Growth Of Proximal Tubule Cells Research Articles

G1 kinases and transforming growth factor-β; signaling are associated with a growth pattern switch in diabetes-induced renal growth

Diabetes mellitus-induced nephromegaly is thought to involve both hyperplastic and hypertrophic proximal tubule cell growth. The temporal relationship between these growth patterns and the mechanisms that mediate them are unknown. Renal growth was assayed in isolated renal proximal tubules harvested from diabetic rats. Diabetes mellitus was induced by streptozotocin. Following the induction of a diabetic state, there was a progressive increase in the kidney:body weight ratio. This was associated with an increase in 5-bromo-2-deoxyuridine incorporation (marker for hyperplastic cell growth) at day 2, which returned to baselines levels by day 4, and an increase in the protein:DNA ratio (marker for hypertrophic cell growth), which was clearly evident by day 10. Thus, diabetes-induced proximal tubule growth involved an initial hyperplastic, followed by a hypertrophic, growth period. During the hyperplastic growth period, both cdk4/cyclin D (cyclin D) and cdk2/cyclin E (cyclin E) kinase activities were increased. The switch between the growth periods was associated with continued activation of cyclin D, but inhibition of cyclin E kinase. The reduction in cyclin E kinase activity correlated with a reduction in cdk2/cyclin E complex abundance and an increased abundance of cyclin kinase inhibitors in cdk2/cyclin E complexes that did form. Also associated with the switch in growth patterns was a change in transforming growth factor-beta (TGF-beta) receptor expression. During the hyperplastic growth period, TGF-beta receptor II expression was decreased, while during the hypertrophic growth period, there was both a return of receptor II expression to baseline levels and increased expression of receptor I. Consistent with an increase in TGF-beta signaling during hypertrophy, there was an increase in Smad 2/3 protein expression and an increase in the abundance of Smad 2/4 complexes. Diabetes-induced proximal tubule growth involves an initial hyperplastic growth period that switches to a hypertrophic growth period within a couple of days. The pattern of G1 kinase activity associated with the growth pattern switch demonstrates that the hypertrophy is mediated by a cell cycle-dependent mechanism. Regulation of TGF-beta receptor expression and signaling activity through the Smad protein cascade possibly plays a role in the growth pattern switch.

TGF-β1 dissociates human proximal tubule cell growth and Na+-H+ exchange activity

Stimulation of proximal tubule cell (PTC) growth in a variety of physiological and pathological renal conditions is preceded by increased renal production of transforming growth factor-beta 1 (TGF-beta 1) and by augmented tubular sodium transport via activated sodium hydrogen exchange (NHE). Since TGF-beta 1 has been shown to be an important paracrine and autocrine regulator of PTC growth, the hypothesis that TGF-beta 1 modulates basal and mitogen-stimulated PTC growth via an effect on NHE activity was examined. Confluent, quiescent, human PTC were incubated for 24 hours in serum-free media containing vehicle (control) or 1 ng/ml TGF-beta 1, in the presence or absence of 100 ng/ml insulin-like growth factor-1 (IGF-I). Under basal conditions, TGF-beta 1 inhibited thymidine incorporation (73.5 +/- 7.3% of control, P < 0.05), but exerted no effect on cellular protein content (97.4 +/- 10.7% of control), an index of hypertrophy. There was no significant alteration of NHE activity, measured as ethylisopropylamiloride (EIPA)-sensitive H+ efflux (2.72 +/- 0.50 vs. control 3.26 +/- 0.68 mmol/liter/min) or 22Na+ influx (2.20 +/- 0.23 vs. control 2.19 +/- 0.19 nmol/mg protein/min). When co-incubated with IGF-I. TGF-beta 1 induced significant PTC hypertrophy (116.9 +/- 8.2% of control, P < 0.05), which was not seen with either agent alone. TGF-beta 1 counteracted the stimulatory effect of IGF-I on DNA synthesis (TGF-beta 1 + IGF-I 103.0 +/- 7.3% vs. IGF-I alone 181.2 +/- 30.3% of control, P < 0.05), but did not affect IGF-I-stimulated EIPA-sensitive 22Na+ influx (3.63 +/- 0.63 vs. IGF-I alone 3.67 +/- 0.50 nmol/mg protein/min, P = NS, both vs. control 2.19 +/- 0.19 nmol/mg protein/min, P < 0.05). Similar results were obtained when NHE activity was measured as EIPA-sensitive H+ efflux. Moreover, the kinetics of NHE activation by the combination of TGF-beta 1 and IGF-I (involving an increase in Vmax) were identical to that previously found for PTC exposed to IGF-I alone. The study demonstrates that TGF-beta 1 elicits distinct PTC growth responses in the presence and absence of IGF-I, without modification of NHE activity. The combination of predominant PTC hypertrophy and enhanced proximal tubule Na+ reabsorption found in many conditions that are associated with renal growth is likely to require the integrated actions of both TGF-beta 1 and IGF-I.

Human renal fibroblasts modulate proximal tubule cell growth and transport via the IGF-I axis

To determine the paracrine interactions involved in the tubulointerstitial response to progressive renal disease, the role of insulin-like growth factor-I (IGF-I) and its binding proteins (IGFBPs) in in vitro interactions between human proximal tubule cells (PTC) and renal cortical fibroblasts (CF) were studied in primary cell culture. PTC growth and transport were increased in the presence of CF-conditioned media (CF-CM), as shown by increased thymidine incorporation, cellular protein content and sodium-hydrogen exchange (NHE) activity, to 185 +/- 31% (P < 0.01), 150 +/- 18% (P < 0.05) and 195 +/- 27% (P < 0.01) of the control values, respectively. IGF-I was produced by cultured CF at a rate of 64.6 +/- 7.5 ng/mg protein/day. Exogenous IGF-I applied to PTC provoked similar enhancement of growth and NHE activity as CF-CM and the stimulatory effect of CF-CM was blocked by specific immunoneutralization of IGF-I receptors. These receptors were threefold more abundant on PTC basolateral versus apical membranes. IGF binding proteins (IGFBP)-2 and IGFBP-3 were secreted by CF at rates of 694 +/- 88 and 1769 +/- 45 ng/mg/day, with the release of IGFBP-3 being enhanced in the presence of PTC-CM (120.0 +/- 9.7% of control, P < 0.01). Moreover, the addition of CF-CM to PTC increased cell-associated IGFBP-3 on PTC surfaces, without changes in IGF-I receptor numbers or affinity and without changes in PTC mRNA for IGFBP-3. Des(1-3)IGF-I, an analog that binds to the IGF-I receptor but not to IGFBPs, provided a less potent stimulus for PTC growth compared with IGF-I, indicating that cell-associated IGFBP-3 facilitates the action of IGF-I on PTC. The results support important paracrine roles for both IGF-I and IGFBPs in the interstitial regulation of proximal tubule growth and transport.

Parathyroid hormone-related protein is an autocrine modulator of rabbit proximal tubule cell growth

Parathyroid hormone-related protein (PTHrP), a likely mediator for humoral hypercalcemia of malignancy, is also synthesized in various normal tissues. In the kidney, PTHrP, mainly detected in proximal and distal tubules, has been shown to stimulate proliferation of rat mesangial cells in culture. Experiments were carried out to investigate the possible mitogenic effect of PTHrP in cultures of rabbit proximal tubule cells (PTC). Immunocytochemical analysis, using antihuman (h)PTHrP antibodies to (38-64) and (107-111) epitopes in the PTHrP molecule, showed strong cytoplasmic staining in PTC and proximal tubule-like LLC-PK1 cells. PTC secreted immunoreactive PTHrP (54.8 +/- 7.0 fmol/10(6) cells) into the culture medium. Human PTHrP(1-141) stimulated proliferation in subconfluent cultures of these cells dose-dependently. This effect was similar to that induced by [Tyr34]hPTHrP(1-34) amide (hPTHrP[1-34]), hPTHrP(1-86), and bovine (b)PTH(1-34), while hPTHrP(38-64) amide, hPTHrP9107-111) amide, and hPTHrP(107-139) amide were ineffective. Addition of anti-hPTHrP neutralizing antibodies to (1-34), (38-64), and (107-111) epitopes of PTHrP decreased PTC growth. The mitogenic effect of these agonists was abolished in confluent PTC. In contrast, [Nle8,18, Tyr34]bPTH(3-34)amide (bPTH[3-34]) increased DNA synthesis in either subconfluent or confluent PTC. In LLC-PK1 cells, which also secreted PTHrP and are devoid of PTH receptors, none of these peptides affected proliferation. Forskolin (10 microM) or H-8 (2 microM), a protein kinase A inhibitor, did not affect basal or hPTHrP(1-34)-stimulated DNA synthesis, respectively, in subconfluent PTC. On the other hand, 10 nM staurosporine and 100 nM calphostin C, protein kinase C (PKC) inhibitors, blunted the effects of hPTHrP(1-34) or bPTH(3-34) on DNA synthesis in these cells. These studies suggest that PTHrP may function as an autocrine factor in the regulation of proximal tubule cell growth by a PKC-mediated mechanism.

Signaling and growth responses of LLC-PK1/Cl4 cells transfected with the rabbit AT1 ANG II receptor.

Angiotensin II (ANG II) receptors of the AT1 subtype are present on the apical and basolateral membranes of renal proximal tubule cells. Cells of the proximal tubulelike cell line, LLC-PK1/Cl4, were transfected with an expression plasmid containing cDNA encoding the rabbit AT1 ANG II receptor. In transfected cells, specific binding of 125I-ANG II was detected on both apical and basolateral membranes; wild-type LLC-PK1/Cl4 cells did not express ANG II receptors. In transfected cells, apical or basolateral ANG II increased both S6 kinase activity and incorporation of [3H]leucine. In cells pretreated with pertussis toxin, the stimulatory effect of apical or basolateral ANG II on [3H]leucine incorporation was abolished. In contrast, ANG II did not affect mitogenesis, determined by [3H]thymidine incorporation. Apical or basolateral ANG II (10(-6) M) stimulated phosphoinositide turnover by 13.4 +/- 4.4% (n = 8) and 16.3 +/- 4.2% (n = 9), respectively. The activity of protein kinase C, determined by phosphorylation of a specific protein kinase C peptide substrate, was also stimulated by ANG II in transfected cells. Apical or basolateral ANG II had no significant effect on cellular adenosine 3',5'-cyclic monophosphate levels. In permeabilized transfected cells, apical ANG II (10(-6) M) inhibited the phosphorylation of a specific peptide substrate of protein kinase A; lower apical concentrations or basolateral ANG II were without significant effect. These results indicate that AT1 ANG II receptors sort to both apical and basolateral membranes in renal epithelial cells and are coupled to activation of phospholipase C. ANG II stimulates protein synthesis by binding to either apical or basolateral receptors; this effect requires coupling to G proteins and may be mediated by activation of S6 kinase. Because high concentrations of ANG II exist in proximal tubule, binding to apical and basolateral receptors may regulate proximal tubule cell growth under physiological conditions.

Insulin and other regulatory factors modulate the growth and the phosphoenolpyruvate carboxykinase (PEPCK) activity of primary rabbit kidney proximal tubule cells in serum free medium

Insulin was observed to modulate the growth and the phosphoenolpyruvate carboxykinase (PEPCK) activity of primary cultures of rabbit renal proximal tubule cells in serum free medium. Insulin was stimulatory to primary proximal tubule cell growth at a concentration of 10(-8) M. In contrast, insulin was inhibitory to a proximal tubule function, PEPCK activity, following a 5-minute incubation period. An insulin dosage as low as 10(-10) M was inhibitory to PEPCK activity, suggesting the involvement of insulin receptors. Although insulin was required at a significantly higher dosage to stimulate the growth of the primary renal proximal tubule cells than to inhibit PEPCK activity, the elevated dosage required in order to observe a growth effect may be explained by the degradation of insulin by the primary renal proximal tubule cells. However the possible involvement of receptors for Insulin-like Growth Factor I (IGF-I) and Insulin-like Growth Factor II (IGF-II) in mediating the effects of insulin cannot be excluded. Other effector molecules were also examined with respect to their effects on PEPCK activity. The possible involvement of cyclic AMP in the control of the PEPCK activity of the primary renal cells was indicated by the stimulatory effects of 8 bromocyclic AMP, isobutyl methylxanthine (a cyclic AMP phosphodiesterase inhibitor), and forskolin (an activator of adenylate cyclase). Phorbol 12-myristate 13-acetate (TPA), which activates protein kinase C, was inhibitory. The actions of these effector molecules and insulin on the PEPCK activity of the primary renal cultures are remarkably similar to their effects on hepatic PEPCK. Several growth factors, fibroblast growth factor (FGF), and transforming growth factor beta (TGF beta) were also examined. FGF was observed to be stimulatory, whereas TGF beta was inhibitory to the PEPCK activity of the primary renal proximal tubule cells.

High glucose induces cell hypertrophy and stimulates collagen gene transcription in proximal tubule

Tubulointerstitial changes in the diabetic kidney correlate closely with the decline in glomerular filtration. In this study, we used a cell culture system of mouse proximal tubule epithelial cells to test the effects of glucose on cell growth, size, and matrix biosynthesis. [3H]thymidine incorporation was significantly inhibited in cells grown in 450 mg/dl glucose, compared with cells grown in 100 mg/dl glucose. The cells grown in the higher glucose concentration were slightly larger, their protein content and the total protein synthetic rate were significantly increased, and they secreted approximately twice as much procollagens type IV and type I. Concordantly, steady-state procollagen mRNA levels were also increased: 2.6-fold for the alpha 1(IV) and 2.2-fold for the alpha 2(I) procollagens. Additionally, nuclear run-off studies demonstrated that procollagen gene transcription rate was stimulated approximately 50%; beta-actin transcription rate was not altered. We used chloramphenicol acetyltransferase (CAT) reporter gene constructs to determine whether the increased transcription rate of alpha 2(I) gene was associated with activation of its enhancer sequence. Cells transfected with the enhancer demonstrated more than fivefold increase in CAT activity when cultured in the high-glucose medium. These studies demonstrate a multitude of effects of high ambient glucose concentrations on proximal tubule cell growth and collagen biosynthesis; cell proliferation is decreased although cell hypertrophy occurs. Procollagen gene transcription rate is stimulated and this response contributes to the observed increase in procollagen mRNA content. Activation of an enhancer sequence may be one possible mode through which high glucose levels increase the transcription of procollagen type I, presumably involving trans-acting factor(s).

Restricted growth of rat kidney proximal tubule cells cultured in serum-supplemented and defined media.

Proximal tubules suitable for in vitro culture were prepared from rat kidney cortex by a Ficoll-gradient centrifugation technique which yielded greater than 94% purity. The tubules were seeded into culture dishes, and cell growth was monitored in both Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum and in a defined medium consisting of 50:50 Ham's F12 and Dulbecco's supplemented with insulin, transferrin, and hydrocortisone. Growth in serum-containing medium was continuous; however, the specific activity of the brush border enzyme alkaline phosphatase decreased rapidly with time, and the culture morphology became fibroblastic by 6 days. Neither collagen-coating of the dishes nor addition of the differentiation inducer hexamethylene-bisacetamide had any significant effect on growth or enzyme activity of the cultured cells. Theophylline, another inducer of differentiation, proved cytotoxic. Growth of proximal tubule cells in defined medium proceeded for 4 days before irreversible growth arrest occurred. Alkaline phosphatase activity and epithelial morphology remained relatively constant throughout the culture period. Additions of the growth factors triiodothyronine, prostaglandin E2, and epidermal growth factor were unable to unblock the growth arrest. If cells cultured in defined medium for 3 days were switched to serum-supplemented medium, continuous growth occurred, but both alkaline phosphatase activity and epithelial morphology were rapidly lost. As a test of the culture method, rabbit proximal tubule cells were cultured under similar conditions in defined medium. Growth was prolific and continuous for up to, but not exceeding, 30 days, and differentiated properties were retained. It was concluded that both rat and rabbit proximal tubule cells have a limited proliferative capacity in vitro but that the capacity of the rat cell to divide is much reduced relative to the rabbit cell.