A urinary biomarker profile for children with HIV-associated renal diseases
Authors: Ángel A. Soler-García, Natella Y. Rakhmanina, Parnell C. Mattison, Patricio E. Ray
Journal: Kidney International (2009)

Human immunodeficiency virus (HIV)-infected children are at risk of developing several types of renal diseases, including HIV-associated nephropathy (HIVAN), which is usually seen during late stages of infection in children with a high viral load. This disease is defined by the presence of proteinuria associated with mesangial hyperplasia and/or global–focal segmental glomerulosclerosis combined with microcystic transformation of the renal tubules. Because HIVAN can have an insidious clinical onset, renal biopsy is the only definitive way of establishing a diagnosis. Given the risk of performing this procedure in HIV-infected children with other AIDS-defining illness, we sought to identify informative biomarkers such as growth factors in the urine of 55 HIV-infected children that might be predictive of the extent and activity of the renal lesions characteristic of HIVAN. We found that the levels of epidermal growth factor were lower in the urine of children with renal disease, whereas levels of fibroblast growth factor-2 and metalloproteinase-2 were higher as compared with those levels in infected children without renal disease. Similar changes were observed in HIV-Tg26 mice correlating with the progression of renal disease in this model of HIVAN. Our findings suggest that this urinary growth factor profile may be useful in facilitating the diagnosis of HIV-infected children at risk of developing HIVAN when interpreted in the appropriate clinical setting. Human immunodeficiency virus (HIV)-infected children are at risk of developing several types of renal diseases, including HIV-associated nephropathy (HIVAN), which is usually seen during late stages of infection in children with a high viral load. This disease is defined by the presence of proteinuria associated with mesangial hyperplasia and/or global–focal segmental glomerulosclerosis combined with microcystic transformation of the renal tubules. Because HIVAN can have an insidious clinical onset, renal biopsy is the only definitive way of establishing a diagnosis. Given the risk of performing this procedure in HIV-infected children with other AIDS-defining illness, we sought to identify informative biomarkers such as growth factors in the urine of 55 HIV-infected children that might be predictive of the extent and activity of the renal lesions characteristic of HIVAN. We found that the levels of epidermal growth factor were lower in the urine of children with renal disease, whereas levels of fibroblast growth factor-2 and metalloproteinase-2 were higher as compared with those levels in infected children without renal disease. Similar changes were observed in HIV-Tg26 mice correlating with the progression of renal disease in this model of HIVAN. Our findings suggest that this urinary growth factor profile may be useful in facilitating the diagnosis of HIV-infected children at risk of developing HIVAN when interpreted in the appropriate clinical setting. Childhood human immunodeficiency virus (HIV)-associated nephropathy (HIVAN) is a renal disease (RD) defined by the presence of persistent proteinuria associated with mesangial hyperplasia and/or global–focal segmental glomerulosclerosis in combination with microcystic transformation of renal tubules and rapid progression to chronic renal failure.1Strauss J. Abitbol C.L. Zilleruelo G. et al.Renal diseases in children with the acquired immunodeficiency syndrome.N Engl J Med. 1989; 321: 625-630Crossref PubMed Scopus (202) Google Scholar, 2Ray P.E. Rakusan T.M. Loechelt B.J. et al.Human Immunodeficiency Virus (HIV)-Associated Nephropathy in the children from the Washington D.C. area: 12 Years' experience.Semin Nephrol. 1998; 18: 396-405PubMed Google Scholar, 3Ray P.E. Xu L. Rakusan T. et al.A 20-year history of childhood HIV-associated nephropathy.Pediatr Nephrol. 2004; 19: 1075-1092Crossref PubMed Scopus (45) Google Scholar It is usually seen during the late stages of HIV infection in children with a high viral load. Black children show a unique susceptibility to develop this RD, and if the US data from the pre-HAART (highly active antiretroviral therapy) era were extrapolated to Africa,4UNAIDS '2007 AIDS Epidemic update' www.unaids.org/en/KnoweldgeCenter/HIVData/EpiUpdate/EpicUpdArchive/2007Google Scholar we speculate that approximately 300,000 children without access to antiretroviral therapy could develop HIVAN. In addition, HIV-infected children can develop other RD or tubular toxicity secondary to HAART and other drugs.1Strauss J. Abitbol C.L. Zilleruelo G. et al.Renal diseases in children with the acquired immunodeficiency syndrome.N Engl J Med. 1989; 321: 625-630Crossref PubMed Scopus (202) Google Scholar, 2Ray P.E. Rakusan T.M. Loechelt B.J. et al.Human Immunodeficiency Virus (HIV)-Associated Nephropathy in the children from the Washington D.C. area: 12 Years' experience.Semin Nephrol. 1998; 18: 396-405PubMed Google Scholar, 3Ray P.E. Xu L. Rakusan T. et al.A 20-year history of childhood HIV-associated nephropathy.Pediatr Nephrol. 2004; 19: 1075-1092Crossref PubMed Scopus (45) Google Scholar Thus, to prevent the severe complications associated with these RD, it is necessary to perform an early diagnosis, and the only way of establishing a definitive diagnosis of HIVAN is to make a renal biopsy. Given the risk of performing this procedure in HIV-infected children, it is necessary to find new biomarkers to identify children at high risk of developing HIVAN. In a recent study using a proteomic approach, we were unable to identify a single urine protein biomarker to identify children with biopsy-proven HIVAN.5Soler-García A.A. Jonson D. Hathout Y. et al.Iron related proteins: candidate urine biomarkers in childhood HIV–associated renal diseases.Clin J Am Soc Nephrol. 2009Crossref Scopus (33) Google Scholar Here, we sought to determine whether growth factors released into the urine of children with HIV-RD reflect the extent and activity of the renal lesions characteristic of this disease. This approach is based on earlier studies showing a progressive accumulation of heparin-binding growth factors in correlation with the development of the renal microcystic tubular lesions that are a characteristic feature of childhood HIVAN.6Ray P.E. Liu X-H. Xu L. et al.Accumulation of bFGF in children with HIV-1 associated Hemolytic Uremic Syndrome.Pediatr Nephrol. 1999; 13: 586-593Crossref PubMed Scopus (50) Google Scholar, 7Liu X.H. Aigner A. Wellstein A. et al.Up-regulation of a fibroblast growth factor binding protein in children with renal disease.Kidney Int. 2001; 59: 1717-1728Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 8Ray P.E. Tassi E. Liu X.H. et al.Role of fibroblast growth factor-binding protein in the pathogenesis of HIV-associated hemolytic uremic syndrome.Am J Physiol Regul Integr Comp Physiol. 2001; 290: R105-R113Crossref Scopus (34) Google Scholar, 9Ray P.E. Bruggeman L. Weeks B. et al.Role of bFGF and its low affinity receptors in the pathogenesis of HIV-associated nephropathy in transgenic mice.Kidney Int. 1994; 46: 759-772Abstract Full Text PDF PubMed Scopus (91) Google Scholar We found elevated urine levels of fibroblast growth factor-2 (FGF-2) and matrix metalloproteinase-2 (MMP-2), in association with reduced urine levels of epidermal growth factor (EGF) in children with HIV-RD. As these growth factors are involved in the processes of renal tubular regeneration and cyst formation,10Grandaliano G. Gesualdo L. Bartoli F. et al.MCP-1 and EGF renal expression and urine excretion in human congenital obstructive nephropathy.Kidney Int. 2000; 58: 182-192Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 11Harada H. Furuya M. Ishikura H. et al.Expression of matrix metalloproteinase in the fluids of renal cystic lesions.J Urol. 2002; 168: 19-22Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 12Rankin C.A. Itoh Y. Tian C. et al.Matrix Metalloproteinase-2 in a murine model of infantile-Type Polycystic Kidney Disease.J Am Soc Nephrol. 1999; 10: 210-217PubMed Google Scholar, 13Smith E.R. Zurakowski D. Saad A. et al.Urinary biomarkers predict brain tumor presence and response to therapy.Clin Cancer Res. 2008; 14: 2378-2386Crossref PubMed Scopus (138) Google Scholar, 14Horikoshi S. Kubota S. Martin G.R. et al.Epidermal growth factor (EGF) expression in the congenital polycystic mouse kidney.Kidney Int. 1991; 39: 57-62Abstract Full Text PDF PubMed Scopus (57) Google Scholar, 15Comperat E. Ferlicot S. Camparo P. et al.Expression of epidermal growth factor receptor and proliferative activity of cysts epithelium in human renal cystic diseases.Urol Int. 2006; 76: 269-273Crossref PubMed Scopus (5) Google Scholar, 16Hayakawa M. Nakjima F. Tsuji A. et al.Cytokine levels in cystic renal masses associated with renal cell carcinoma.J Urol. 1998; 159: 1459-1464Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 17Rall L.B. Scott J. Bell G.I. Mouse prepro-epidermal growth factor synthesis by the kidney and other tissues.Nature. 1985; 313: 228-231Crossref PubMed Scopus (441) Google Scholar, 18Gupta G.K. Milner L. Linshaw M.A. et al.Urinary basic fibroblast growth: a noninvasive marker of progressive cystic renal disease in a child.Am J Med Genet. 2000; 93: 132-135Crossref PubMed Scopus (7) Google Scholar, 19Villanueva S. Cespedes C. Gonzalez A. et al.bFGF induces an earlier expression of nephrogenic proteins after ischemic acute renal failure.Am J Physiol Regul Integr Comp Physiol. 2006; 291: R1677-R1687Crossref PubMed Scopus (61) Google Scholar, 20Li Z. Jerebtsova M. Liu X-H. et al.Novel cystogenic role of basic fibroblast growth factor in developing rodent kidneys.Am J Physiol Renal Physiol. 2006; 291: F289-F296Crossref PubMed Scopus (18) Google Scholar our findings suggest that this urine profile might be useful in identifying children undergoing the tubular interstitial and microcystic lesions characteristic of HIVAN. As shown in Figure 1, we found elevated levels of cystatin C, γ-glutamyl transpeptidase (γ-GT), and lysozyme in a group of children with established HIV-RD. The urinary lysozyme levels were also significantly elevated in HIV-infected children with trace proteinuria (P<0.05) (Figure 1a), suggesting that the brush border of renal proximal tubular cells was injured in these patients. On the basis of these findings, we hypothesized that the urinary excretion of EGF, which is synthesized mainly by renal epithelial cells, would be reduced in children with HIVAN. To determine the urinary levels of EGF in HIV-positive and -negative children of different ages without RD, EGF was measured in the following groups of control children: (1) <2 years of age, with functionally immature kidneys with respect to glomerular function; (2) 2–12 years of age, with functionally mature pre-pubescent kidneys; and (3) >12 years, with mature pubescent kidneys. No significant differences in urinary EGF levels were found between HIV-positive or -negative children of similar age without RD (data not shown). However, children <2 years showed the highest urinary EGF levels when compared with all other age control groups (Table 1).Table 1Average urinary levels of EGF, FGF-2, and MMP-2 in children without renal diseaseBiomarkerControl group age (years)*<22–12>12EGF/UCR (pg/mg)96,045±7751*40,238±296729,485±6468FGF-2/UCR (pg/mg)5.86±1.324.9±1.142.33±0.39MMP-2 (ng/mg)2.93±0.860.93±0.231.10±0.22EGF, epidermal growth factor; FGF-2, fibroblast growth factor-2; MMP-2, matrix metalloproteinase-2; UCR, urinary creatinine ratio.All values are expressed as the mean±s.e. of the mean, and expressed as a ratio of the urinary creatinine value. (n=8–24 children per group). Non-parametric analysis of variance *P<0.05 and 0.001 when compared with the EGF groups of children of 2–12 years and >12 years of age, respectively. No statistically significant differences were found between the groups of children >2 years of age for EGF, FGF-2, and MMP-2 (P=0.0648; 0.1716; and 0.5668, respectively). Open table in a new tab EGF, epidermal growth factor; FGF-2, fibroblast growth factor-2; MMP-2, matrix metalloproteinase-2; UCR, urinary creatinine ratio. All values are expressed as the mean±s.e. of the mean, and expressed as a ratio of the urinary creatinine value. (n=8–24 children per group). Non-parametric analysis of variance *P<0.05 and 0.001 when compared with the EGF groups of children of 2–12 years and >12 years of age, respectively. No statistically significant differences were found between the groups of children >2 years of age for EGF, FGF-2, and MMP-2 (P=0.0648; 0.1716; and 0.5668, respectively). The urinary EGF concentration was significantly lower in children >2 years with biopsy-proven HIVAN (n=6), when compared with their corresponding age-matched HIV controls and trace proteinuria groups (Figure 2a, P<0.0001 and Figure 2b, P<0.05). Moreover, two children <2 years with HIVAN also showed a reduction in their urinary EGF levels of approximately 32 and 45%, relative to the mean values of age-matched controls. These changes were similar to the low values reported in children of similar age with congenital obstructive nephropathy.10Grandaliano G. Gesualdo L. Bartoli F. et al.MCP-1 and EGF renal expression and urine excretion in human congenital obstructive nephropathy.Kidney Int. 2000; 58: 182-192Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar In summary, only one child within six children >2 years with biopsy-proven HIVAN showed normal urinary EGF levels. As shown in Figure 2c, urine samples harvested from HIV-infected children with and without RD stimulated the growth of cultured renal tubular epithelial CV-1 cells by an average 88 and 145% (P<0.001), respectively, when compared with the cells exposed to control media containing 1% fetal bovine serum. Moreover, urine samples harvested from children with HIVAN increased the growth of CV-1 cells by an average of 57.6% when compared with the control samples (P<0.05) (Figure 2c). EGF-neutralizing antibodies were able to suppress the growth-promoting activity of urine samples harvested from the HIV-infected controls (HIV-C; P<0.001) and trace proteinuria (HIV-T; P<0.01) groups, respectively (Figure 2d and e). In contrast, EGF antibodies failed to exert a complete growth inhibition of CV-1 cells exposed to urine supernatants harvested from children with HIVAN (P>0.05) (Figure 2f). These data suggest that other growth factors that are present in the urine of children with HIVAN (that is, FGF-2), may act in synergy with EGF to enhance the proliferation of tubular epithelial cells in these patients.6Ray P.E. Liu X-H. Xu L. et al.Accumulation of bFGF in children with HIV-1 associated Hemolytic Uremic Syndrome.Pediatr Nephrol. 1999; 13: 586-593Crossref PubMed Scopus (50) Google Scholar, 7Liu X.H. Aigner A. Wellstein A. et al.Up-regulation of a fibroblast growth factor binding protein in children with renal disease.Kidney Int. 2001; 59: 1717-1728Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 8Ray P.E. Tassi E. Liu X.H. et al.Role of fibroblast growth factor-binding protein in the pathogenesis of HIV-associated hemolytic uremic syndrome.Am J Physiol Regul Integr Comp Physiol. 2001; 290: R105-R113Crossref Scopus (34) Google Scholar, 9Ray P.E. Bruggeman L. Weeks B. et al.Role of bFGF and its low affinity receptors in the pathogenesis of HIV-associated nephropathy in transgenic mice.Kidney Int. 1994; 46: 759-772Abstract Full Text PDF PubMed Scopus (91) Google Scholar Children <2 years show the highest urinary levels of FGF-2, but these changes were not statistically significant when compared with all other age control groups (Table 1). All children >2 years with biopsy-proven HIVAN (n=6) showed high abnormal urinary FGF-2 levels, when compared with their corresponding age-matched HIV controls and trace proteinuria groups (Figure 3a and b). Similarly, in both children <2 years with HIVAN, the urinary FGF-2 levels increased by an average of 2.5- to 11-fold over their age-matched controls, and above the normal values reported for children of similar age.18Gupta G.K. Milner L. Linshaw M.A. et al.Urinary basic fibroblast growth: a noninvasive marker of progressive cystic renal disease in a child.Am J Med Genet. 2000; 93: 132-135Crossref PubMed Scopus (7) Google Scholar Children <2 years showed the highest urinary levels of MMP-2, when compared with all other age control groups (Table 1). MMP-2 was elevated in the urine of children >2 years with biopsy-proven HIVAN (n=6) in comparison with their respective age-matched control (P<0.001) and trace proteinuria (P<0.05) groups (Figure 3c and d). In a similar way, urinary MMP-2 increased in both children <2 years with HIVAN by an average of 0.5- to 68-fold over their age-matched controls and above the normal values reported by others.13Smith E.R. Zurakowski D. Saad A. et al.Urinary biomarkers predict brain tumor presence and response to therapy.Clin Cancer Res. 2008; 14: 2378-2386Crossref PubMed Scopus (138) Google Scholar Finally, no significant differences in the urinary levels of MMP-9 were detected between HIV-infected children in the control, trace proteinuria, and HIVAN groups, respectively (>0.05) (Figure 3e). In summary, all children with biopsy-proven HIVAN showed abnormally elevated urinary levels of both MMP-2 and FGF-2 relative to their corresponding age-matched control groups. Figure 4a shows a representative gelatin zymographic analysis of urine samples harvested from HIV-infected children with and without RD, including seven children with HIVAN. The collagenolytic activity in these urine samples was inhibited in the presence of 20 mM 1, 10-phenanthroline, confirming the presence of metalloproteinases activity (Figure 4a). MMP-2 was detected as 72 and 64 kDa collagenolytic bands corresponding to the latent and active forms of this metalloproteinase.13Smith E.R. Zurakowski D. Saad A. et al.Urinary biomarkers predict brain tumor presence and response to therapy.Clin Cancer Res. 2008; 14: 2378-2386Crossref PubMed Scopus (138) Google Scholar The activity of MMP-2 was increased in the urine of children with established RD relative to the control group (Figure 4b). MMP-9 was detected as 92 and 82 kDa collagenolytic bands corresponding to the latent and active forms of this metalloproteinase (Figure 4c). HIV-infected children with trace proteinuria and established RD showed an increased urinary MMP-9 activity; however, these changes were not statistically significant (Figure 4c). As shown in Figure 5, the urinary levels of EGF in a child with HIVAN increased in correlation with the recovery of renal function in response to antiretroviral therapy (Figure 5a and b). In contrast, the urinary concentration of FGF-2 and MMP-2 decreased in correlation with the recovery of renal function. However, the urinary levels of EGF, FGF-2, and MMP-2 remained within abnormal values throughout the follow-up period (Figure 5c and d). As shown in Figure 6, representative urine samples harvested from wild-type (WT) littermate control mice showed an increased EGF-binding activity when compared with the samples corresponding HIV-Tg mice with severe RD (Figure 6a). When adjusted for the urinary creatinine concentration, EGF activity was approximately fivefold greater in the urine of WT mice relative to the urine of HIV-Tg mice with RD. In support of these findings, northern blots analysis showed a significant reduction of EGF mRNA expression of approximately one- to twofold in the renal medulla of HIV-Tg26 mice with early and late RD when compared with WT control littermate mice (Figure 6b). In a similar manner, the collagenolytic activity of MMP-9 and MMP-2 was increased in the urine of HIV-Tg26 mice with RD by an average of 10- to 50-fold, relative to WT and HIV-Tg26 mice without RD (Figure 6c). Finally, in an earlier study, we found a significant accumulation of FGF-2 in the kidney of HIV-Tg26 mice with RD in association with the presence of the tubular microcystic lesions characteristic of HIVAN.9Ray P.E. Bruggeman L. Weeks B. et al.Role of bFGF and its low affinity receptors in the pathogenesis of HIV-associated nephropathy in transgenic mice.Kidney Int. 1994; 46: 759-772Abstract Full Text PDF PubMed Scopus (91) Google Scholar Taken together, all these findings strongly suggest that the urinary profile of EGF, MMP-2, and FGF-2 changes in correlation to the progression of the renal tubulointerstitial and microcystic lesions characteristic of HIVAN. Childhood HIV-associated nephropathy is characterized by the presence of mesangial hyperplasia and/or focal segmental glomerulosclerosis in association with the microcystic transformation of renal tubules and rapid progression of the RD.1Strauss J. Abitbol C.L. Zilleruelo G. et al.Renal diseases in children with the acquired immunodeficiency syndrome.N Engl J Med. 1989; 321: 625-630Crossref PubMed Scopus (202) Google Scholar, 2Ray P.E. Rakusan T.M. Loechelt B.J. et al.Human Immunodeficiency Virus (HIV)-Associated Nephropathy in the children from the Washington D.C. area: 12 Years' experience.Semin Nephrol. 1998; 18: 396-405PubMed Google Scholar, 3Ray P.E. Xu L. Rakusan T. et al.A 20-year history of childhood HIV-associated nephropathy.Pediatr Nephrol. 2004; 19: 1075-1092Crossref PubMed Scopus (45) Google Scholar Earlier studies have shown that cytokines play a role in the development of these renal lesions.21Conaldi P.G. Botelli A. Wade-Evans A. et al.HIV-persistent infection and cytokine induction in mesangial cells: a potential mechanism for HIV-associated glomerulosclerosis.AIDS. 2000; 14: 2045-2047Crossref PubMed Scopus (22) Google Scholar, 22O'Donnel M.P. Chao C.C. Gekker G. et al.Renal cell cytokine production stimulates HIV-1 expression in chronically HIV-1 infected monocytes.Kidney Int. 1998; 53: 593-597Abstract Full Text PDF PubMed Scopus (18) Google Scholar, 23Kimmel P.L. Cohen D.J. Abraham A.A. et al.Upregulation of MCH class II, interferon-alpha and interferon-gamma receptor protein expression in HIV-associated nephropathy.Nephrol Dial Transplant. 2003; 18: 285-292Crossref PubMed Scopus (30) Google Scholar, 24Ross M.J. Fan C. Ross M.D. et al.HIV-1 infection initiates an inflammatory cascade in human renal tubular epithelia cells.J Acquir Immune Defic Syndr. 2006; 42: 1-11Crossref PubMed Scopus (63) Google Scholar In the present study, we found a reduced excretion of EGF in the urine of children with HIV-RD in association with increased urinary levels of FGF-2 and MMP-2, when compared with HIV-positive or -negative children without RD. Overall, six out of seven children of all ages with biopsy-proven HIVAN showed abnormal urinary values of all three candidate biomarkers in samples collected when their blood urea nitrogen and serum creatinine levels were still within the normal range. In contrast, none of the HIV-positive or -negative control children without RD or trace proteinuria showed abnormal urinary levels for all three candidate biomarkers. These findings suggest that the urinary profile of EGF, FGF-2, and MMP-2 may allow the identification of children at high risk of developing HIVAN. Both EGF and FGF-2 are powerful mitogenic growth factors for renal tubular epithelial cells.14Horikoshi S. Kubota S. Martin G.R. et al.Epidermal growth factor (EGF) expression in the congenital polycystic mouse kidney.Kidney Int. 1991; 39: 57-62Abstract Full Text PDF PubMed Scopus (57) Google Scholar, 15Comperat E. Ferlicot S. Camparo P. et al.Expression of epidermal growth factor receptor and proliferative activity of cysts epithelium in human renal cystic diseases.Urol Int. 2006; 76: 269-273Crossref PubMed Scopus (5) Google Scholar, 16Hayakawa M. Nakjima F. Tsuji A. et al.Cytokine levels in cystic renal masses associated with renal cell carcinoma.J Urol. 1998; 159: 1459-1464Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 17Rall L.B. Scott J. Bell G.I. Mouse prepro-epidermal growth factor synthesis by the kidney and other tissues.Nature. 1985; 313: 228-231Crossref PubMed Scopus (441) Google Scholar, 18Gupta G.K. Milner L. Linshaw M.A. et al.Urinary basic fibroblast growth: a noninvasive marker of progressive cystic renal disease in a child.Am J Med Genet. 2000; 93: 132-135Crossref PubMed Scopus (7) Google Scholar, 19Villanueva S. Cespedes C. Gonzalez A. et al.bFGF induces an earlier expression of nephrogenic proteins after ischemic acute renal failure.Am J Physiol Regul Integr Comp Physiol. 2006; 291: R1677-R1687Crossref PubMed Scopus (61) Google Scholar, 20Li Z. Jerebtsova M. Liu X-H. et al.Novel cystogenic role of basic fibroblast growth factor in developing rodent kidneys.Am J Physiol Renal Physiol. 2006; 291: F289-F296Crossref PubMed Scopus (18) Google Scholar They play important roles during renal development and in the pathogenesis of renal cystic disorders. EGF is predominately synthesized by renal tubular epithelial cells,14Horikoshi S. Kubota S. Martin G.R. et al.Epidermal growth factor (EGF) expression in the congenital polycystic mouse kidney.Kidney Int. 1991; 39: 57-62Abstract Full Text PDF PubMed Scopus (57) Google Scholar, 15Comperat E. Ferlicot S. Camparo P. et al.Expression of epidermal growth factor receptor and proliferative activity of cysts epithelium in human renal cystic diseases.Urol Int. 2006; 76: 269-273Crossref PubMed Scopus (5) Google Scholar, 16Hayakawa M. Nakjima F. Tsuji A. et al.Cytokine levels in cystic renal masses associated with renal cell carcinoma.J Urol. 1998; 159: 1459-1464Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 17Rall L.B. Scott J. Bell G.I. Mouse prepro-epidermal growth factor synthesis by the kidney and other tissues.Nature. 1985; 313: 228-231Crossref PubMed Scopus (441) Google Scholar and in the presence of renal tubular injury, its synthesis and release into the tubular lumen is expected to decrease. This notion is supported by the findings in HIV-Tg26 mice with RD, which showed a significant reduction in EGF mRNA expression in correlation with the presence of renal tubular injury and progression of the RD. A similar global reduction of mRNA and protein EGF expression was found by Horikoshi et al.14Horikoshi S. Kubota S. Martin G.R. et al.Epidermal growth factor (EGF) expression in the congenital polycystic mouse kidney.Kidney Int. 1991; 39: 57-62Abstract Full Text PDF PubMed Scopus (57) Google Scholar in mice with polycystic kidney disease (cpk). EGF is needed for the normal growth and regeneration of renal epithelial cells,14Horikoshi S. Kubota S. Martin G.R. et al.Epidermal growth factor (EGF) expression in the congenital polycystic mouse kidney.Kidney Int. 1991; 39: 57-62Abstract Full Text PDF PubMed Scopus (57) Google Scholar, 15Comperat E. Ferlicot S. Camparo P. et al.Expression of epidermal growth factor receptor and proliferative activity of cysts epithelium in human renal cystic diseases.Urol Int. 2006; 76: 269-273Crossref PubMed Scopus (5) Google Scholar, 16Hayakawa M. Nakjima F. Tsuji A. et al.Cytokine levels in cystic renal masses associated with renal cell carcinoma.J Urol. 1998; 159: 1459-1464Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 17Rall L.B. Scott J. Bell G.I. Mouse prepro-epidermal growth factor synthesis by the kidney and other tissues.Nature. 1985; 313: 228-231Crossref PubMed Scopus (441) Google Scholar and we have confirmed that EGF present in the urine of HIV-infected children can stimulate the growth of cultured human renal tubular epithelial cells. However, as antibodies against EGF were unable to suppress the complete mitogenic activity of the urine samples harvested from children with HIVAN, other growth factors present in these urine samples (that is, FGF-2) might play a synergistic role with EGF in this process.6Ray P.E. Liu X-H. Xu L. et al.Accumulation of bFGF in children with HIV-1 associated Hemolytic Uremic Syndrome.Pediatr Nephrol. 1999; 13: 586-593Crossref PubMed Scopus (50) Google Scholar, 7Liu X.H. Aigner A. Wellstein A. et al.Up-regulation of a fibroblast growth factor binding protein in children with renal disease.Kidney Int. 2001; 59: 1717-1728Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 8Ray P.E. Tassi E. Liu X.H. et al.Role of fibroblast growth factor-binding protein in the pathogenesis of HIV-associated hemolytic uremic syndrome.Am J Physiol Regul Integr Comp Physiol. 2001; 290: R105-R113Crossref Scopus (34) Google Scholar, 9Ray P.E. Bruggeman L. Weeks B. et al.Role of bFGF and its low affinity receptors in the pathogenesis of HIV-associated nephropathy in transgenic mice.Kidney Int. 1994; 46: 759-772Abstract Full Text PDF PubMed Scopus (91) Google Scholar In summary, taken in consideration all these findings, we propose that the progressive reduction in the urinary levels of EGF in HIV-infected children with RD reflect the presence of diffuse renal tubular injury characteristic of childhood HIVAN. In support of this notion, we have found that at least three markers of tubular injury, cystatin C, γ-glutamyl transpeptidase, and lysozyme, were elevated in the urine of patients with RD in correlation with the decreased EGF levels. Fibroblast growth factor-2 is a heparin-binding growth factor that lacks a classic signal peptide for secretion, but is released into the circulation by non-conventional pathways and injured endothelial cells.3Ray P.E. Xu L. Rakusan T. et al.A 20-year history of childhood HIV-associated nephropathy.Pediatr Nephrol. 2004; 19: 1075-1092Crossref PubMed Scopus (45) Google Scholar, 6Ray P.E. Liu X-H. Xu L. et al.Accumulation of bFGF in children with HIV-1 associated Hemolytic Uremic Syndrome.Pediatr Nephrol. 1999; 13: 586-593Crossref PubMed Scopus (50) Google Scholar, 7Liu X.H. Aigner A. Wellstein A. et al.Up-regulation of a fibroblast growth factor bindi