among independent isolates of human proximal tubule cell cultures. Seventeen independent isolates of cells were assessed, totaling 260 individual determinations of spontaneous electrical properties. On average, these cell monolayers displayed an apical-negative PD of 1.5 +/- 0.1 mV, an Isc of 2.7 +/- 0.2 microA/cm2, and an RT of 480 +/- 19 ohms x cm2. Each independent cell isolate, however, displayed electrical values within a narrow range, in some cases allowing isolates to be distinguished from one another. The individual isolates were also assessed for Na-coupled glucose transport, Na+,K(+)-ATPase activity, cAMP stimulation by parathyroid hormone (PTH), forskolin stimulation of Isc, and ouabain inhibition. With the exception of a strong correlation between Na+,K(+)-ATPase activity and Isc, these parameters, in contrast to electrical properties, were found to be consistent and did not reveal distinctions among the isolates. HPT cell cultures seem to consistently retain important features of proximal tubule differentiation while maintaining the variability, as demonstrated by electrical properties, that might be expected of cells isolated from a random population.

5-methyltetrahydrofolate urinary excretion: modeling by cultured human kidney cells.

Initial attempts to model urinary folate reabsorption using cultures of HPT cells on porous filter inserts produced disappointing results in that large amounts of 5M were transported across the epithelial monolayer in a nonspecific manner. Since the impermeable molecule inulin was also transported, there apparently existed a significant leakage pathway in the way that the cultured cells were used for transport studies. 5M was bound to the AP membrane and taken up into the HPT cell by specific processes, while inulin was excluded, suggesting that the HPT cells were nevertheless operating functionally. TER values from cultured HPT cells plateaued at a high level when cells became confluent, suggesting an epithelial layer with functional tight junctions. However, when growth media were removed and replaced with transport buffers, there was an immediate loss of TER that fully recovered if the transport buffers were preincubated for 60 min. Under these conditions, transport studies showed the expected results--no movement of inulin through the cell layer and much reduced transfer of folate through the paracellular pathway. These results suggest that a transient opening of tight junctions occurs when growth media are replaced with biological buffers (or with fresh growth media), but that recovery of tight junction function occurs with time.

Folate transport and binding by cultured human proximal tubule cells.

Because mechanisms for the renal regulation of folate excretion are poorly understood, a cell culture system representative of the human proximal tubule (HPT) was used for studies of renal folate transport. After confluent cultures of HPT cells were incubated with 3H-labeled folic acid (PteGlu), binding to the apical membrane was determined by an acid removal procedure, and transport was subsequently measured in solubilized cells. Although PteGlu binding was almost all specific (suppressed by excess unlabeled PteGlu), HPT cells transported PteGlu by specific and nonspecific processes. Specific PteGlu binding and transport were both saturable processes, reaching maxima of 0.5 and 0.1 pmol/mg protein, respectively, with half-maximal constants of 12 and 50 nM, respectively. The PteGlu analogues methotrexate and 5-methyltetrahydrofolic acid (5-CH3-H4PteGlu) inhibited both the binding and transport of PteGlu, with 5-CH3-H4PteGlu being more potent (lower half-maximal inhibitory concentration). In contrast, 5-formyltetrahydrofolic acid significantly reduced PteGlu transport at concentrations (100-250 nM) that had no effect on binding. These data suggest that the HPT cells will serve as a good model for studies of renal folate reabsorption. Initial characterization of the transport of folate by HPT cells suggests two distinct processes, binding to a high-affinity membrane folate-binding protein followed by a structurally specific transfer into the cell.

Use of human proximal tubule cell cultures to study folate transport and binding

Use of human proximal tubule cell cultures to study folate transport and binding

Cadmium nephrotoxicity in human proximal tubule cell cultures.

Human proximal tubule kidney cells grown in a serum-free tissue culture medium were exposed to concentrations of CdCl2 in a range of 0.5 to 10 micrograms/ml. Cells were observed from 1 to 20 d upon initiation of cadmium in the culture fluid. Both confluent and subconfluent populations of cells were treated and evaluated for cytotoxicity. Both populations exhibited a concentration-dependent toxicity to ionic cadmium. For cells treated with 2.0 to 10 micrograms/ml Cd, the decreases in cell numbers were largely irreversible. However, cells treated with Cd in a range of 0.5 to 1.0 micrograms/ml exhibited a partial recovery of cell number and control morphology. In this range, recovery was more efficient in the subconfluent cultures. Fine structural alterations in Cd-treated tubule cells included condensation of nuclear chromatin, loss of microvilli structure, disorganization of lateral membrane interdigitation, as well as decreased uptake of aminoglycoside antibiotics as evidenced by decreased numbers of myeloid bodies in these cells. The results of this study imply that use of a human proximal tubule culture system has potential in discerning structural and functional effects of cadmium as well as other nephrotoxic metals and compounds on the human kidney.

An electrophysiological freeze fracture assessment of cadmium nephrotoxicity in vitro.

Human proximal tubule cell cultures exposed to doses of cadmium chloride (CdCl2) between 0.05 microgram/ml and 0.5 microgram/ml exhibited alterations in cell membrane structure and transport function. At these Cd concentrations, cell numbers were not significantly altered from control values in either nonreplicating confluent, or actively replicating subconfluent cultures. Transmission electron microscopy revealed few alterations in cultures treated with 0.05 microgram/ml Cd. Tight junctions were intact; organelles and myeloid body formation appeared normal. Freeze fracture analysis confirmed the integrity of the tight junctions as well as increased numbers of vesicles or pits along the lateral cell membrane, indicating increased endocytotic activity. Cells exposed to 0.1 microgram/ml Cd were characterized by decreased numbers of microvilli and inhibited myeloid body formation. Cd doses of 0.5 micrograms/ml elicited nuclear chromatin condensation, fragmented sealing strands in 5 to 10% of the tight junction profiles, sparse microvilli, and inhibited myeloid body formation. Electrophysiologic assessments of transport function by Ussing chamber analysis revealed decreases in transepithelial potentials for all three concentrations, with significant differences at Cd concentrations of 0.5 to 0.1 micrograms/ml. Cells treated with 0.5 micrograms/ml Cd also exhibited slight decreases in electrical resistance, consistent with the minimal fragmentation of sealing strands observed in freeze fracture replicas. Resistance in cultures treated with 0.1 or 0.05 micrograms/ml Cd remained within control values and indicated that drops in potential difference and short circuit current in these cells reflected true alterations in ion transport.