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Abstract
Chronic kidney disease (CKD) which is characterized by progressive loss of renal function and renal fibrosis is a worldwide public health problem. Hepatocyte growth factor (HGF) is a polypeptide that exhibits multiple functions including antifibrotic effects on kidneys. The present study was aimed at evaluating HGF levels and studying its association with markers of inflammation and oxidative stress in patients of predialysis and dialysis CKD. A total of 80 subjects including 20 healthy controls, 40 patients of CKD stage 1 to stage 5 (predialysis), and 20 CKD patients with end-stage renal disease on hemodialysis were recruited. HGF, high-sensitivity C-reactive protein (hsCRP), malondialdehyde (MDA), and ferric reducing ability of plasma (FRAP) were measured in all the subjects. HGF levels were significantly higher in all patients with CKD compared to controls. The levels were found to be lower in patients on dialysis than in the predialysis group; however, the difference was not statistically significant. hsCRP, MDA, and FRAP were significantly higher in all patients with CKD than in controls. HGF levels did not show a significant correlation with the markers studied. HGF levels were increased in response to renal injury in CKD patients. The levels were higher in predialysis patients of CKD than in CKD patients on dialysis. HGF levels may be used as an indicator of renal fibrosis in patients with CKD.
Introduction 
Kidney failure is a global public health problem with increasing incidence and preva lence, economic burden, and poor outcomes.[1] Further, the increased prevalence of the earlier stages of chronic kidney disease (CKD) and the associated complications including progression of renal dysfunction and increased risk of cardiovascular disease (CVD) indicate the need for development of strategies to prevent the increased morbidity and mortality in patients with CKD.[2]
Hepatocyte growth factor (HGF), which is a polypeptide growth factor, was initially identified as a molecule that promotes hepatocyte growth and hepatic regeneration. However, it is now found to exhibit pleiotropic functions such as mitogenic, motogenic, and morphogenic effects on multiple tissues including kidneys.[3],[4] One of the important physiological functions of HGF is its role in the regeneration of various organs, and HGF levels were found to be increased in response to acute injuries and diseases of various tissues.[5] The actions of HGF are mediated through its receptor c-Met (mesenchymalepithelial transition factor) which is expressed on the surfaces of various cells.[6] In the kidneys, HGF is produced by mesangial cells, endothelial cells, and macrophages.[5] Studies have shown that HGF acts as a renotropic growth factor and the expression of HGF was found to be increased after acute renal injury.[7] CKD which is characterized by progressive loss of parenchymal cells and renal fibrosis ultimately progresses to end-stage renal disease (ESRD) that requires renal replacement therapy. Transforming growth factor-β (TGF-β) is known to play an important role in fibrotic disorders including renal fibrosis. On the other hand, HGF suppresses the expression of TGF-β and acts as an antifibrotic factor, thereby inhibiting renal fibrosis and preventing the progression of renal dysfunction.[8] Hence, administration of HGF may be considered a treatment option in patients with chronic renal disease.[8] Almost all forms of CKD progress to ESRD over a period of time, leading to increased morbidity and mortality. Inflammation and oxidative stress are the important processes that accelerate the progression of renal disease.[9] Studies have suggested that HGF suppresses inflammation,[10] and exerts antioxidant effects.[11]
In this background, the present study was conducted to measure hepatocyte growth levels in patients with CKD [predialysis and on maintenance hemodialysis (HD)] and compare them with those of healthy controls and also to study the association of HGF with markers of inflammation [high-sensitivity C-reactive protein (hsCRP)] and oxidative stress [malondialdehyde (MDA) and ferric reducing ability of plasma (FRAP)].
Materials and Methods Subjects
The study was conducted from May 2016 to December 2017 and included a total of 60 patients diagnosed with CKD stage 1 to stage 5 based on the Kidney Disease Outcomes Quality Initiative guidelines[12] attending our nephrology facility and maintenance dialysis unit and 20 apparently healthy controls after obtaining institutional ethics committee clearance and informed consent from all the participants. The subjects were further classified into three groups as Group 1 (healthy controls, n = 20), Group 2 (CKD stage 1 to stage 5, n = 40), and Group 3 (CKD stage 5D/ESRD, n = 20). Patients in pediatric age group (<18 years),with history of alcohol abuse, acute renal failure, acute-on-CKD, smoking, pregnancy, diabetes mellitus, vasculitis, liver disease, and malignancy, and those not willing were excluded from the study.
Sample collection
Six milliliters of fasting venous blood samples was collected from all the subjects. The samples were allowed to clot and serum was separated by centrifuging the samples at 3000 revolutions per minute for 15 minutes. The serum samples were stored in appropriately labeled aliquots at –80°C until biochemical analysis.
Methods
Urea and creatinine were measured by enzymatic timed fixed end-point method and modified Jaffe’s rate reaction method, respectively. hsCRP was estimated by immunoturbidimetry. MDA, as a measure of lipid peroxidation, was estimated using thiobarbituric acid reactive substances method,[13] and FRAP was measured as an indicator of total antioxidant status using the method described by Benzie and Strain.[14] HGF levels were assayed by enzyme-linked immunosorbent assay using commercial kit.
Statistical Analysis The numerical data were expressed as mean ± standard deviation (for data in normal distribution) and median and interquartile range (for data in nonnormal distribution). Unpaired Student’s t-test or Mann–Whitney U-test was used for comparison of means between controls and cases. Comparison of means across multiple groups was done by analysis of variance (ANOVA for parametric data) and Kruskal–Wallis test (for nonparametric data) followed by posthoc analysis. The association between the parameters studied was tested using Pearson’s correlation coefficient for parametric data and Spearman’s correlation coefficient for non-parametric data. Statistical analysis was performed using Microsoft Excel spreadsheets and IBM SPSS Statistics version 25.0 (IBM Corp., Armonk, NY, USA). P <0.05 was considered statistically significant.
Results The present study included 20 healthy controls (11 males and 9 females) and 60 patients with CKD (35 males and 25 females). [Table 1] shows the parameters studied in healthy controls and all CKD patients. Serum HGF levels were found to be significantly higher in CKD patients than in controls. Similarly, hsCRP, MDA, and FRAP levels were significantly higher in CKD patients when compared to controls. [Table 2] shows the comparison of parameters between controls, predialysis patients, and dialysis CKD patients. HGF, hsCRP, and MDA showed a significant difference across the study groups. Predialysis CKD patients had significantly higher HGF levels than controls. HGF levels did not show a significant difference between other groups studied. hsCRP levels increased progressively across the groups and both the groups of CKD patients had significantly higher hsCRP levels than controls. However, although the levels were higher in dialysis patients compared to predialysis patients, the difference was not statistically significant. MDA levels also increased progressively with increasing renal impairment with patients on dialysis having significantly higher levels compared to controls and predialysis patients. FRAP increased progressively across the study groups; however, the difference was significant between controls and predialysis groups. The association of HGF with the parameters studied was assessed using correlation analysis. HGF did not show a significant correlation with any of the markers studied [Table 3].
Table 1: Demographic and biochemical parameters in controls and patients with CKD.
Table 2: Demographic and biochemical parameters in controls, predialysis patients, and dialysis patients with CKD. Discussion Progressive fibrosis involving tissues such as liver, kidney, and lung results in chronic diseases and is an important cause of increased morbidity and mortality. The number of patients with CKD and on maintenance HD is increasing globally. Several factors including hypertension, diabetes mellitus, and proteinuria contribute to the progression of renal dysfunction while HGF protects against renal fibrosis. In the present study, serum HGF levels were found to be significantly higher (P <0.001) in patients with CKD when compared to healthy controls [Table 1] and [Figure 1]. When further analyzed, predialysis patients with CKD had significantly higher serum HGF levels than controls (P <0.001). Although patients undergoing HD had higher HGF levels when compared to controls, the difference was not statistically significant (P = 0.078). Zayet reported that patients in all stages of CKD had significantly higher levels of HGF when compared to controls and the levels were highest in the group of patients including patients in CKD stage V and on HD.[15] Serum HGF levels in CKD patients on HD in the present study were found to be lower than predialysis CKD patients; however, the difference was not statistically significant (P = 0.566) [Table 2] and [Figure 2]. During early stages of chronic renal injury, there is an upregulation of expression of HGF, thus resulting in higher levels of HGF and compensatory regenerative responses in CKD patients. However, with progression of renal disease, HGF levels decline gradually since there is an overexpression of TGF-β which is a potent fibrogenic cytokine and an inhibitor of HGF expression.[5]
Figure 1: Parameters studied in controls and patients with chronic kidney disease. CKD: Chronic kidney disease.
Figure 2: Parameters studied in controls, predialysis patients, and dialysis patients with chronic kidney disease. CKD: Chronic kidney disease.CKD is associated with a pro-inflammatory state and hsCRP levels were measured as a marker of inflammation in CKD patients in the present study. It was found that hsCRP levels were significantly higher in all CKD patients when compared to controls (P = 0.003) [Table 1] and [Figure 1]. Further, both predialysis and dialysis CKD patients had significantly higher hsCRP levels when compared to controls (P = 0.007 and 0.008, respectively). Studies have shown that inflammatory stress is evident from earlier stages of CKD and the inflammatory stress increases as CKD stage progresses.[16] Similar pattern was observed in the present study with significantly higher hsCRP levels in predialysis patients than in controls and the levels increasing further in CKD patients on HD [Table 2] and [Figure 2]. The increase in hsCRP levels was statistically significant even though the study population was relatively small indicating the importance of inflammation in the progression of CKD as well as development of CKD-associated complications since inflammation plays an important role in atherogenesis. The levels were higher in patients on dialysis than in predialysis patients; however, the difference was not statistically significant (P = 0.739). Romão et al observed that hsCRP levels did not show a significant difference between patients with CKD and those on HD, and HD procedure could have partially corrected the inflammatory process in patients with severe CKD.[16] The association between renal function impairment and inflammation is well established. CKD itself is a low-grade inflammatory state, and the presence of factors such as oxidative stress, malnutrition, and volume overload may further play a role in the inflammatory process of CKD.[9] The increase in CRP levels in CKD patients could be a result of decreased renal clearance and increased hepatic synthesis secondary to chronic stimulation by interleukin-6 (IL-6) and other uremia-related factors.[17]
In the present study, MDA and FRAP were measured as markers of pro-oxidant and antioxidant status, respectively. Both MDA and FRAP levels were found to be significantly higher in patients with CKD than in controls (P = 0.002 and 0.020 for MDA and FRAP, respectively) [Table 1] and [Figure 1]. Serum MDA levels increased progressively with progression of renal dysfunction, and patients on HD had significantly higher MDA levels than controls (P <0.001) and predialysis CKD patients (P = 0.015) [Table 2] and [Figure 2]. Durak et al,[18] and Tbahriti et al,[19] reported that patients with chronic renal disease had significant oxidative stress as evidenced by increased lipid peroxidation which is further exacerbated by HD. FRAP levels in predialysis and dialysis patients in the present study were higher compared to controls; however, the difference was statistically significant between the predialysis and control groups (P = 0.003) while not significant between other groups [Table 2] and [Figure 2]. Increased FRAP levels in CKD patients were reported in earlier studies, and the increase in FRAP levels was attributed to uric acid levels which were also increased.[20] The antioxidant capacity is expected to be lowered in patients with CKD. However, 60% of the total antioxidant capacity as measured by FRAP assay is contributed by uric acid,[14] and CKD patients in the present study had increased uric acid levels which could be the reason for the increased FRAP levels observed in them. Oxidative stress occurs due to an imbalance of oxidant and antioxidant mechanisms. Renal dysfunction is commonly associated with oxidative stress. The oxidative stress in uremic state is a result of an increased production of reactive oxygen species and occurs even in early CKD. CKD per se and the presence of comorbidities such as diabetes mellitus, hypertension, and dyslipidemia contribute to increased oxidative stress in uremic patients.[9] The increased free radicals during oxidative stress oxidatively modify membrane lipids causing their peroxidation and formation of various lipid peroxidation products including MDA.[21]
When the association of serum HGF with hsCRP, MDA, and FRAP was evaluated in the present study, HGF did not show a significant correlation with any of the parameters [Table 3]. This could be due to factors such as small sample size and nonhomogenous sample. Studies on larger population may give more information on the correlation of HGF with other parameters.
In conclusion, the present study showed that serum HGF levels were increased in response to renal injury indicating compensatory response. However, with progressing renal fibrosis, the levels decreased. Increased hsCRP levels and MDA levels indicate the presence of inflammation and oxidative stress. Although HGF did not correlate with changes in other markers of CKD, it may be used as an indicator of fibrosis in patients with CKD.
Conflict of interest: None declared.
References 
Correspondence Address:
Vinapamula S Kiranmayi
Department of Biochemistry, Sri Venkateswara Institute of Medical Sciences, Tirupati - 517507, Andhra Pradesh
India
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/1319-2442.367826
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