Hepatocyte growth factor in glycerol-induced acute renal failure

Hepatocyte growth factor (HGF) facilitates the regeneration of injured kidney in acute renal failure (ARF). Here we investigated the HGF production in glycerol-induced ARF rats. HGF mRNA expression levels were elevated in liver, spleen, and lung 6-24 h after glycerol injection. Tissue HGF protein levels determined by an enzyme-linked immunosorbent assay also increased in liver and spleen, whereas they decreased in the injured kidney 24 h after injection. Immunohistochemical studies showed that the number of HGF-producing cells did not increase in the liver. HGF receptor/c-Met mRNA levels were elevated only in the kidney. These results indicate that HGF supplied in an endocrine manner may play an important role in the regenerating process following ARF.     

Fasting suppresses pulsatile luteinizing hormone (LH) secretion and enhances orderliness of LH release in young but not older men

BACKGROUND: Studies identifying genes that are differentially expressed following induction of acute ischemic injury have been useful in delineating the pathophysiology of acute renal failure. METHODS: A differential cDNA library screening technique was used to identify genes that are differentially expressed in rat kidney following induction of acute ischemic renal injury. RESULTS: Levels of mRNA with a high homology to that coding for Siva, a human proapoptotic protein, were increased approximately 4.5-fold in kidneys obtained from rats within 12 hours following ischemia, compared to kidneys from sham-operated rats. A partial cDNA sequence for the rat protein (rat Siva) was determined that overlaps 92% of the human open reading frame. The cDNA sequence predicts a protein 177 amino acids in length with 76% homology to human Siva. Levels of rat Siva in kidneys were elevated at one, five and seven days post-ischemia were not different from those in kidneys from sham-operated controls. In situ hybridization demonstrated that rat Siva mRNA was expressed in cells lining damaged sections in the S3 segment of the proximal tubule at 12 hours and one day post-ischemia. At five and seven days, Siva mRNA was located in epithelial cells of regenerating tubules including in papillary proliferations. TdT-mediated dUTP-biotin nick end-labeling (TUNEL)-positive cells colocalized with cells containing Siva mRNA. CD27, the receptor for Siva was localized by immunohistochemistry to sloughed cells in the lumens of damaged S3 segments at 12 hours post-ischemia and to cells within papillary proliferations at five days post-injury. CONCLUSIONS: Siva that is produced within the kidney could be a mediator of apoptosis post-ischemia via an interaction with CD27.     

Hepatocyte growth factor-stimulated renal tubular mitogenesis: effects on expression of c-myc, c-fos, c-met, VEGF and the VHL tumour-suppressor and related genes

Hepatocyte growth factor (HGF/SF) is a potent renal proximal tubular cell (PTEC) mitogen involved in renal development. HGF/SF is the functional ligand for the c-met proto-oncogene, and germline c-met mutations are associated with familial papillary renal cell carcinoma. Somatic von Hippel-Lindau disease tumour-suppressor gene (VHL) mutations are frequently detected in sporadic clear cell renal cell carcinomas (RCC), and germline VHL mutations are the commonest cause of familial clear cell RCC. pVHL binds to the positive regulatory components of the trimeric elongin (SIII) complex (elongins B and C) and has been observed to deregulate expression of the vascular endothelial growth factor (VEGF) gene. HGF/SF has similarly been reported to up-regulate expression of the VEGF gene in non-renal experimental systems. To investigate the mechanism of HGF/SF action in PTECs and, specifically, to examine potential interactions between the HGF/c-met and the VHL-mediated pathways for renal tubular growth control, we have isolated untransformed PTECs from normal kidneys, developed conditions for their culture in vitro and used these cells to investigate changes in mRNA levels of the VHL, elongin A, B and C, VEGF, c-myc, c-fos and c-met genes after HGF/SF exposure. Significant elevations in the mRNA levels of VEGF, c-myc, c-fos, c-met and elongins A, B and C, but not VHL, were detected after HGF/SF stimulation of human PTECs (P < 0.02), with a consistent order of peak levels observed over successive replicates (c-fos at 1 h, VEGF at 2-4 h, c-myc, at 4 h, followed by c-met and all three elongin subunits at 8 h). This study highlights the spectrum of changes in gene expression observed in PTECs after HGF/SF stimulation and has identified possible candidate mediators of the HGF/SF-induced mitogenic response. Our evidence would suggest that the changes in PTEC VEGF expression induced by HGF/SF are mediated by a VHL-independent pathway.     

Potential role of hepatocyte growth factor in the maintenance of renal structure: anti-apoptotic action of HGF on epithelial cells

BACKGROUND: Mesangial cells (MC) are known to secrete various vasoactive substances that may control endothelial and epithelial cell growth. Therefore, the cell-cell interactions among these cells may be important in the control of renal function. However, the exact mechanisms of maintaining the cell-cell interactions are not yet understood. We have focused on the role of hepatocyte growth factor (HGF) in the regulation of cell-cell interactions, since HGF has many protective functions in the kidney. To investigate the role of HGF in renal injury, we examined (1) the effects of HGF on epithelial injury induced by serum deprivation, and (2) the role of local HGF production in the maintenance of renal structure. METHODS: Apoptotic changes in epithelial cells were assessed by nuclear morphology and DNA fragmentation assay. Transfection of human HGF vector into epithelial cells was performed by a highly efficient viral-liposome method. The effects of secreted HGF on the growth of renal cells were examined using a co-culture system. RESULTS: The addition of recombinant HGF (rHGF) stimulated the growth of rat and porcine epithelial cells. Moreover, the decrease in number of epithelial cells by serum deprivation was significantly attenuated by rHGF. Interestingly, apoptotic changes in epithelial cells induced by serum deprivation were also significantly attenuated by rHGF (P < 0.01). As a model of gene therapy, the effects of overexpression of human HGF gene in epithelial cells on apoptosis induced by serum deprivation were examined. Transfection of human HGF vector into epithelial cells also attenuated epithelial cell death induced by serum deprivation through the inhibition of apoptosis, accompanied by increased HGF production (P < 0.01). In addition, HGF also prevented endothelial injury induced by tumor necrosis factor-alpha and dexamethasone. Given the presence of a local HGF system, we measured local HGF secreted from renal cells. Immunoreactive HGF was observed in the conditioned medium of MC, but not epithelial cells, while the specific receptor of HGF, c-met, was expressed in epithelial cells. Of importance, co-culture of MC with epithelial cells resulted in a significant increase in number of epithelial cells, which was significantly abolished by neutralizing anti-HGF antibody. CONCLUSIONS: Overall, these results demonstrate that local production of HGF in MC may maintain the growth of epithelial and endothelial cells through its anti-apoptotic action.     

Development of a limited sampling approach in pharmacokinetic studies: experience with the antiepilepsy drug tiagabine

To examine the mechanisms involved in the progression of mercury chloride (HgCl2)-induced acute tubular necrosis (ATN), we investigated the histopathological changes and the expression of inducible nitric oxide synthase (iNOS) mRNA and protein in renal cortices of rats at 20 hours after exposure to HgCl2. The expression of iNOS mRNA was significantly augmented in renal cortices of rats with HgCl2-induced acute renal failure (ARF). Likewise, the induction of iNOS protein was observed in damaged proximal tubule epithelial cells of rats with HgCl2-induced ARF. Pretreatment of rats with iNOS inhibitor aminoguanidine, however, suppressed the development of proximal tubule epithelial cell injury and prevented an increase in blood urea nitrogen and serum creatinine as well as resulting in a marked fall in iNOS mRNA and protein in rats with HgCl2-induced ARF. These observations indicate that the induction of iNOS may play a role in the progression of HgCl2-induced ATN through the exacerbation of proximal tubule epithelial cell damage.     

Hepatocyte growth factor may act as a pulmotrophic factor on lung regeneration after acute lung injury

Hepatocyte growth factor (HGF) has been shown to have hepatotrophic and renotropic functions for regeneration of the liver and kidney through its mitogenic, motogenic, and morphogenic properties. To examine the involvement of HGF in lung regeneration after acute injury, we analyzed changes of HGF mRNA, HGF activity, and HGF receptors in the rat lung after lung insult and measured HGF concentration in sera of patients with various lung diseases. Following the onset of acute lung injury induced by intratracheal hydrochloride injection, a compensatory DNA synthesis occurred in the bronchial epithelium with a peak at 24 h and in the alveolar epithelium with a peak at 48 h. Expression of HGF mRNA in the rat lung remarkably increased only 3 h after the treatment and HGF activity in the lung also increased to about 3-fold at 6 h later. HGF receptors in the lung but not in the other noninjured organs were down-regulated 12 h later. These marked increases in HGF mRNA and HGF activity and the concomitant down-regulation of HGF receptor occurred before the marked compensatory DNA synthesis in bronchial and alveolar epithelial cells. HGF concentration in sera of patients with various lung diseases, as measured by radioimmunoassay, was much higher than that in healthy donors. These results suggest that HGF is newly produced in the lung after acute lung injury and may have a role in regeneration of the lung.     

Induction of hepatocyte growth factor in the liver, kidney and lung following total body irradiation in rat

Hepatocyte growth factor (HGF) has been shown to have a pleiotropic function to act as a potent organotropic factor in the regeneration of injury in various organs, including the liver, kidney and lung. To examine the involvement of HGF in radiation injury, the authors analysed the changes in HGF mRNA and HGF protein levels in the rat organs (liver, lung, kidney) and plasma following 6 Gy of total body irradiation. Expression of HGF mRNA in the liver and kidney increased 6-48 h after total body irradiation and returned to previous values 1 week later. HGF protein levels in lung and liver showed 1.3-2-fold elevations 1-2 weeks after irradiation (P < 0.05). HGF levels in plasma stayed at undetectable levels up to 1 month after total body irradiation. The labelling index determined 2 weeks and 1 month after total body irradiation indicated no enhancement of regeneration. Thus, total body irradiation induced transient HGF elevation in these organs without enhancement of regeneration.     

Enhanced expression of the protooncogenes c-myc and c-fos in normal and malignant renal growth

The protooncogenes c-myc and c-fos play an important role in growth and differentiation of renal tissue. They are highly expressed during embryogenesis in the mitotically active tubule epithelium, while in terminally differentiated tubule cells of the kidney the expression is completely shut off. Furthermore, induction of cell proliferation in cultured renal cells by addition of growth factors is preceded by enhanced expression of c-myc and c-fos. Increased expression of these protooncogenes is also obtained by treatment of kidney cells in culture with the potent nephrocarcinogen N-dimethylnitrosamine and also with the nephrotoxin and possibly nephrocarcinogen S-(1,2-dichlorovinyl)-L-cysteine. Finally, the expression of c-myc and c-fos is induced after unilateral nephrectomy during compensatory renal growth in the remaining kidney and also during regenerative cell proliferation after in vivo application of the strong nephrotoxins folic acid and mercury chloride.     

Thrombin increases clusterin mRNA in glomerular epithelial and mesangial cells

Clusterin, a multifunctional protein with complement blocking activity, and fibrin, a product of thrombin's enzymatic activity, are present in the kidney during acute and chronic renal failure. The role of thrombin in regulating clusterin mRNA in the kidney is not known. The effect of thrombin on clusterin mRNA expression was examined in rat glomerular mesangial and glomerular epithelial cells, and cultured human renal proximal tubular epithelial cells by northern blot. Thrombin (10(-8) M) increased clusterin mRNA levels two- to fourfold in glomerular mesangial, glomerular epithelial, and proximal tubule epithelial cells. This was a specific effect of thrombin receptor activation because peptides corresponding to the tethered ligand of the thrombin receptor were also able to increase clusterin mRNA levels. Epidermal growth factor, insulin-like growth factor-1, and transforming growth factor-beta 1 had little or no effect on clusterin mRNA levels. The protein kinase C inhibitor RO-32-0432 (1 microM) inhibited the thrombin-induced increase in clusterin mRNA, suggesting that thrombin receptor activation may regulate renal clusterin mRNA levels through protein kinase C.     

Gland atrophy following retrograde injection of methyl violet as a treatment in chronic obstructive parotitis

BACKGROUND: The growth and progression of prostate cancer depends on the stromal-epithelial interaction which is under paracrine control. Hepatocyte growth factor (HGF), produced by mesenchymal cells, is a multifunctional growth factor stimulating the movement and growth of epithelial cells including cancer cells. We therefore assessed the relationship between the invasive potential of prostate cancer and HGF in vitro. METHODS: Three human prostate cancer cell lines were used including PC-3 and DU145 (androgen-independent), and LNCaP (androgen-dependent). We studied the expression of the HGF receptor c-met proto-oncogene (c-met) by Western blot analysis, and also determined the effects of HGF on cell scattering, and the mechanisms of invasion and proliferation, by microscopic observation, the matrigel invasion chamber assay, and the MTT assay. RESULTS: c-met was detected in PC-3 and DU145 cells, but not in the LNCaP cells. There was increased cell motility in the scatter assay and an increased cell invasive potential in the matrigel invasion chamber assay by stimulation with HGF only with DU145 cells. CONCLUSION: HGF plays an important role in the invasion and metastasis of the DU145 cell line through a paracrine mechanism mediated by the c-metreceptor. In the PC-3 cell line, the lack of downstream signal transduction after the c-met receptor is suggested.     

Connections between the tendons of the musculus flexor digitorum profundus involving the synovial sheaths in the carpal tunnel

We earlier described a model of fulminant hepatic failure (FHF) in the rat where partial hepatectomy is combined with induction of right liver lobes necrosis. After this procedure, lack of regenerative response in the residual viable liver tissue (omental lobes) was associated with elevated plasma hepatocyte growth factor (HGF) and transforming growth factor beta (TGF-beta1) levels and delayed expression of HGF and c-met mRNA in the remnant liver. Here, we investigated whether syngeneic isolated hepatocytes transplanted in the spleen will prolong survival and facilitate liver regeneration in FHF rats. Inbred male Lewis rats were used. Group I rats (n = 46) received intrasplenic injection of 2 x 10(7) hepatocytes and 2 days later FHF was induced. Group II FHF rats (n = 46) received intrasplenic injection of saline. Rats undergoing partial hepatectomy of 68% (PH; n = 30) and a sham operation (SO; n = 30) served as controls. In 20 FHF rats (10 rats/group), survival time was determined. The remaining 72 FHF rats (36 rats/group) were used for physiologic studies (liver function and regeneration and plasma growth factor levels). In Group I rats survival was longer than that of Group II controls (73 +/- 22 hr vs. 33 +/- 9 hr; P < 0. 01). During the first 36 hr, Group I rats had lower blood ammonia, lactate, total bilirubin, PT, and PTT values, lower activity of liver enzymes, and higher monoethylglycinexylidide (MEGX) production than Group II rats. In Group I rats, livers increased in weight at a rate similar to that seen in PH controls and showed distinct mitotic and DNA synthetic activity (incorporation of bromodeoxyuridine and proliferation cell nuclear antigen expression). Plasma HGF and TGF-beta1 levels in these rats decreased and followed the pattern seen in PH rats; additionally, c-met expression in the remnant liver was accelerated. Hepatocyte transplantation prolonged survival in FHF rats and facilitated liver regeneration. Even though the remnant liver increased in weight four times reaching 30% of the original liver mass, the transplant-bearing rats expired due to inability of the regenerating liver to support the rat.     

Hepatocyte growth factor promotes motor neuron survival and synergizes with ciliary neurotrophic factor

Hepatocyte growth factor (HGF) has been shown to function as a potent mitogen for a variety of cells, transducing its signal through the c-met tyrosine kinase receptor. Ciliary neurotrophic factor (CNTF) is a cytokine that has been shown to promote survival of motor neurons. We show here that c-met mRNA is present in the embryonic rat spinal cord. Peak expression of c-met (at E14) coincides with the period of naturally occurring cell death in motor neurons, suggesting a possible role of HGF in the regulation of this process. Utilizing a neuron-enriched culture system, we established that HGF, like CNTF, stimulates choline acetyltransferase (CAT) activity in motor neurons. When co-administered to motor neuron cultures, saturating concentrations of HGF and CNTF produced a synergistic increase in CAT levels. We show that this synergy reflects enhanced motor neuron survival. Exposure of motor neuron cultures to the cytostatic agent vincristine markedly decreased CAT levels; co-treatment with HGF and CNTF (but not either factor alone) restored CAT activity to control levels. Our findings indicate that HGF is a survival factor for motor neurons, that it acts synergistically with CNTF, and that HGF and CNTF can together be neuroprotective in the face of vincristine toxicity.     

Expression of fetal kidney growth factors in a kidney tumor line: role of FGF2 in kidney development

To identify growth factors which may play a role in kidney organogenesis, we have analyzed culture supernatants from the pediatric kidney tumor cell line G401. G401 cells were found to secrete fibroblast growth factor 2 (FGF2), a potent mitogen for mesenchymal cells, OP-1/BMP7, an epithelial cell growth inhibitor, and midkine (MK). Northern blotting confirmed expression of FGF2, OP-1/BMP7 and MK mRNA, as well as Wnt5A mRNA in G401 cells. In situ hybridization and immunocytochemistry on human fetal kidney demonstrated FGF2 expression in epithelial cells of the branching ureteric bud epithelium, nephron precursors ("S-shaped bodies"), proximal tubule epithelium and the parietal epithelium of the glomerulus. FGF2 protein in condensed "caps" of induced mesenchymal cells was also detected by immunocytochemistry. FGF2 protein was found to be concentrated in nuclei, particularly in proximal tubule epithelial cells. Recombinant FGF2 was found to act as a mitogen on primary mouse fetal kidney cell cultures. The results demonstrate G401 cells secrete a variety of fetal kidney growth factors and that FGF2 may act as a mitogen for fetal kidney cells and thus could play a role in the morphogenesis of the kidney.