Thyroid hormone upregulates Na,K-ATPase alpha and beta mRNA in primary cultures of proximal tubule cells

In vivo studies have demonstrated that thyroid hormone regulates the activity of Na,K-ATPase in the mammalian kidney. However, it is still unclear whether upregulation of Na,K-ATPase by thyroid hormone is mediated through the direct action on renal tubule cells or through other mediators, such as an increase in glomerular filtration rate. Using primary cultures of rabbit renal proximal tubule cells, studies were undertaken to elucidate this problem. We found that Na,K-ATPase activity was increased by 26 +/- 8%, 30 +/- 9%, 39 +/- 9% after 24-h treatment with T3 of 10(-11), 10(-9), 10(-7) M, respectively. We further demonstrated that 24-h incubation of T3 (10(-7) M) enhanced alpha- and beta-protein abundance by 44 +/- 29% and 31 +/- 16%, and alpha- and beta-mRNA levels by 84 +/- 27% and 65 +/- 11%, respectively. The time course studies revealed that the significant increase in Na,K-ATPase activity, alpha- and beta-protein and mRNA abundance didn't appear until 24-h of T3 treatment. Our data indicate that thyroid hormone directly upregulates Na,K-ATPase in proximal tubule cells via a pretranslational mechanism.     

Colloidal silica-coated tissue culture dishes for primary cell cultures: growth of rabbit renal proximal tubule cells

The use of colloidal silica as a substratum for primary cultures of differentiated cells has significant advantages over classic tissue culture polystyrene. In this report, the growth and the level of expression of differentiated function of primary rabbit renal proximal tubule (RPT) cell cultures on colloidal silica is examined, using hormonally defined serum-free medium. Primary RPT cells grew to confluence more rapidly on colloidal silica than on tissue culture polystyrene (TC+). Moreover, following three passages, the RPT cells increased in number threefold more than parallel cultures on TC+. The morphology of primary RPT cells on colloidal silica were found by means of transmission electron microscopy to possess a polarized morphology with a brush border, and differentiated markers were retained even after passaging, including the Na+/glucose cotransport system and Glut 7.     

Urinary acidification: CO2 transport by the rabbit proximal straight tubule

Proximal straight tubules from rabbit kidneys were perfused in vitro in order to study transport of bicarbonate. Total CO2 content was measured in perfused and collected tubule fluid, using microcalorimetry. When the initial perfusate and bath contained 25 mM bicarbonate, the concentration of total CO2 decreased in the collected tubule fluid, indicating net reabsorption of bicarbonate. When the initial perfusate contained no bicarbonate and the bath contained 25 mM bicarbonate, total CO2 appeared in the collected tubule fluid. The rate at which total CO2 appeared in the tubule fluid was rapid, indicating a high permeability. Proximal straight tubules from superficial and juxtamedullary nephrons were compared and found to differ in permeability to CO2 and in transport rate. This functional heterogeneity may affect urinary acidification when there is redistribution of renal blood flow.     

Paracrine stimulation of human renal fibroblasts by proximal tubule cells

Paracrine stimulation of human renal fibroblasts by proximal tubule cells. BACKGROUND: Interstitial fibrosis strongly predicts the degree and progression of renal failure in human renal disorders. Since active fibrosis tends to initially occur in a peritubular distribution, the possibility that human proximal tubule cells (PTC) relay fibrogenic signals to neighboring cortical fibroblasts was examined in vitro. METHODS: Cell proliferation (cell counts and thymidine incorporation), total collagen synthesis (proline incorporation), matrix metalloproteinase (MMP) activity (gelatin zymography), and autocrine secretion of insulin-like growth factor-I (IGF-I) were measured in primary cultures of human cortical fibroblasts cocultured with PTC or exposed to PTC-conditioned media (PTCCM). RESULTS: Cell numbers and thymidine incorporation rates were increased in cortical fibroblasts cocultured with PTC (136.4+/-7.3% and 119.3+/-8.2% of control values, respectively, P < 0.05) or incubated in PTC-CM (114.0+/-5.9%, P < 0.05 and 146.7+/-13.3%, P < 0.05, respectively). PTC-CM stimulated cortical fibroblast collagen synthesis (13.5+/-1.0% vs. 10.8+/-0.7%, respectively, N = 24, P < 0.05) and MMP-2 and MMP-9 secretion. Cortical fibroblast secretion of IGF-I binding protein-3 (IGFBP-3), which in turn modulates the autocrine and paracrine actions of IGF-I, was enhanced in the presence of PTC-CM compared with control (1162.2+/-94.2 vs. 969.1+/-58.9 ng/mg protein/day, P < 0.05), but no change was observed in cortical fibroblast secretion of IGFBP-2 (260.9+/-38.8 vs. 290.9+/-36.6 ng/mg protein/day, P = NS) or IGF-I (56.7+/-6.6 vs. 57.0+/-6.8 ng/mg protein/day, P = NS). Human PTC secreted transforming growth factor-beta1 (TGF-beta1) and the AB heterodimer of platelet-derived growth factor (PDGF-AB) in a time-dependent fashion and the augmentation of cortical fibroblasts mitogenesis, collagen synthesis and IGFBP-3 secretion induced by PTC-CM was replicated by exogenous TGF-beta1 and PDGF. Furthermore, the stimulatory effects of PTC on cortical fibroblasts were potentiated in transiently acidified PTC-CM (which activated latent TGF-beta1), and were abrogated by neutralizing antibodies specifically directed against TGF-beta1 and PDGF-AB. Cortical fibroblasts in turn released a soluble factor(s) into cortical fibroblast-conditioned media that reciprocally stimulated PDGF-AB production by PTC (4.79+/-1.55 vs. 0.78+/-.06 ng/mg protein/day, P < 0.05). CONCLUSIONS: PTC modulate the biological behavior of neighboring cortical fibroblasts in the human kidney through paracrine mechanisms, which include the production and release of PDGF-AB and TGF-beta1. Renal insults that result in proximal tubule injury may perturb this paracrine interaction, thereby culminating in excessive fibroblast proliferation and interstitial fibrosis.     

Cytotoxic effect of Shiga toxin-1 on human proximal tubule cells

BACKGROUND: Cytolytic Shiga toxins (Stx) are believed to be largely responsible for renal damage in post-diarrheal hemolytic-uremic syndrome (D + HUS). Despite the general belief that endothelial cells are the primary target of Stx, there is evidence that proximal tubules may be a site of toxin action. We hypothesized that cultured proximal tubular cells are sensitive to the cytotoxic effects of Stx. METHODS: Cultured human proximal tubular cells were exposed to Stx-1 in the presence and absence of a variety of inflammatory factors likely to be elevated in the kidney or serum of patients with D + HUS. Cell survival, protein synthesis, total cell levels and synthesis of Stx receptors (GB3), and Stx binding were measured. RESULTS: Proximal tubules were extremely sensitive to the cytotoxic effect of Stx-1 with an LD50 at least equal to, if not less than, that seen with Vero cells. Interleukin-1 (IL-1), lipopolysaccharide (LPS), and butyrate (but not tumor necrosis factor or interleukin-6) up-regulated proximal tubule sensitivity to Stx-1. IL-1 increased Stx-1 binding, but did not alter total cell levels or synthesis of GB3, the glycosphingolipid receptor for Stx-1. In contrast, LPS and butyrate, despite increasing Stx-1 sensitivity, had no effect on Stx-1 binding. CONCLUSIONS: These studies indicate that proximal tubules are exquisitely sensitive to Stx-1 cytotoxicity and that inflammatory factors can increase toxin responsiveness through a variety of mechanisms. It is suggested that proximal tubules may be an important early target of Stx-1 action in D + HUS.     

Determination of folate transport pathways in cultured rat proximal tubule cells

Deficiency of the vitamin folic acid has recently been linked with increased incidence of neural tube defects and of cardiovascular disease, through elevated plasma homocysteine levels. The kidney has an important role in conserving folate to counteract development of deficiency. Urinary folate excretion is regulated by the degree of reabsorption of folate by the proximal tubule cell. To evaluate an in vitro model for studies of the regulation of urinary folate excretion, the present studies examined the transport of 5-methyltetrahydrofolate (5-CH3-H4PteGlu), the primary form of folate in the glomerular filtrate, by normal rat proximal tubule (RPT) cells in confluent monolayer cultures. Specific binding of 5-CH3-H4PteGlu to the apical membrane was saturable (K(D) = 27 nM), but intracellular transport was not saturated up to 100 nM concentrations. 5-CH3-H4PteGlu transport was decreased 50% by concentrations of folic acid that completely blocked 5-CH3-H4PteGlu binding by the apical folate receptor. Probenecid (10 mM), an anion exchange (reduced folate carrier) inhibitor, reduced 5CH3-H4PteGlu transport by 50% without significantly affecting binding. Aspirin (3 mM) did not alter 5-CH3-H4PteGlu transport, but significantly enhanced the inhibition due to probenecid. Similarly, indomethacin (5 microM) potentiated the inhibition of 5-CH3-H4PteGlu transport by probenecid. These data suggest that RPT cells take up 5-CH3-H4PteGlu by both the folate receptor and the reduced folate carrier, implying a role for both pathways in regulating urinary folate excretion.     

Shiga toxin-1 regulation of cytokine production by human proximal tubule cells

BACKGROUND: Interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) levels are elevated in kidneys of patients with post-diarrheal hemolytic uremic syndrome (D+HUS) and may contribute to renal dysfunction. The renal cellular sources of these inflammatory cytokines in D+HUS are largely unknown, however, the proximal tubule has emerged as a potentially important candidate. Since Shiga toxin-1 (Stx-1) has been implicated in the genesis of D+HUS, we examined the effect of Stx-1 on cytokine production by human proximal tubule cells. METHODS: Stx-1 cytotoxicity, protein synthesis inhibition, and effect on IL-1, IL-6, and TNF protein release and mRNA levels were determined. The effect of another protein synthesis inhibitor, cycloheximide (CHX), on these parameters was also evaluated. RESULTS: Stx-1 greatly increased TNF release and mRNA levels while CHX, at concentrations that produced similar inhibition of protein synthesis, had no effect on TNF production. In contrast, Stx-1 and CHX caused comparable elevations in IL-1 release and mRNA accumulation. Stx-1 and CHX also stimulated IL-6 mRNA accumulation, but only at concentrations that either were cytotoxic or substantially blocked protein synthesis. Finally, lipopolysaccharide, which is likely to be elevated in the circulation of patients with D+HUS, had no effect alone, but synergized with Stx-1 to increase IL-1 production. CONCLUSIONS: These results indicate that Stx-1 stimulates proximal tubule inflammatory cytokine production and that this effect is due partially to nonspecific induction of mRNA levels as well as activation of Stx-1-specific mechanisms.     

Predominant expression of monoamine oxidase B isoform in rabbit renal proximal tubule: regulation by I2 imidazoline ligands in intact cells

Previous studies have shown that a subpopulation of the catecholamine-degrading enzymes monoamine oxidase (MAO) A and B holds a previously unknown regulatory site, the I2-imidazoline binding site (I2BS). In the present work, we characterized the isoforms of monoamine oxidases expressed in the rabbit renal proximal tubule, defined their relationship with I2BS, and investigated the ability of I2BS ligands to inhibit enzyme activity in intact cells. Two findings indicate that MAO-B is the predominant isoform expressed in the renal proximal tubule cells: 1) Western blot performed with an anti-MAO-A/MAO-B polyclonal antiserum revealed a single 55-kDa band corresponding to MAO-B; 2) enzyme assays showed an elevated MAO-B activity ([14C]beta-phenylethylamine oxidation: Vmax = 1.31 +/- 0.41 nmol/min/mg protein), whereas MAO-A activity was only detectable ([14C]5-HT oxidation: Vmax = 80.3 +/- 19 pmol/min/mg protein). Photoaffinity labeling with the I2BS ligand [125I]2-(3-azido-4-iodophenoxy)-methylimidazoline revealed a single 55-kDa band, which indicates that MAO-B of the renal proximal tubule cells holds the I2 imidazoline binding site. [3H]Idazoxan binding studies and enzyme assays showed that, in intact cells, I2BS ligands bind to and inhibit MAO-B. Indeed, the increase in the accessibility of intracellular compartment by cell permeabilization did not enhance [3H]idazoxan binding, which indicates that, in intact cells, intracellular I2BS are fully occupied by imidazoline ligands. In addition, enzyme assays showed that incubation of proximal tubule cells with imidazoline ligands leads to a complete, dose-dependent inhibition of MAO activity. These data show the predominant expression of MAO-B in rabbit renal proximal tubule and its regulation by imidazoline ligands in intact cells.     

Induction of rabbit renal mixed-function oxidases by phenobarbital: cell specific ultrastructural changes in the proximal tubule

Administration of phenobarbital (60 mg/kg) daily for 4 days to male rabbits resulted in induction of renal cytochrome P-450 (3.5-fold) and a corresponding increase in ethoxycoumarin-O-deethylase and benzphetamine-N-demethylase activity (17- and 4-fold, respectively). Kidney weight to body weight ratio and renal ethoxyresorufin-O-deethylase were not affected by phenobarbital pretreatment. Numerous focal areas of proliferation of smooth endoplasmic reticulum (SER) were evident in proximal tubule cells from phenobarbital treated rabbits while proximal tubular cells from control rabbits had only small and sparcely located aggregates of SER. Phenobarbital-induced SER proliferation was specifically localized to the S3 segment of the proximal tubule. Proliferation was not observed in S2 cells of the proximal tubule, cells of Henle's loop, distal tubules, or collecting tubules in rabbits pretreated with phenobarbital. These data demonstrate the biochemical heterogeneity of cell types within the proximal tubules of rabbits. Furthermore, induction of mixed-function oxidases specifically in S3 cells of the proximal tubule may be of toxicological significance in the metabolic activation of certain nephrotoxicants.     

Transforming growth factor beta (TGF beta) activity in urine of patients with glomerulonephritis

Several lines of experimental evidence have shown that transforming growth factor beta (TGF beta) may play major role in glomerular diseases, mediating the inflammatory response through glomerulosclerosis. In the present study we evaluated TGF beta activity in occasional urine samples from 7 normal individuals and from 15 patients (10 with focal glomerular sclerosis and 5 with membranous glomerulonephritis) using a CCL-64 mink lung cell growth inhibition assay. Urinary TGF beta activity (reported in relation to urine creatinine concentration, Ucr, mean +/- SD) was higher in patients with focal glomerular sclerosis (mean = 17.32 +/- 15.75/10 micrograms Ucr) and patients with membranous glomerulonephritis (mean = 17.78 +/- 11.53/10 micrograms Ucr) than in normal individuals (mean = 0.8 +/- 0.44/10 micrograms Ucr). We also observed that TGF beta activity in urine from patients with focal glomerular sclerosis correlated with their plasma creatinine levels (r = 0.85), suggesting that TGF beta activity may be correlated with other indices of disease progression. Our data suggest that measurement of urinary TGF beta activity could be a useful noninvasive procedure for the evaluation of renal TGF beta production, which may be useful to assess prognosis and to evaluate therapeutic efficacy in patients with renal disease.     

Bcl-2 overexpression is associated with resistance to paclitaxel, but not gemcitabine, in multiple myeloma cells

Multiple myeloma (MM) is an incurable disease despite an initial response-rate of >60% with conventional or high-dose chemotherapy using glucocorticosteroids (i.e. dexamethasone), or alkylating agents (i.e. melphalan). Although these agents are capable of inducing complete remission (CR) in >50% of MM patients, resistance develops rapidly, in >90% of patients, within 2 years of treatment. Therefore, there is a need for new drugs for the treatment of relapsing and refractory MM patients. Gemcitabine (GEM) is a pyrimidine analog that blocks DNA synthesis, whereas, paclitaxel (TAX) is a mitotic spindle poison that promotes microtubular aggregation. Since it appears that these two drugs have different cellular targets, we examined the effect of each drug individually for several parameters and for possible synergy. We studied the cytotoxic effect of TAX and GEM on MM cells expressing varying levels of the antiapoptotic protein bcl-2, which is overexpressed in the majority of myeloma cell from MM patients. We found that both drugs are cytotoxic by inducing apoptosis, however, the extent of apoptosis with TAX, but not with GEM was dependent on the levels of bcl-2 expression. We further investigated the effect of TAX and GEM on the cell cycle distribution and on the levels of bcl-2. The results indicate that the two drugs have different modes of action with respect to each parameter tested. TAX induced arrest of the cells in the G2/M phase of the cell cycle, regardless of bcl-2 levels, however, apoptosis was induced in mitotic cells expressing relatively low levels of bcl-2. In contrast, GEM caused apoptosis of cells in the S-phase, regardless of level of bcl-2 expression. A major difference between TAX and GEM was in their effects on the levels of bcl-2. Whereas, TAX induced an early downregulation of bcl-2 (only in the cells with relatively low levels of bcl-2), treatment with GEM did not affect bcl-2 levels. The effects of TAX on both the cell cycle and bcl-2 were detected very early (4-8 h) and preceded the onset of apoptosis. GEM and TAX act synergistically, at low doses (IC50 of 0.5 microM for GEM and 0.025 microM for TAX), to effectively kill bcl-2 overexpressing cells that are resistant to higher doses (0.25 microM) of TAX alone. Therefore, we have initiated a phase II clinical trial of TAX and GEM for MM patients refractory to current therapy.     

Inhibition of proximal convoluted tubule transport by dopamine

BACKGROUND: Dopamine can produce a natriuresis and diuresis independent of changes in renal hemodynamics. However, previous studies have failed to demonstrate an inhibition of transport by dopamine in intact proximal convoluted tubules. METHODS: Rabbit proximal convoluted tubules were perfused in vitro with an ultrafiltrate-like solution and bathed in a serum-like albumin solution. RESULTS: In the present study, the addition of 10-5 M dopamine to the lumen or bath of proximal convoluted tubules perfused in vitro had no effect on transport. In proximal convoluted tubules, addition of 10-6 M bath norepinephrine increased the rate of volume absorption from 0.65 +/- 0.08 to 0.93 +/- 0.08 nl/mm. min (P < 0.01). Addition of 10-5 M luminal dopamine in the presence of bath norepinephrine inhibited the rate of volume absorption to 0.72 +/- 0.10 nl/mm. min (P = 0.01). The inhibition in the rate of volume absorption by luminal dopamine in the presence of bath norepinephrine was completely blocked by the DA1 antagonist, SCH 23390. The DA1 agonist luminal 10-5 M fenoldopam also inhibited volume absorption in the presence of bath norepinephrine, but the DA2 agonist luminal 10-5 M quinpirole was without effect. Bath 10-5 M dopamine had no effect on volume absorption in the presence of bath norepinephrine. CONCLUSION: Dopamine has no direct epithelial action on the proximal convoluted tubule. However, luminal dopamine antagonizes the stimulation in transport produced by norepinephrine. These studies suggest that luminal dopamine may play a role to modulate sodium transport in the presence of renal nerve activity.     

Pregnancy-dependent growth of mammary tumors is associated with overexpression of insulin-like growth factor II

We demonstrate that although IGF-II gene expression is approximately 3-fold higher in 9,10-dimethyl-1,2-benzanthracine (DMBA)-induced rat mammary tumors (MTs) than in nonneoplastic breast tissue, IGF-II mRNA abundance in DMBA-induced MTs is approximately 130-fold higher in pregnant as compared to nonpregnant hosts. This correlated with accelerated tumor growth in pregnant hosts. Immunohistochemical studies of DMBA-induced MTs with an anti-IGF-II antibody showed an intense staining of tumor cells for IGF-II, whereas a very low staining signal was observed for normal epithelial cells in the lobules. A similar immunostaining pattern was observed in three of three human ductal cancers and adjacent normal breast tissue obtained during pregnancy. DMBA-induced MTs expressed high levels of type I receptor for IGFs as determined by Northern blots. In vitro studies confirmed that IGF-II is a mitogen for neoplastic epithelial cells derived from DMBA-induced MTs. These results demonstrate that hormonal changes associated with pregnancy accelerate breast cancer cell proliferation in the DMBA-induced MT model and suggest that this acceleration is mediated by up-regulation of IGF-II expression within neoplasms.     

Voltage and cosubstrate dependence of the Na-HCO3 cotransporter kinetics in renal proximal tubule cells

The voltage dependence of the kinetics of the sodium bicarbonate cotransporter was studied in proximal tubule cells. This electrogenic cotransporter transports one Na+, three HCO3-, and two negative charges. Cells were grown to confluence on a permeable support, mounted on a Ussing-type chamber, and permeabilized apically to small monovalent ions with amphotericin B. The steady-state, di-nitro-stilbene-di-sulfonate-sensitive current was shown to be sodium and bicarbonate dependent and therefore was taken as flux through the cotransporter. Voltage-current relations were measured as a function of Na+ and HCO3- concentrations between -160 and +160 mV under zero-trans and symmetrical conditions. The kinetics could be described by a Michaelis-Menten behavior with a Hill coefficient of 3 for HCO3- and 1 for Na+. The data were fitted to six-state ordered binding models without restrictions with respect to the rate-limiting step. All ordered models could quantitatively account for the observed current-voltage relationships and the transinhibition by high bicarbonate concentration. The models indicate that 1) the unloaded transporter carries a positive charge; 2) the binding of cytosolic bicarbonate to the transporter "senses" 12% of the electric field in the membrane, whereas its translocation across the membrane "senses" 88% of the field; 3) the binding of Na+ to the cotransporter is voltage independent.     

Moxonidine inhibits Na+/H+ exchange in proximal tubule cells and cortical collecting duct

The lateral arm free flap can be harvested as a fascial flap or fasciocutaneous flap. In this report we describe the use of the lateral arm fascial flap for degloving injuries of the fingers and for skin loss on the dorsum of the hand with exposure of tendons and bones. Concomitant reconstruction of a missing phalanx with a portion of the distal humerus is also described. The use of the fascial flap allows a large area of tissue to be harvested, and still, the donor site can be closed primarily. The fascia is thin and pliable and so conforms well to the contour of the fingers. Its bulk does not interfere with finger motion, and its undersurface creates a gliding surface for tendons. Complications in the reported cases were negligible.     

Endogenous production of angiotensin II modulates rat proximal tubule transport

There is evidence that angiotensin II is synthesized by the proximal tubule and secreted into the tubular lumen. This study examined the functional significance of endogenously produced angiotensin II on proximal tubule transport in male Sprague-Dawley rats. Addition of 10(-11), 10(-8), and 10(-6) M angiotensin II to the lumen of proximal convoluted tubules perfused in vivo had no effect on the rate of fluid reabsorption. The absence of an effect of exogenous luminal angiotensin II could be due to its endogenous production and luminal secretion. Luminal 10(-8) M Dup 753 (an angiotensin II receptor antagonist) resulted in a 35% decrease in proximal tubule fluid reabsorption when compared to control (Jv = 1.64 +/- 0.12 nl/mm.min vs. 2.55 +/- 0.32 nl/mm.min, P < 0.05). Similarly, luminal 10(-4) M enalaprilat, an angiotensin converting enzyme inhibitor, decreased fluid reabsorption by 40% (Jv = 1.53 +/- 0.23 nl/mm.min vs. 2.55 +/- 0.32 nl/mm.min, P < 0.05). When 10(-11) or 10(-8) M exogenous angiotensin II was added to enalaprilat (10(-4) M) in the luminal perfusate, fluid reabsorption returned to its baseline rate (Jv = 2.78 +/- 0.35 nl/mm.min). Thus, addition of exogenous angiotensin II stimulates proximal tubule transport when endogenous production is inhibited. These experiments show that endogenously produced angiotensin II modulates fluid transport in the proximal tubule independent of systemic angiotensin II.