Abstract
Hepatocyte growth factor (HGF) facilitates the regeneration of injured kidney in acute renal failure (ARF). HGF is produced as a single-chain precursor by cells of mesenchymal origin and is converted to a biologically active, heterodimeric molecule by proteol ytic processing.
We studied HGF mRNA and protein levels in systemic organs of glycerol-induced ARF rats, a model of crush syndrome. HGF protein concentration of tissue homogenate was measured by ELISA. Both mRNA and protein levels were increased in liver and spleen at 24 hours after the glycerol injection whereas HGF protein level was decreased in the injured kidney. Expression of HGF receptor/c-met mRNA was elevated only in the kidney. These results suggest that HGF supplied in an endocrine manner may play an important role in the regenerating process following ARF.
Next, we measured serum HGF concentration by ELISA in 8 ARF patients caused by crush syndrome and the molecular size of serum HGF was determined byimmun oblotting. Although serum HGF levels elevated in all patients, the HGF levels did not associate with their prognoses. While a single-chain molecule was predominantly observed in sera from chronic renal failure patients and healthy subjects, the majority of serum HGF was a heterodimeric form in 7 ARF patients. In one patient who developed disseminated intravascular coagulation syndrome and had a poor prognosis, a single-chain molecule was predominant although the serum HGF concentration was equivalent. These data suggest that the activity of proteolytic processing may be also an important factor for the expression of the biological function of HGF.
INTRODUCTION
Hepatocyte growth factor (HGF) has mitogenic activity for various epithelial cells including renal epithelial cells [[1]] and accelerates the tissue regeneration in acute renal failure (ARF) [2-4]. HGF is produced as a single-chain precursor by cells of mesenchymal origin, is converted to a biologically active a-, b-heterodimeric molecule by proteolytic processing [[5]], and expresses the function by binding to its receptor, c-met protoonco-gene product of the target cells. HGF is thought to function in a paracrine and/or endocrine manner [[6]].
In Study 1, to investigate HGF production in ARF, we studied HGF mRNA expression and protein levels in kidney and the other major HGF-producing organs of glycerol-induced ARF rats, a model for ARF caused by crush syndrome. We also studied c-met mRNA expression in these organs. In Study 2, we studied the serum HGF in ARF patients caused by crush syndrome. Although the elevation of serum HGF levels has been reported in ARF patients [[7]], there has been no clinical study concerning the processing conditions of increased levels of serum HGF. In order to examine proteolytic activation of HGF in ARF, we analyzed serum HGF molecular size by immunoblot analysis.
METHODS
Study 1
ARF was induced in male Sprague-Dawley rats (250–300 g BW) by intramuscular injection to the hind limbs with 10 mL/kg BW of 50% glycerol after overnight dehydration. Total RNA was isolated from spleen, kidney, liver, and lung at 24 hours after the glycerol injection, and 15 mg of total RNA for each organ was analyzed by Northern blot hybridization using 1.4 kb EcoR1 fragment of rat HGF cDNA or 0.8 kb Nde1 fragment of rat c-met cDNA as a probe.
For the measurement of tissue HGF concentrations, to avoid the influence of HGF secreted from platelets, the whole body of the anesthetized rat was perfused with ice-cold saline from the aorta. Spleen, kidney, liver, and lung were homogenized with 2M NaCl, 20mM Tris-HCl, pH 7.4, 1mM EDTA, 1mM PMSF, and 0.1 % Tween-80. After centrifugation the supernatant was assayed for HGF by an ELISA kit (Institute of Immunology, Tochigi, Japan).
Study 2
Table 1 shows the profiles of eight ARF patients caused by crush syndrome. The ARF patients were treated by either intermittent or continuous hemodialysis, in which nafamostat mesilate was used as an anticoagulant. Blood sampling was on the fourth or fifth day after the causal accidents. Two healthy subjects and two chronic renal failure (CRF) patients treated with hemodialysis were studied as controls. The serum HGF levels determined by an ELISA kit (Otsuka, Tokushima, Japan) were 0.31 and 0.50 ng/mL in CRF patients and 0.20 and 0.19 ng/mL in the healthy subjects. The serum HGF was partially purified by heparin affinity column and SDS-polyacrylamide gel electrophresis was performed under reducing condition. HGF was detected by Western blot analysis using mouse anti-human HGF a-chain monoclonal antibody(Otsuka, Japan).
Table 1. Patient Data on Serum HGF and ARF Treatment
RESULTS
Study 1
BUN levels were 16 ± 2 before the glycerol injections, and elevated to 85 ± 16 and 102 ± 20 mg/dL at 24 hours and 48 hours after the injections, respectively. Serum creatinine levels were 0.9 ± 0.2 before the injections, and elevated to3.4 ± 1.0 and 3.7 ± 1.1 mg/dL at 24 hours and 48 hours after the injections, respectively. By Northern blot analysis, weak expression of HGF mRNA was observed in lung and kidney of the normal control rat (Figure 1A). At 24 hours after the glycerol injection, the expression levels increased, particularlyin spleen and liver while kidney showed no change. On the other hand, c-met mRNA expression was observed in lung, liver and kidney of the normal rat and was markedly enhanced in kidney at 24 hours after the glycerol injection, while no remarkable changes were observed in the other organs (Figure 1B).
Published online:
07 July 2009Figure 1. Northern blot analysis of HGF (A) and c-met (B) mRNA in the acute renal failure (ARF) rat tissues. Total RNA was isolated from spleen (S), kidney(K), liver (Li) and lung (Lu) of normal rats and glycerol-induced ARF rats at 24 hours after glycerol injection; 15 mg of total RNA for each organ was analyzed by Northern blot hybridization as described in the method.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/irnf20/2001/irnf20.v023.i03-04/jdi-100104741/20150417/images/medium/irnf_a_11378954_uf0001_b.gif"})
Figure 1. Northern blot analysis of HGF (A) and c-met (B) mRNA in the acute renal failure (ARF) rat tissues. Total RNA was isolated from spleen (S), kidney(K), liver (Li) and lung (Lu) of normal rats and glycerol-induced ARF rats at 24 hours after glycerol injection; 15 mg of total RNA for each organ was analyzed by Northern blot hybridization as described in the method.
Consistent with the results of Northern blot analysis, tissue HGF concentration was high in kidney and lung among the four organs studied in normal rats. HGF content was significantly reduced to approximately 30% of the normal level in kidney at 24 hours after the glycerol injection, and showed a gradual recovery thereafter (Figure 2). Although there was no significant change in the lung HGF levels, tissue HGF concentrations more than doubled the control values in liver and spleen at 24 hours after injection.
Published online:
07 July 2009Figure 2. Tissue HGF concentrations in spleen, kidney, liver and lung of ARF rats. HGF concentration in each organ was determined in duplicate by an ELISA and was expressed per mg protein. Results are mean ± SD.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/irnf20/2001/irnf20.v023.i03-04/jdi-100104741/20150417/images/medium/irnf_a_11378954_uf0002_b.gif"})
Figure 2. Tissue HGF concentrations in spleen, kidney, liver and lung of ARF rats. HGF concentration in each organ was determined in duplicate by an ELISA and was expressed per mg protein. Results are mean ± SD.
Study 2
A major band was observed at approximately 60 kDa in the lanes of the ARF patients, excluding Patient 8 (Figure 3). This band corresponded to that of the recombinant human HGF, indicating an a-chain of HGF. In contrast, a major band was apparent at approximately 90 kDa in the lanes of CRF patients, normal subjects, and one of the ARF patients. Based on its molecular size, the band was suspected to be a single-chain HGF before processing, and it was confirmed by the finding that the larger band disappeared and the a-chain band was enhanced after a 24-hour incubation of the serum. Thus, the serum HGF was converted by the processing activity in 7 of the 8 ARF patients, whereas it was not converted in the CRF patients nor in the healthy subjects. The one exception was Patient 8 who expired due to multiple organ failure.
Published online:
07 July 2009Figure 3. Immunoblot analysis of serum HGF. Lane A is recombinant human HGF. The serum in Lane B, which is from CRF Patient 2, was preincubated for 24 hours.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/irnf20/2001/irnf20.v023.i03-04/jdi-100104741/20150417/images/medium/irnf_a_11378954_uf0003_b.gif"})
Figure 3. Immunoblot analysis of serum HGF. Lane A is recombinant human HGF. The serum in Lane B, which is from CRF Patient 2, was preincubated for 24 hours.
DISCUSSION
The unique multi-functional characteristics of HGF are expressed during organ development and reorganization after injuries [[1]]. Several studies indicate the important role of HGF in recovery from ARF [2-4]. Although HGF appears to be supplied to the injured tissues in a paracrine or endocrine manner, there have been no studies concerning HGF production in the systemic organs in ARF. In the present study using the glycerol-induced ARF model, we checked HGF production in the four major HGF-producing organs at mRNA and protein levels, and demonstrated the importance of HGF supplied through the endocrine manner, particularly from liver and spleen. The glycerol-induced ARF model is thought to mimic ARF caused by crush syndrome, and is characterized by necrosis of proximal tubules and obstruction of the distal nephron by casts. Although liver may have a minor damage in this ARF model, the target of HGF produced in liver and spleen appeared to be the injured kidney, not liver or spleen because the expression levels of the HGF receptor, c-met were increased only in the kidney.
Concerning HGF localized in the kidney, local cellular injury and necrosis may account for the decrease in the protein levels at 24 hours after the glycerol injection. Under such circumstances HGF is supplied in an endocrine manner in order to supplement the insufficient local HGF supply.
In Study 2 the elevation of serum HGF levels observed in ARF patients was consistent with previous reports [[7]]. There was no obvious relationship between patients' serum HGF levels and their prognoses in this limited number of the cases. Although the serum HGF level may reflect the production of HGF, it is evident that this HGF level does not necessarily show the biological activity because serum HGF concentrations are determined by enzyme immunoassay in which antibodies used detect both a biologically active heterodimeric form and an inactive single-chain form. In fact, there was a case where the majority of serum HGF remained as a single-chain form. Disseminated intravascular coagulation syndrome (DIC) may explain the low processing activity because the HGF activator has been reported to be stimulated bythromb in [[8]], an. Patient 8 had the highest DIC score among the patients studied. Thus, HGF supplied in an endocrine manner likely plays an important role in ARF, and likely requires an active enzyme system for activating the growth factor. This activity may be low in some patients. We conclude that administration of the active form of recombinant HGF will be an appropriate and effective treatment for ARF patients, particularly those with low HGF processing activity.
| Case | Sex | Age | Complications | Treatment for ARF* | Serum HGF (ng/ml) | Prognosis† |
|---|---|---|---|---|---|---|
| 1 | M | 24 | HD | 0.69 | Completed HD on day 11 | |
| 2 | M | 26 | Pulmonary edema | CHD | 0.39 | Completed HD on day 35 |
| 3 | F | 21 | pneumonia | HD | 0.57 | Completed HD on day 15 |
| 4 | F | 49 | CHD | 6.20 | Completed HD on day 25 | |
| 5 | F | 71 | colitis | HD | 0.66 | Completed HD on day 17 |
| 6 | F | 73 | atelectasis | CHD | 0.40 | Completed HD on day 10 |
| 7 | M | 69 | GI bleeding | HD | 1.15 | Completed HD on day 8 |
| 8 | M | 79 | sepsis | CHD | 1.20 | Completed HD on day 9 |
HD = intermittent hemodialysis; CHD = continuous hemodialysis.
*Treatment at time of blood sampling. CHD was changed to HD in Patient 2, 4 and 6 afterwards.
†The day is after the occurrence of the causal accidents.
REFERENCES
- Matsumoto K, Nakamura T. Hepatocyte growth factor: Molecular structure, roles in liver regeneration, and other biological functions. Crit Rev Onco 1992; 3: 27–54 [PubMed], [Google Scholar]
- Igawa T, Matsumoto K, Kanda S, Saito Y, Nakamura T. Hepatocyte growth factor may function as a renotropic factor for regeneration in rats with acute renal injury. Am J Physiol 1993; 265: F61–F69 [PubMed], [Web of Science ®], [Google Scholar]
- Miller S B, Martin D R, Kissane J, Hammerman M R. Hepatocyte growth factor accelerates recovery from acute ischemic renal injury in rats. Am J Physiol 1994; 266: F129–F134 [PubMed], [Web of Science ®], [Google Scholar]
- Kawaida K, Matsumoto K, Shimazu H, Nakamura T. Hepatocyte growth factor prevents acute renal failure and accelerates renal regeneration in mice. Proc Natl Acad Sci USA 1994; 91: 4357–4361 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Naka D, Ishii T, Yoshiyama Y, Miyazawa K, Hara H, Hishida T, Kitamura N. Activation of hepatocyte growth factor by proteolytic conversion of a single chain form to a heterodimer. J Biol Chem 1992; 267: 20114–20119 [PubMed], [Web of Science ®], [Google Scholar]
- Yanagita K, Nagaike M, Ishibashi H, Niho Y, Matsumoto K, Nakamura T. Lung may have an endocrine function producing hepatocyte growth factor in response to injury of distal organs. Biochem Biophys Res Commun 1992; 182: 802–809 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Ishibashi K, Sasaki S, Sakamoto H, Nakamura T, Marumo F. Expression of hepatocyte growth factor (HGF) and its receptor mRNA in kidney after renal ischemia or uninephrectomy. J Am Soc Nephrol 1991; 2: 648 [Google Scholar]
- Shimomura T, Kondo J, Ochiai M, Naka D, Miyazawa K, Morimoto Y, Kitamura N. Activation of the zymogen of hepatocyte growth factor activator bythrombin. J Biol Chem 1993; 268: 22927–22932 [Google Scholar]