|Year : 2018 | Volume
| Issue : 4 | Page : 271-275
The effect of co-infection with hepatitis B and hepatitis C viruses on the prevalence of proteinuria and loss of renal function: a single-center experience
Amin R Soliman, Rabab M Ahmed, Mahmoud Soliman, Ahmed Abdallah, Bahaa Zayed
Department of Internal Medicine, Faculty of Medicine, Cairo University, Cairo, Egypt
|Date of Submission||10-Jul-2018|
|Date of Acceptance||12-Aug-2018|
|Date of Web Publication||14-Jan-2019|
Rabab M Ahmed
Department of Internal Medicine, Cairo University, Cairo
Source of Support: None, Conflict of Interest: None
Introduction and aim of the work Patients infected with hepatitis B virus (HBV) or hepatitis C virus (HCV) are at increased risk of renal disease. This study compares factors associated with proteinuria and doubling of serum creatinine level in patients who were infected with HCV or HBV alone with those who were coinfected with HCV and HBV.
Materials and methods The study was performed on 1243 patients who were diagnosed with HBV and/or HCV at the Cairo University Hospitals. All the included subjects underwent urine analysis for proteinuria and serum creatinine level. Clinical characteristics were recorded at baseline and at last follow-up.
Results Of 1243 patients, 293 (23.6%) patients had proteinuria. Subset analysis of the patients with proteinuria showed that 10.6% were HBV infected, 63.8% were HCV infected, and the remaining 25.6% were coinfected with both HBV and HCV. Overall, coinfection with both viruses (P=0.01), lower serum albumin (P=0.001), hypertension (P=0.01), and diabetes (P=0.001) were associated with an increase in risk of proteinuria. Coinfection (P=0.001), presence of HBV (P=0.001), and increasing HCV RNA level in patients with HCV and in coinfected patients (P=0.05) was associated with doubling of serum creatinine level.
Conclusion The patients coinfected with HBV and HCV are at greater risk of clinically significant proteinuria and loss of renal function owing to complex virological profile. Progressive loss of renal function in that population is associated with markers of viral activity such as proteinuria and increasing HCV RNA levels among HCV-infected patients.
Keywords: co-infection, creatinine, hepatitis, proteinuria
|How to cite this article:|
Soliman AR, Ahmed RM, Soliman M, Abdallah A, Zayed B. The effect of co-infection with hepatitis B and hepatitis C viruses on the prevalence of proteinuria and loss of renal function: a single-center experience. Egypt J Intern Med 2018;30:271-5
|How to cite this URL:|
Soliman AR, Ahmed RM, Soliman M, Abdallah A, Zayed B. The effect of co-infection with hepatitis B and hepatitis C viruses on the prevalence of proteinuria and loss of renal function: a single-center experience. Egypt J Intern Med [serial online] 2018 [cited 2019 Jun 24];30:271-5. Available from: http://www.esim.eg.net/text.asp?2018/30/4/271/249966
| Introduction|| |
In Egypt, ∼15% of the population is infected with hepatitis C virus (HCV) and 8% is infected with hepatitis B virus (HBV). Both viruses represent a major endemic medical health problem in our country ,. They are not only associated with end-stage liver disease and hepatocellular carcinoma, the most frequent causes of death in patient with liver disease, but also with various extrahepatic manifestations ,,,,. In addition to symptomatic disease, clinically silent glomerular disease has been described in these patients in the form of elevated serum creatinine, microscopic hematuria, and/or proteinuria ,. Proteinuria has been shown to be an early diagnostic marker of kidney damage and can predict the progression of renal disease. Protein overload can upregulate complement cascade and various proinflammatory, vasoactive, and fibrogenic genes in proximal tubular cells leading to apoptosis of proximal tubules ,,,,,,,,,,. Many studies, such as the ALLHAT , INSIGHT ,, and LIFE , have shown that increasing proteinuria and deterioration of renal function are associated with poorer outcomes from vascular disease, higher incidence of cardiovascular events, and fatal and nonfatal stroke in both diabetic and nondiabetic patients. Microalbuminuria is also associated with a failure of nocturnal dipping in blood pressure, insulin resistance, and abnormal vascular responses to various stimuli ,,. Therefore, reducing proteinuria is a well-known renoprotective approach in nephrology. Proteinuria is a modifiable risk factor and can be managed by treating its cause. In this line of concept, the Kidney Disease Improving Global Outcomes clinical practice guidelines recommend screening for kidney disease at the time of HCV diagnosis ,. Complex virological profiles may be present in infected patients, and dually infected patients are an extremely heterogeneous population . This study assesses factors associated with proteinuria and doubling of serum creatinine level among patients infected with HBV or HCV alone or coinfected with both viruses.
| Materials and methods|| |
This is a prospective study conducted at the Cairo University Hospitals, Egypt, over 2 years. Ethical and Research Committee of the National Research Centre approved the study protocol. A total of 1243 patients who were diagnosed with viral hepatitis (HCV or HBV alone or coinfected with both) were enrolled. All patients had agreed to take part in the study and signed a declaration of informed consent form.
Clinical characteristics were recorded at baseline and at last follow-up. Co-morbid conditions such as diabetes mellitus and hypertension were recorded. Type 2 diabetes was diagnosed using the criteria proposed by the American Diabetes Association. Fasting and 2-h postprandial glucose levels in venous blood were measured with an autoanalyzer (Automated Beckman analyzer Au 680; Beckman Coulter Diagnostics Company, USA). Hypertension was defined as mean systolic blood pressure greater than or equal to 140 mmHg, mean diastolic blood pressure greater than or equal to 90 mmHg, a diagnosis of hypertension, or current use of antihypertensive medications. Serum creatinine (mg/dl) and albumin (g/dl) were measured by creatinine kinetic Jaffe uncompensated method and albumin BCP method, respectively, by using serum sample with an autoanalyzer (Automated Beckman analyzer Au 680). A serum albumin level less than 3.5 g/dl was considered ‘low’.
Urine analyses were done in all subjects using reagent-strip tests (Albustix™; McGuff Company (McGuff), USA). Patients were considered proteinuric if the reading was equal to or greater than 1. Patients were included in the analyses if they had at least two creatinine measures obtained at a minimum of 3 months apart. Patients were included if they had urine analysis for proteinuria at entry and on last seen visit. The serum samples were analyzed for hepatitis B surface antigen (HBsAg) and anti-HCV antibody using commercially available fourth generation ELISA kits (Dialab, Wiener Neudorf, Austria). The specificity of the kit is 99.87% and sensitivity is 100%. The test is very simple and cost-effective. HCV-infected patients who were positive for HCV antibody were further confirmed for the presence of HCV RNA by PCR as per the routine methodology used in our hospital.
Continuous data are expressed as means±SD. Characteristics for patients were compared using either χ2-test or Student’s t-test. Multivariate binary logistic regression assessed the association between clinical variables, the presence of proteinuria, and doubling of serum creatinine level. For non-normal data, a Mann–Whitney U-test was performed. P less than 0.05 was considered statistically significant. All statistics were carried out using SPSS, version 16 (SPSS; SPSS Inc., Chicago, Illinois, USA).
| Results|| |
Proteinuria in the study population
Of the 1243 patients, 79% were males, with a mean age of 39.2±8.4 years. A total of 293 (23.6%) patients had proteinuria at both initial and follow-up urine analyses studies. The remaining patients (950) with positive virology but without proteinuria were considered the control group. Their age and sex were comparable to patients with positive virology and proteinuria. The prevalence of proteinuria was the highest among patients infected with HCV alone (63.8%), followed by coinfection (25.6%) and HBV alone (10.6%) ([Table 1]).
Factors associated with proteinuria
Results of multivariate logistic regression analyses on independent factors associated with proteinuria in patients with viral hepatitis are shown in [Table 2]. In proteinuric patients, coinfection with both viruses further increased the risk of proteinuria when compared with mono infection. The odds of having proteinuria in coinfected state was 1.20 when compared with HBV alone (P=0.08, not statistically significant) and 1.50 when compared with HCV alone (P=0.01, statistically significant). Low serum albumin level had a significant overall effect on proteinuria with an odd ratio (OR) of 1.96. When serum albumin level was compared against the type of infection, neither HBV alone nor coinfection influenced the OR significantly from the overall OR (1.6 and 1.9, respectively); however, such comparison showed an OR of 2.4 for HCV alone. Hypertension had an overall OR of 1.5, but the type of infection did not alter the OR significantly. Diabetes was the most significant factor associated with proteinuria (OR=3.2), and again, the type of infection did not alter the OR significantly from the overall OR.
|Table 2 Factors associated with increasing proteinuria in the 293 patients|
Click here to view
Factors associated with doubling of serum creatinine level
HBV infection, with or without HCV coinfection, was associated with serum doubling (P<0.001; [Table 3]). Hypertension in HCV-infected patients with or without HCV coinfection was associated with an increased risk of doubling of creatinine level (OR=2.70); it was not found to be statistically significant. Increasing HCV RNA also had significant association with serum creatinine doubling in HCV-infected patients and in coinfected patients (P=0.05) ([Table 3]).
|Table 3 Factors associated with doubling of serum creatinine level in patients with proteinuria|
Click here to view
| Discussion|| |
Proteinuria and renal diseases are often associated with diabetes mellitus and hypertension, but when we talk about endemic countries of hepatitis, we must also consider HCV and HBV as causative factors of proteinuria and kidney functions deterioration . Approximately one-fourth of our cases in this study (23.6%), infected with either HCV or HBV alone or with both viruses, had proteinuria. This result was expected as it has been known that chronic HCV had a well-recognized association with renal and extrahepatic manifestations . In addition, HBV infection is also associated with renal disease, and treatment of HBV improves renal outcome . Proteinuria is thought to be caused by either the deposition of immune complexes or the presence of the virus itself. In our study, the prevalence of proteinuria was higher in patients infected with HCV. The prevalence of nephropathy in HBV infection is reported to be 3–20%, and in HCV infection, it is ∼38% . A study from an endemic area of southern Taiwan demonstrated a significant association between proteinuria and HCV (10.2%) but not HBV infections .
In our study, 25.6% of our patients (293 patients) had coinfection and proteinuria. Coinfection is not uncommon in endemic countries because of the shared modes of transmission, and the prevalence of dual infection varies from country to country. In Egypt, the dual infection prevalence is reported to be 0.7% whereas in India, it is 16%. In Turkey, it is 2.6%, and data from Spain, Italy, Japan, Taiwan, and Iran, have demonstrated that ∼10–15% of patients with chronic HBV infection are also infected with HCV, and ∼2–10% of anti-HCV-positive patients are also HBsAg positive ,,,,,. These patients with dual infection have a higher risk of progression of their renal disease and increasing proteinuria owing to various patterns of viral replication and great variations of immune profiles.
Our study showed that, in patients with viral hepatitis, diabetes mellitus is the most significant factor associated with proteinuria followed by low serum albumin and hypertension. Coinfection is associated with 1.5 times the risk of proteinuria compared with HCV mono infection and 1.2 times the risk of proteinuria compared with HBV mono infection. This comes in agreement with different studies analyzing factors associated with proteinuria ,,,,,,,.
In our cases, patients infected with HBV with concurrent infection with HCV (coinfection) or even without HCV were associated with an increased risk of proteinuria and doubling of creatinine levels. Proteinuria in HBV can be mediated by immune reactions, for example, glomerular deposition of immune complexes, or virus-induced specific immunological effector mechanisms (specific T-lymphocyte or antibody), or indirect effects from virus-induced cytokines/mediators on renal tissue.
HBV antigens are also expressed in renal tubular epithelial cells . They can upregulate complement-mediated inflammatory pathways and contribute to the pathogenesis of nephropathy. Finally, HBV infection has been shown to induce apoptotic damage to the renal tubular cells ,,.
The work by Chen et al.  on 17 758 patients with chronic HBV infection and randomly selected 71 032 matched controls without HBV to assess the risk of ESRD found significant associations of end stage renal disease (ESRD) with HBV in men of any age and women younger than 60 years and concluded that HBV-infected patients should have targeted monitoring for the development of ESRD.
However, our results are in partial agreement with the study by Lee et al.  that found HCV infection, but not HBV infection, was associated significantly with prevalence and CKD stage, and are in agreement with another study that found HCV was associated with 40% greater risk of renal insufficiency among persons who had a positive HCV antibody test as compared with those who had a negative one .
Increasing HCV RNA level in HCV-infected patients and in coinfected patients was associated with an increasing risk of doubling of creatinine level. This can be explained by renal parenchymal expression of CD81 and SR-B1 receptors that allow the binding of HCV to the cell surface and endocytosis of HCV RNA and related proteins found in mesangial cells, tubular epithelial cells, and endothelial cells of glomerular and tubular capillaries causing direct mesangial injury. In addition, toll-like receptors may have a role in recognizing molecular patterns associated with microbial pathogens like HCV RNA and induce an immune response .
Another explanation is kidney injury owing to systemic immune response that is mediated by cryoglobulins .
Finally, the non-immunological aspects of HCV-related kidney injury cannot be ruled out because HCV-positive patients often have insulin resistance and hyperinsulinemia, and a higher prevalence of diabetes, which itself causes proteinuria. The HCV core protein directly reduces expression of insulin receptor substrate proteins 1 and 2 and increases the expression of tumor necrosis factor-α in hepatic cells, which upregulate the expression of angiotensin II type 1 receptors in mesangial cells, thus enhancing the deleterious effects of angiotensin II in the kidney .
Hypertension in HCV-infected patients was associated with risk of doubling of serum creatinine level. This can be explained by the use of angiotensin-converting enzyme inhibitors and angiotensin-receptor blockers, which are commonly used in arterial hypertension treatment and can cause elevation of serum creatinine level above the patient’s baseline level. Angiotensin II constricts both the afferent and efferent arterioles, but it preferentially increases efferent arteriole resistance. This lowers intraglomerular pressure and reduces the glomerular filtration rate .
| Conclusion|| |
Patients coinfected with HBV and HCV are at a greater risk for clinically significant proteinuria and loss of renal function. Progressive loss of renal function is associated with markers of viral activity such as proteinuria among HBV-infected patients and increasing HCV RNA levels among HCV-infected patients. Thus, a careful evaluation of the HBV and HCV viremia levels is mandatory for a correct diagnosis and proper therapeutic approach.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Gomaa A, Allam N, Elsharkway A, El Kassas M, Waked I. Hepatitis C infection in Egypt: prevalence, impact and management strategies. Hepat Med 2017; 9:17–25.
Khaled IA, Mahmoud OM, Saleh AF, Bioumie EE. Prevalence of HBV genotypes in Egypt among hepatitis patients. Mol Biol Rep 2011; 38:4353–4357.
Morales JM, Kamar N, Rostaing L. Hepatitis C and renal disease: epidemiology, diagnosis, pathogenesis and therapy. Contrib Nephrol 2012; 176:10.
Cacoub P, Comarmond C, Domont F, Savey L, Desbois AC, Saadoun D. Extrahepatic manifestations of chronic hepatitis C virus infection. Ther Adv Infect Dis 2016; 3:3–14.
Kappus MR, Richard K. Extrahepatic manifestations of acute hepatitis B virus infection. Gastroenterol Hepatol (N Y) 2013; 9:123–126.
Han SH. Extrahepatic manifestations of chronic hepatitis B. Clin Liver Dis 2004; 8:403–418.
Baig S, Alamgir M. The extrahepatic manifestations of hepatitis B virus. J Coll Physicians Surg Pak 2008; 18:451–457.
Radhakrishnan J, Uppot RN, Colvin RB. Case records of the Massachusetts General Hospital. Case 5-2010. A 51-year-old man with HIV infection, proteinuria, and edema. N Engl J Med 2010; 362:636.
McGuire BM, Julian BA, Bynon JS Jr, Cook WJ, King SJ, Curtis JJ et al.
Brief communication: Glomerulonephritis in patients with hepatitis C cirrhosis undergoing liver transplantation. Ann Intern Med 2006; 144:735.
Abbate M, Zoja C, Remuzzi G. How does proteinuria cause progressive renal damage? J Am Soc Nephrol 2006; 17:2974–2984.
Peruzzi L, Trusolino L, Amore A, Gianoglio B, Cirina P, Basso G et al.
Tubulointerstitial responses in the progression of glomerular diseases: albuminuria modulates alpha v beta 5 integrin. Kidney Int 1996; 50:1310–1320.
Wang Y, Chen J, Chen L, Tay YC, Rangan GK, Harris DC. Induction of monocyte chemoattractant protein-1 in proximal tubule cells by urinary protein. J Am Soc Nephrol 1997; 8:1537–1545.
Burton CJ, Combe C, Walls J, Harris KP. Secretion of chemokines and cytokines by human tubular epithelial cells in response to proteins. Nephrol Dial Transplant 1999; 14:2628–2633.
Drumm K, Bauer B, Freudinger R, Gekle M. Albumin induces NF-kappaB expression in human proximal tubule-derived cells (IHKE-1). Cell Physiol Biochem 2002; 12:187–196.
Morigi M, Macconi D, Zoja C, Donadelli R, Buelli S, Zanchi C et al.
Protein overload-induced NF-kappaB activation in proximal tubular cells requires H2O2 through a PKC-dependent pathway. J Am Soc Nephrol 2002; 13:1179–1189.
Nakajima H, Takenaka M, Kaimori J-Y., Nagasawa Y, Kosugi A, Kawamoto S et al.
Gene expression profile of renal proximal tubules regulated by proteinuria. Kidney Int 2002; 61:1577–1587.
Tang S, Leung JCK, Abe K, Wah Chan K, Chan LYY, Mao Chan T et al.
Albumin stimulates interleukin-8 expression in proximal tubular epithelial cells in vitro and in vivo. J Clin Invest 2003; 111:515–527.
Wohlfarth V, Drumm K, Mildenberger S, Freudinger R, Gekle M. Protein uptake disturbs collagen homeostasis in proximal tubule-derived cells. Kidney Int Suppl 2003; 84:S103–S109.
Donadelli R, Zanchi C, Morigi M, Buelli S, Batani C, Tomasoni S et al.
Protein overload induces fractalkine upregulation in proximal tubular cells through nuclear factor kappaB- and p38 mitogen-activated protein kinase-dependent pathways. J Am Soc Nephrol 2003; 14:2436–2446.
Zandi-Nejad K, Eddy AA, Glassock RJ, Brenner BM. Why is proteinuria an ominous biomarker of progressive kidney disease? Kidney Int Suppl 2004; 92:S76–S89.
ALLHAT Officers and Coordinators for the ALLHAT Collaborative Research Group. Major outcomes in high-risk hypertensive patients randomized to angiotensin-converting enzyme inhibitor or calcium channel blocker vs diuretic: the antihypertensive and lipid-lowering treatment to prevent heart attack trial (allhat). JAMA 2002; 288:2981–2997.
Brown MJ, Castaigne A, Ruilope LM, Mancia G, Rosenthal T, de Leeuw PW et al.
INSIGHT: international nifedipine GITS study intervention as a goal in hypertension treatment. J Hum Hypertens 1996; 10 (Suppl 3):S157–S160.
Segura J, Campo C, Ruilope L. Effect of proteinuria and glomerular filtration rate on cardiovascular risk in essential hypertension. Kid Internat 2004; 66 (Suppl 92): S45–S49.
Dahlöf B, Devereux RB, Kjeldsen SE, Julius S, Beevers G, de Faire U et al.
Cardiovascular morbidity and mortality in the Losartan Intervention for Endpoint Reduction in Hypertension Study (LIFE): a randomised trial against atenolol. Lancet 2002; 359:995–1003.
Levy J. Proteinuria, renal impairment, and death. BMJ 2006; 332:1402–1403.
Adinolfi L, Zampino R, Restivo L, Lonardo A, Guerrera B, Marrone A et al.
Chronic hepatitis C virus infection and atherosclerosis: clinical impact and mechanisms. World J Gastroenterol 2014; 20:3410–3417.
Aghemo A, Prati G, Rumi M, Soffredini R, D’Ambrosio R, Orsi E et al.
Sustained virological response prevents the development of insulin resistance in patients with chronic hepatitis C. Hepatology 2012; 56:1681–1687.
Levey AS, Atkins R, Coresh J, Cohen EP, Collins AJ, Eckardt KU et al.
Chronic kidney disease as a global public health problem: approaches and initiatives − a position statement from Kidney Disease Improving Global Outcomes. Kidney Int 2007; 72:247.
Castillo I, Martinez-Ara J, Olea T, Bartolomé J, Madero R, Hernández E et al.
High prevalence of occult hepatitis C virus infection in patients with primary and secondary glomerular nephropathies. Kidney Int 2014; 86:619.
Caccamo G, Saffioti F, Giovanni R. Hepatitis B virus and hepatitis C virus dual infection. World J Gastroenterol 2014; 20:14559–14567.
Hill NR, Fatoba ST, Oke JL, Hirst JA, O’Callaghan C, Lasserson DS et al.
Global prevalence of chronic kidney disease − a systematic review and meta-analysis. PLoS One 2016; 11:e0158765.
Tang S, Lai FM, Lui YH, Tang CS, Kung NN, Ho YW et al.
Lamivudine in hepatitis B-associated membranous nephropathy. Kidney Int 2005; 68:1750–1758.
Kong X-L., Ma X-J., Su H, Xu D-M. Relationship between occult hepatitis B virus infection and chronic kidney disease in a Chinese population-based cohort. Chronic Dis Transl Med 2016; 2:55–60.
Huang JF, Chuang WL, Dai CY, Ho CK, Hwang SJ, Chen SC et al.
Viral hepatitis and proteinuria in an area endemic for hepatitis B and C infections: another chain of link? J Intern Med 2006; 260:255–262.
Pallas JR, Farinas-Alvarez C, Prieto D, Delgado-Rodríguez M. Coinfections by HIV, hepatitis B and hepatitis C in imprisoned injecting drug users. Eur J Epidemiol 1999; 15:699–704.
Reddy GA, Dakshinamurthy KV, Neelaprasad P, Gangadhar T, Lakshmi V. Prevalence of HBV and HCV dual infection in patients on haemodialysis. Indian J Med Microbiol 2005; 23:41–43.
] [Full text]
Aroldi A, Lampertico P, Montagnino G, Patrizia P, Margherita V, Maria CR. et al.
Natural history of hepatitis B and C in renal allograft recipients. Transplantation 2005; 79:1132–1136.
Kalinowska-Nowak A, Bociąga-Jasik M, Garlicki A. Prevalence of hepatotropic viruses HBV and HCV in HIV-infected patients from Southern region of Poland. Acta Virologica 2000; 44:23–28.
Irshad M, Peter S. Spectrum of viral hepatitis in thalassemic children receiving multiple blood transfusions. Indian J Gastroenterol 2002; 21:183–184.
Viswanathan V, Tilak P, Kumpatla S. Risk factors associated with the development of overt nephropathy in type 2 diabetes patients: a 12 years observational study. Indian J Med Res 2012; 136:46–53. [Full text]
Deray G, Buti M, Gane E, Jia JD, Lik Yuen Chan H, Craxi A et al.
Hepatitis B virus infection and the kidney: renal abnormalities in HBV patients, antiviral drugs handling, and specific follow-up. Advances Hepatol 2015; 2015:596829.
Bhimma R, Hammond MG, Coovadia HM, Adhikari Catherine M, Connolly A. HLA class I and II in black children with hepatitis B virus-associated membranous nephropathy. 61 2002; 61:1510–1515.
Perico N, Cattaneo D, Bikbov B, Remuzzi G. Hepatitis C infection and chronic renal diseases. CJASN 2009; 4:207–220.
Chen YC, Su YC, Li CY, Wu CP, Lee MS. A nationwide cohort study suggests chronic hepatitis B virus infection increases the risk of end-stage renal disease among patients in Taiwan. Kidney Int 2015; 87:1030–1038.
Lee JJ, Lin MY, Yang YH MHA, Yang YH, Lu SN, Chen HC et al.
Association of hepatitis C and B virus infection with CKD in an endemic area in Taiwan: a cross-sectional study. Am J Kidney Dis 2010; 56:23–31.
Dalrymple LS, Koepsell T, Sampson J, Dominitz JA, Young B, Kestenbaum B. Hepatitis C Virus Infection and the Prevalence of Renal Insufficiency. CJASN 2007; 2:4715–4721.
Wörnle M, Schmid H, Banas B, Merkle M, Henger A, Roeder M et al.
Novel role of toll-like receptor 3 in hepatitis C-associated glomerulonephritis. Am J Pathol 2006; 168:370–385.
Fabrizi F. Hepatitis C virus, cryoglobulinemia, and kidney: novel evidence. Scientifica 2012; 2012:128382.
Cohen DL, Townsend RR. What should the physician do when creatinine increases after starting an angiotensin-converting enzyme inhibitor or an angiotensin receptor blocker? JCH 2008; 10:803–804.
[Table 1], [Table 2], [Table 3]