Hepatocyte growth factor is a potent mitogen for cultured rabbit renal tubular epithelial cells.

Hepatocyte growth factor (HGF), which is a potent growth factor of adult rat hepatocytes in primary culture, also strongly stimulated DNA synthesis of rabbit renal tubular epithelial cells in secondary culture. Its mitogenic activity was dose-dependent, being detectable at 3 ng/ml and maximal at 30 ng/ml. Over 20% of the cells were shifted to the S-phase by HGF alone, judging by the labeling index. HGF had additive effects with EGF, acidic fibroblast growth factor (a-FGF), and insulin. Transforming growth factor-beta 1 (TGF-beta 1) strongly inhibited DNA synthesis of renal tubular cells stimulated by HGF. The growth of renal tubular epithelial cells was also regulated by cell density: DNA synthesis stimulated by HGF was high at lower cell density and was strongly suppressed at high cell density. These results suggest that HGF may act as a renotropic factor in compensatory renal growth or renal regeneration in vivo.     

Renotropic stimulation in rat kidney cell culture

A circulating renotropic factor specific for renal cells has been described in rats. The addition of sera obtained from unilaterally nephrectomized (uni) rats 24h after operation compared to sham-operated (sham) rats augments 3H-thymidine incorporation into the DNA of incubating kidney slices approximately 10%-30%. Attempting to amplify the sensitivity of the assay for this renotropic agent, we replaced slices with primary rat kidney cultures. The assay system was based on one previously used for rabbits. The cultured cells were synchronized in their growth phase by a period of protein-free starvation. Compared to sera from sham rats, sera from uni rats showed significant stimulation of thymidine incorporation into DNA, 35.5% +/- 9.3 (SEM), p less than .0001, at 16 h; 63.3% +/- 10.0 (SEM), p less than .001, at 24 h; and 19.5% +/- 6.5 (SEM), p less than .01, at 48 h post operation. Accordingly, the maximal stimulation at 24 h was greater than that previously found using the kidney slice assay. Measurable renotropic activity occurred earlier and over a shorter duration than in rabbits. Stimulation was similar when a D-valine medium, relatively specific for renal epithelial cells, replaced DME medium. We conclude that growth synchronized, primary rat renal cells in culture verify the presence of a circulating renotropin arising 24 h post uni.     

Biological properties of a renotropic protein present in plasma of uninephrectomized rats.

The biological characteristics of a kidney growth factor (KGF) from uninephrectomized rat plasma have been studied. A crude preparation of this factor [Nephrol. Dial. Transplant. 4: 334-338, 1989] was further purified by hydrophobic interaction HPLC and gel filtration. KGF was found to be a heat- and trypsin-resistant protein. This factor stimulated dose-dependently DNA synthesis by the mouse kidney in vivo, and by either rat renal tubules or serum-deprived LLC-PK1 cells, in vitro. KGF also increased protein synthesis in these cells, in a dose-dependent manner. Moreover, KGF stimulated sodium uptake by these cells, associated with the maximal increase of protein synthesis. Our findings indicate that KGF is a potent renotropic protein which can play a key role in the renal compensatory growth after uninephrectomy.     

Tumor necrosis factor causes amplification of arachidonic acid metabolism in response to interleukin 1, bradykinin, and other agonists

Tumor necrosis factor stimulated prostaglandin E2 synthesis in Swiss 3T3 fibroblasts. Interleukin 1 also stimulated prostaglandin synthesis. Simultaneous addition of tumor necrosis factor and interleukin 1 synergistically stimulated prostaglandin synthesis, even when both growth factors were added at what would be supramaximal concentrations by themselves. Several small peptides and nonpeptides rapidly stimulate prostaglandin synthesis in these cells. Pretreatment with tumor necrosis factor synergistically enhanced prostaglandin synthesis in response to bradykinin, bombesin, thrombin, norepinephrine, and platelet-activating factor. Thus, tumor necrosis factor stimulates prostaglandin synthesis and greatly amplifies prostaglandin synthesis in response to other agonists. This finding may have significance in chronic inflammatory diseases such as rheumatoid arthritis in which several hormones and growth factors may synergistically augment eicosanoid synthesis.     

Inhibition of compensatory renal growth by indomethacin

Renal prostaglandins may be important in the modulation of compensatory renal growth. Reductions in renal mass are associated with increased synthesis of these substances by the remaining kidney, and inhibition of prostaglandin synthesis diminishes renal function in partially nephrectomized animals and in patients with reduced functioning renal mass. We examined the effects of uninephrectomy and treatment with indomethacin on renal prostaglandin E2 and 6-keto prostaglandin F1 alpha concentrations in adult male Sprague Dawley rats. The renal content of these prostaglandins was significantly increased in the remaining kidney two days following uninephrectomy (p less than 0.01). Treatment with 5 mg/kg/day of indomethacin over this period abolished the compensatory increase in renal prostaglandin synthesis and significantly attenuated compensatory increases in renal mass, protein and RNA concentrations (p less than 0.05). No alterations in kidney weight, protein or RNA concentrations were found in intact animals treated with the same dose of indomethacin. These findings suggest renal prostaglandins may participate in the biological events leading to compensatory renal growth.     

Biochemical and Molecular Responses to Loss of Renal Mass: Membranes and Protein Synthesis: Further Studies on a Renotropic System in Rats

Based upon many investigations, the existence of short-lived, specific, circulating substances which incite and/or regulate compensatory renal growth has been proposed. In our studies, we find that sera and plasma from unilaterally nephrectomized rats compared to sera and plasma from sham-operated rats stimulate the incorporation of ³H-thymidine monophosphate, ³H-thymidine and ¹⁴C-uridine into the DNA of incubating rat kidney fragments. While extracts from growing rat kidneys are not excitatory, they produce a relative enhancement to incorporation of isotope into DNA when combined with sera from uninephrectomized rats—more than the sera do alone. The above is found also for the incorporation of ¹⁴C-uridine into RNA of incubating rat kidney fragments. Sera from uninephrectomized rats fail to stimulate DNA synthesis in liver slices from rats but do so in the presence of extracts from growing kidneys. Renotropic factors in sera and extracts do not appear to work by diluting the isotopes, by enhancing transport, or by effecting overall metabolism of the renal cells. The above described serum and liver factors may play a role in compensatory renal growth.     

Anti-epidermal growth factor antibody inhibits compensatory renal hyperplasia but not hypertrophy after unilateral nephrectomy in mice.

The effect of anti-epidermal growth factor (EGF) antibody on the compensatory renal growth including hyperplasia and hypertrophy was investigated. The antibody used in this study blocked the stimulatory effect of EGF on the DNA synthesis of cultured rat hepatocytes. When this antibody was injected into mice intravenously after unilateral nephrectomy, the labeling index of the renal cortical tubular cells decreased significantly at the second day after uninephrectomy, but the antibody caused no decrease in remaining kidney weight. Immunohistochemical study revealed that injected anti-mouse EGF rabbit IgG was positively stained at the renal cortical tubular cells. EGF would thus appear importantly essential to compensatory renal hyperplasia.     

Renotropic functions of hepatocyte growth factor in renal regeneration after unilateral nephrectomy.

Hepatocyte growth factor (HGF), a most potent growth factor for mature hepatocytes may act as a trigger for liver regeneration. We reported that HGF strongly stimulates DNA synthesis of rabbit renal tubular cells in secondary culture (Igawa, T., Kanda, S., Kanetake, H., Saitoh, Y., Ichihara, A., Tomita, Y., and Nakamura, T. (1991) Biochem. Biophys. Res. Commun. 174, 831-838). To investigate whether or not HGF is involved in renal regeneration, we examined changes in HGF mRNA, HGF activity, and HGF receptor in the rat kidney following unilateral nephrectomy or treatment with carbon tetrachloride (CCl4). In the intact kidney, the HGF mRNA increased markedly reaching a maximum 6 h after unilateral nephrectomy, followed by an increase of HGF activity at 12 h after the surgery. The marked increase in HGF mRNA and HGF activity was also found in the kidney of rats treated with CCl4. Results of in situ hybridization suggested that cells producing HGF in the kidney are endothelial cells. The number of HGF receptors on renal plasma membranes decreased to 30% of the normal value 12 h after unilateral nephrectomy, with no change in the Kd value. The HGF receptor was greatly diminished 24 h after the operation, and recovery to 60% of the normal level was evident 1 week after the operation. Because the decrease in HGF binding may result from internalization of the HGF receptor, the HGF may bind to its receptor in vivo and act as a mitogen for renal epithelial cells. HGF may function as a renotropic factor during renal regeneration after kidney injury.     

The role of adenosine 3′:5′-cyclic monophosphate in the division of WI 38 cells. The cellular response to prostaglandin E1 and the effects of an cyclic adenosine 3′:5′-cyclic monophosphate analogue and prostaglandin E1 on cell division

Inhibition of growth and DNA synthesis was observed in WI 38 cells incubated with 8-methylthioadenosine 3':5'-cyclic monophosphate or prostaglandin E(1). The effect of both compounds on cell growth was reversible. On removal of these compounds from culture media the cells initiated DNA synthesis and divided. In addition, prostaglandin E(1) stimulated cyclic AMP formation in these cells to over 40 times the normal basal value. The increase in cyclic AMP concentration in WI 38 cells after addition of prostaglandin E(1) showed a marked variation. Cells that had recently been treated with trypsin and plated at a lower cell density exhibited a smaller response to addition of prostaglandin E(1) than cells that had divided and reached confluence.     

Inhibition of compensatory renal growth by indomethacin

Renal prostaglandins may be important in the modulation of compensatory renal growth. Reductions in renal mass are associated with increased synthesis of these substances by the remaining kidney, and inhibition of prostaglandin synthesis diminishes renal function in partially nephrectomized animals and in patients with reduced functioning renal mass. We examined the effects of uninephrectomy and treatment with indomethacin on renal prostaglandin E₂ and 6-keto prostaglandin F_1α concentrations in adult male Sprague Dawley rats. The renal content of these prostaglandins was significantly increased in the remaining kidney two days following uninephrectomy (p<0.01). Treatment with 5 mg/kg/day of indomethacin over this period abolished the compensatory increase in renal prostaglandin synthesis and significantly attenuated compensatory increases in renal mass, protein and RNA concentration (p<0.05). No alterations in kidney weight, protein or RNA concentrations were found in intact animals treated with the same dose of indomethacin. These findings suggest renal prostaglandins may participate in the biological events leading to compensatory renal growth.     

Requirement for prostaglandin F2 alpha in 17 beta-estradiol stimulation of DNA synthesis in rabbit endometrial cultures

We have hypothesized that two of the endogenously synthesized endometrial prostaglandins (PGs), prostaglandin F2 alpha (PGF2 alpha), and prostaglandin E1 (PGE1), play a regulatory role in growth control of the rabbit endometrium. PGF2 alpha increases DNA synthesis and PGE1 inhibits that effect. Primary cultures of rabbit endometrial cells were used to examine the possible role of these PGs in the mechanism of action of 17 beta-estradiol on DNA synthesis. Towards this end, binding, second messenger and DNA synthesis experiments were performed. 17 beta-estradiol stimulation resulted in a time dependent (optimal: approximately 6 h) and 17 beta-estradiol concentration dependent (optimal: approximately 10(-7) M 17 beta-estradiol in phenol red-containing medium) increase in [3H]PGF2 alpha binding. Scatchard type analysis of the binding data revealed an increase in receptor number while the receptor affinity for [3H]PGF2 alpha remained the same as in the control treated cultures. This 17 beta-estradiol stimulated increase in PGF2 alpha receptor allowed a suboptimal concentration of PGF2 alpha (10(-9) M) to increase intracellular levels of inositol polyphosphates, while by itself this concentration of PGF2 alpha caused no significant change in intracellular inositol polyphosphate levels. 17 beta-estradiol, alone among the several studied steroid hormones, could increase [3H]PGF2 alpha binding. Proliferation studies revealed that, in these primary cultures of rabbit endometrium, 17 beta-estradiol could increase DNA synthesis but not in the presence of indomethacin, unless PGF2 alpha was added to the medium at a concentration (10(-10) M) near or above what is normally accumulated in the medium by these cultures. In the absence of 17 beta-estradiol stimulation, addition of these same low concentrations of PGF2 alpha had no effect on DNA synthesis. Apparently, through its effect on the PGF2 alpha receptor, 17 beta-estradiol enhances the PGF2 alpha stimulated DNA synthesis response approximately 100 fold. The DNA synthesis induced by 17 beta-estradiol can be inhibited by PGE1, as can PGF2 alpha-induced DNA synthesis. We propose that 17 beta-estradiol may be mediating its mitogenic effect through an alteration of the prostaglandin agonist:antagonist control of proliferation in rabbit endometrial cultures. In addition we suggest that, if 17 beta-estradiol acts to increase PGF2 alpha, receptors as part of its mode of action, this may be of importance in other tissues possessing both prostaglandin and 17 beta-estradiol receptors.     

Effects of the rat renotropic system on 14C-uridine incorporation into RNA and RNA precursors.

We used the incorporation of ¹⁴C-uridine into RNA of incubating kidney fragments from normal control rats to evaluate RNA metabolism. Sera from unilaterally nephrectomized rats (uni) obtained 20 hrs post-operatively stimulate ¹⁴C-uridine incorporation into RNA significantly more than sera from sham-operated rats (sham). Differently, sera from uni and sham rats have little influence on specific activities of endogenous uridine nucleotide pool in renal fragments. Renal extracts were obtained by homogenizing kidneys in saline. Extracts from kidneys of uni and sham rats 20 hrs post operation depress incorporation markedly, and each depresses to a similar extent, but kidney extracts dilute the specific activities of uridine pools. Correcting for the latter dilution demonstrates that kidney extracts alone have little effect on ¹⁴C-uridine incorporation into RNA. We then followed the results when these sera and extracts were combined. Compared to fragments incubating in sham sera and sham extracts, substitution of uni extracts or both uni extracts and uni sera enhances ¹⁴C-uridine into renal RNA, whether or not results are corrected for changes in the specific activities of the uridine pools. We conclude that after uninephrectomy there is a concurrent elevation in circulating renotropin and a tissue activating factor in the remaining kidney. The tissue factor can only form an excitor to ¹⁴C-uridine incorporation into RNA when serum is present. The rat renotropic system that enhances incorporation of ³H-thymidine into DNA also can stimulate ¹⁴C-uridine incorporation into renal RNA.     

Triacylglycerol lipase mediated release of arachidonic acid for prostaglandin synthesis in rabbit kidney medulla microsomes

The effect of triarachidonin on the synthesis of prostaglandins in rabbit kidney medulla microsomes was examined. Medulla microsomes were incubated with triarachidonin in 0.1 M--Tris/HCl buffer (pH 7.0) containing reduced glutathione and hydroquinone and the formed prostaglandin E2, prostaglandin F2 alpha and prostaglandin D2 were measured by high-pressure liquid chromatography using 9-anthryldiazomethane for derivatization. The addition of triarachidonin (1-10 microM) stimulated prostaglandin formation in a dose-dependent manner. Under our incubation conditions rabbit kidney medulla was found to produce prostaglandin E2 mainly. When arachidonic acid, instead of triarachidonin, was added to the incubation mixture of microsomes, the identical profile of prostaglandin products was obtained. When the pH of the reaction mixture was changed from 7.0 to 8.0, the rate of triarachidonin-induced prostaglandin E2 formation was approximately 60% of that observed at pH 7.0. Studies utilizing Ca2+ and EGTA revealed that triacylglycerol lipase of kidney medulla is independent of Ca2+. The addition of epinephrine made the stimulatory effect of triarachidonin on prostaglandin E2 formation more pronounced. These results suggest that epinephrine-activated triacylglycerol lipase is present in the renomedullary microsomes, and this enzyme activity is a potential mediator of release of arachidonic acid for prostaglandin synthesis in the kidney medulla.     

Compensatory renal growth in human fetuses with unilateral renal agenesis

To determine whether compensatory growth of the kidney occurs during fetal life we studied 20 human specimens with a unilateral kidney as an isolated defect. The mean combined kidney weight to body weight ratio x 100 in controls was 0.76 +/- 0.14 (SD) and in the solitary kidney cases (after doubling the kidney weight) was 1.26 +/- 0.35 (SD). This significant increase leads us to hypothesize that the increased weight may be due to an induced negative feedback system involving a renotropic factor. From histologic studies a uniform increase in all nephron elements was found. Why should a fetus with adequate placental clearance of metabolic wastes need increased renal size?     

Evidence for cAMP-independent inhibition of S-phase DNA synthesis by prostaglandins

Two prostaglandins, prostaglandin E1 (PGE1) and prostaglandin B1 (PGB1), block S-phase DNA synthesis in synchronous cultured baby hamster kidney (BHK) cells. The prostaglandin inhibition of DNA synthesis does not appear to require elevated levels of cAMP. In BHK-21 cells that have been "desensitized" to prostaglandin stimulation of adenylate cyclase and, therefore, have control levels of cAMP, PGE1 retains its inhibitory effect on the incorporation of tritiated thymidine into DNA. When BHK cells are exposed to PGB1 (a prostaglandin that does not elicit a cAMP response), DNA synthesis is also blocked. In nonsynchronous cells exposed for 1 h to PGE and then incubated for 1 h with PGE removed, a rebound of DNA synthesis occurs, therefore providing evidence that a transient rise of cAMP in itself is not capable of causing a cascade of reactions that block the synthesis of DNA. In addition, the concentration of PGE required for inhibition of DNA synthesis is significantly less than that required for cAMP generation. Addition of 1 x 10(-8) M PGE to BHK cells can be shown to significantly inhibit DNA synthesis within 30 min, with half-maximal inhibition seen at 3 x 10(-7) M PGE. Cyclic AMP levels for controls were 4.9 +/- 0.2 and 4.6 +/- 0.1 for 1 x 10(-6) M PGE1. These findings suggest that the prostaglandins can act independently of cAMP at physiological concentrations; and, therefore, it is possible that prostaglandins have a physiological role in the control of cell growth during S-phase.     

Mechanism of prostaglandin biosynthesis in rabbit kidney medulla. A rate-limiting step and the differential stimulatory actions of L-adrenaline and glutathione.

Microsomal prostaglandin synthase (EC 1.14.99.1) from rabbit kidney medulla was assayed with [5,6,8,9,11,12,14,15-3H]-and [1-14C]-arachidonic acid as the substrate. The ratios of prostaglandin F2 alpha to prostaglandin E2 and to prostaglandin D2 were determined by both 3H and 14C labelling. When 3H was used as a label the ratios were much higher than with 14C labelling indicating that the removal of hydrogen at C-9 or C-11 was the rate-limiting step in the biosynthesis of prostaglandin E2 or prostaglandin D2. This finding shows that the octatritiated arachidonic acid is not the appropriate substrate marker for studying the regulation of the synthesis of different prostaglandins by various agents. When the enzyme assay was carried out in the presence of SnCL2, which was capable of accumulating exclusively prostaglandin F2alpha at the expenses of prostaglandin E2 and prostaglandin D2, the addition of L-adrenaline to the microsomal fraction either alone or with reduced glutathione equally stimulated the formation of prostaglandin F2alpha, whereas the addition of reduced glutathione to the microsomal fraction either alone or with L-adrenaline produced no additional effect. These results suggest that endoperoxide is formed as the common intermediate for the biosynthesis of three different prostaglandins in rabbit kidney medulla, and that L-adrenaline stimulates the synthesis of endoperoxide, whereas reduced glutathione facilitates the formation of prostaglandins from endoperoxide.     

Transforming growth factor-beta-like activity is secreted by rabbit renal cortical tubular cells in primary culture.

The activity of an autocrine growth factor in a medium conditioned by cultured rabbit renal cortical tubular cells was investigated. Little stimulatory growth activity for tubular cells was observed in the conditioned medium, and inhibitory activity was seen only in acidified conditioned medium. This factor stimulated the colony formation of NRK 49F cells in soft agar only with epidermal growth factor and inhibited the DNA synthesis of primary cultured rat hepatocytes, and its molecular weight was about 25 kDa. The factor was neutralized by the specific antibody to transforming growth factor (TGF)-beta 1. These results indicate that renal tubular epithelial cells can produce latent TGF-beta in primary culture.     

Effect of phorbol ester on prostaglandin regulation of proliferation in rabbit endometrial cells

We have proposed that two of the endogenously synthesized endometrial prostaglandins, prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E1 (PGE1), play a regulatory role in growth control of the endometrium. PGF2 alpha increases DNA synthesis and PGE1 inhibits that effect. Primary cultures of rabbit endometrial cells were used here to examine the effects of the tumor-promoting, diacylglycerol mimicking, phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), on the prostaglandin control of cell proliferation. TPA treatment of these cultures results in: a decrease in control levels of proliferation and complete inhibition by TPA of PGF2 alpha stimulated DNA synthesis; a reduction in [3H]PGF2 alpha binding with short term treatment but an increase to above control binding level with long term treatment; an inhibition of the normal PGF2 alpha stimulated inositol polyphosphate synthesis; and a small increase in accumulation of PGF2 alpha in the culture media. Furthermore, in this culture system, TPA does not down regulate [3H]PGE1 binding; it does not alter the normal PGE1 stimulation of cAMP synthesis; and it has no effect on the normal endogenous PGE1 synthesis by these cultures. The above results are consistent with our previous observations that PGF2 alpha works through the intracellular messengers inositol polyphosphate/diacylglycerol whereas PGE1 works through cAMP.     

Mastoparan, a novel mitogen for Swiss 3T3 cells, stimulates pertussis toxin-sensitive arachidonic acid release without inositol phosphate accumulation

Mastoparan, a basic tetradecapeptide isolated from wasp venom, is a novel mitogen for Swiss 3T3 cells. This peptide induced DNA synthesis in synergy with insulin in a concentration-dependent manner; half-maximum and maximum responses were achieved at 14 and 17 microM, respectively. Mastoparan also stimulated DNA synthesis in the presence of other growth promoting factors including bombesin, insulin-like growth factor-1, and platelet-derived growth factor. The synergistic mitogenic stimulation by mastoparan can be dissociated from activation of phospholipase C. Mastoparan did not stimulate phosphoinositide breakdown, Ca2+ mobilization or protein kinase C-mediated phosphorylation of a major cellular substrate or transmodulation of the epidermal growth factor receptor. In contrast, mastoparan stimulated arachidonic acid release, prostaglandin E2 production, and enhanced cAMP accumulation in the presence of forskolin. These responses were inhibited by prior treatment with pertussis toxin. Hence, mastoparan stimulates arachidonic acid release via a pertussis toxin-sensitive G protein in Swiss 3T3 cells. Arachidonic acid, like mastoparan, stimulated DNA synthesis in the presence of insulin. The ability of mastoparan to stimulate mitogenesis was reduced by pertussis toxin treatment. These results demonstrate, for the first time, that mastoparan stimulates reinitiation of DNA synthesis in Swiss 3T3 cells and indicate that this peptide may be a useful probe to elucidate signal transduction mechanisms in mitogenesis.     

Hepatocyte growth factor inhibits intrinsic antibacterial activity of Madin-Darby canine kidney cells

We investigated whether or not polarized renal epithelial cells produce antibacterial factors, which aid in host defense at the cell surface of renal epithelium. A model of polarized Madin-Darby canine kidney (MDCK) epithelial cells grown on filters was used to test for the presence of apically or basolaterally secreted factors on the growth of non-virulent (XL1-Blue) and uropathogenic (J96) strains of Escherichia coli (E. coli). Growth of both XL1-Blue and J96 strains of E. coli in medium on the apical and basolateral surface of MDCK cells was inhibited as compared to bacterial growth in medium not exposed to MDCK cells. The inhibition of bacterial growth was similar in both apical and basolateral surface medium. Pretreatment of MDCK cells with hepatocyte growth factor (HGF) blunted the inhibition of XL1-Blue and J96 growth in apical and basolateral surface medium as compared to growth in medium on the surfaces of untreated MDCK cells. Immunofluorescent analysis demonstrated the presence of beta-defensin isoforms 1-3 in MDCK cells, with isoform 1 being the most prevalent form observed. HGF treatment reduced the amount of immunoreactive beta-defensin-1 in MDCK cells. These data demonstrate that polarized renal epithelium produce antibacterial factors. The renotropic growth factor HGF inhibits these antibacterial factors. beta-defensins may contribute to this antibacterial activity and play an important role in renal epithelial resistance to bacterial infections.