Antiviral Therapy 13:

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1 Antiviral Therapy 13: Short communication Cystatin C as a marker of renal function is affected by HIV replication leading to an underestimation of kidney function in HIV patients Stefan Mauss 1 *, Florian Berger 1, Dieter Kuschak 2, Julia Henke 1, Petra Hegener 1, Eva Wolf 3, Stephan Schauseil 2 and Guenther Schmutz 1 1 Center for HIV and Hepatogastroenterology, Duesseldorf, Germany 2 Medical Laboratories Duesseldorf, Duesseldorf, Germany 3 MUC-Research, Munich, Germany *Corresponding author: stefan.mauss@center-duesseldorf.de Background: Broad use of tenofovir and an ageing HIVinfected population have created an interest in renal function in HIV patients. Serum cystatin C is a newer marker of renal function and might be more sensitive than creatinine. Methods: Patients were enrolled consecutively in an observational study. HIV-seropositive patients naive to antiretroviral therapy (n=261) were compared with healthy volunteers undergoing check-up procedures (n=193). Estimated glomerular filtration rate (egfr) was derived using creatinine-based Modification of Diet in Renal Disease (MDRD) and Cockcroft Gault formulas or cystatin C-based calculations. HIV-seropositive patients starting antiretroviral therapy (n=92) were followed prospectively after enrolment. Results: MDRD showed a higher median egfr in antiretroviral- naive HIV-seropositive patients compared with controls (104 versus 93 ml/min; P<0.001). Cockcroft Gault gave similar results (118 versus 106 ml/min; P<0.001). By contrast, cystatin C levels in HIV-seropositive individuals were higher, resulting in a lower median egfr compared with controls (99 versus 120 ml/min; P<0.001). Cystatin C was positively correlated with HIV RNA (r=0.33, P<0.01) and inversely correlated with CD4 + T-cell count (r=-0.29, P<0.01). Initiating antiretroviral therapy (n=92) decreased cystatin C levels and led to an increased cystatin C-based egfr from median 84 to 103 ml/min at week 24 (P<0.001). Serum creatinine was not substantially altered. Conclusions: Correlation of cystatin C with HIV RNA and CD4 + T cell count, plus decrease of cystatin C after suppression of HIV replication, suggest an increase of cystatin C levels by active HIV infection. This might result in overestimation of renal impairment, particularly in treatment-naive patients. Therefore, use of cystatin C to calculate GFR in HIV-seropositive individuals should not be recommended without further validation. Introduction There is an increasing need to assess renal function because of ageing among the HIV-infected population and the presence of potentially nephrotoxic antiretrovirals [1]. Formulas that provide estimated glomerular filtration rate (egfr) based on serum creatinine are well established, but might underestimate mild to moderate impairment of renal function [2]. In addition, creatinine-based formulas have not been specifically validated in HIV-seropositive populations. Recently, serum cystatin C has been reported to be a more sensitive marker of modest renal impairment [3,4]. Cystatin C is a non-glycosylated, low molecular weight (13 kda), basic protein produced by most nucleated cells. Cystatin C is freely filtered at the glomerulus and its production rate seems to be remarkably constant; therefore, its serum concentration has been proposed as a marker of GFR [5 8]. However, the use of cystatin C has not been systematically validated in HIV-infected individuals. Methods HIV-seropositive, treatment-naive, Caucasian patients were consecutively enrolled in a single centre. Healthy Caucasian individuals undergoing routine check-up procedures served as controls. The first cross-sectional 2008 International Medical Press

2 S Mauss et al. This pdf is for personal use only. To obtain commercial reprints, Table 1. Baseline data of HIV-seropositive antiretroviral therapy-naive patients and healthy controls Variable HIV-seropositive Control P-value Total, n Male gender, n (%) 226 (86) 90 (47) <0.001 BMI, kg/m ( ) 23.9 ( ) Age, years 37 (20 70) 46 (18 85) <0.001 Creatinine, mg/dl 0.85 ( ) 0.83 ( ) Cystatin C, mg/l 0.81 ( ) 0.70 ( ) <0.001 egfr MDRD, ml/min 104 (63 196) 93 (56 161) <0.001 egfr CG, ml/min 118 (61 330) 106 (52 211) <0.001 egfr cystatin, ml/min 99 (39 194) 120 (43 194) <0.001 Data shown are median (range) unless otherwise indicated. The Mann Whitney U test was used. BMI, body mass index; CG, Cockcroft Gault; egfr, estimated glomerular filtration rate; MDRD, Modification of Diet in Renal Disease. data set from HIV-seropositive patients and healthy controls was used for a comparison of cystatin C, creatinine levels and their respective egfrs. In the second data set, all patients starting antiretroviral therapy from April 2004 to August 2007 with a follow-up 24 weeks were longitudinally evaluated for the effect of antiretroviral therapy on egfr (n=92). Serum creatinine and cystatin C were analysed as part of the clinical routine from the same blood sample. Creatinine was measured on a Roche Modular P model (Roche, Mannheim, Germany) using a kinetic Jaffe test based on the Jaffe reaction, as modified by Bartels et al. [9]. Cystatin C was determined by particle-enhanced nephelometric immunoassays on a ProSpec machine (Dade Behring, Siemens Medical Solutions, Eschborn, Germany). A range of mg/l was covered with a detection limit for N latex cystatin C of 0.05 mg/l [10]. The dimension of egfr is given in ml/min with the understanding that these values are normalized to an average body surface area of 1.73 m 2. Formulas used for egfr by serum creatinine included the abbreviated Modification of Diet in Renal Disease (MDRD) formula (egfr MDRD [ml/min]=186 serum creatinine [mg/dl] exp age [years] exp ; if female) [11,12] and the Cockcroft Gault equation (egfr Cockcroft Gault [ml/min]=140- age [years] weight [kg]/72 serum creatinine [mg/dl]; 0.85 if female) [13]. The serum cystatin C-based equation was egfr cystatin C [ml/min]=74.835/(cystatin C [mg/l] exp 1.333) for the commercially available N latex cystatin C test used. For statistical analyses Mann Whitney U, Wilcoxon signed-rank, Spearman s rank and Friedman tests were used (SPSS GmbH version 15.0; SPSS, Munich, Germany). In logistic regression analyses, sex, age, HIV status and body mass index (BMI) were used as independent variables. Cystatin C, creatinine and their respective egfrs were outcome variables. The effect of each single variable was assessed after adjusting for the other three variables (StatView version 5.01; SAS Institute Inc., Cary, NC, USA). Results In total, 261 HIV-seroposititive treatment-naive individuals and 193 healthy controls were consecutively enrolled. Controls were older, had a higher BMI and were mostly female (Table 1). Compared with controls, the distribution of serum cystatin C levels showed a shift towards higher levels in HIV-seropositive patients compared with healthy controls (P<0.001; Table 1). In univariate analyses, both the MDRD and Cockcroft Gault formulas showed a higher median egfr in HIV-seropositive individuals compared with controls. By contrast, egfr based on cystatin C showed a significantly lower median egfr in HIV-seropositive individuals compared with controls (Table 1). In logistic regression analyses, sex, age, BMI and HIV status were entered as independent variables. After adjustment for the other three variables, the regression model showed a trend towards an association between HIV seropositivity with lower serum creatinine (odds ratio [OR] 0.56, 95% confidence interval [CI] , P=0.07) and a higher likelihood of a normal egfr as calculated by MDRD (OR 1.71, 95% CI , P=0.09) or Cockcroft Gault (OR 1.83, 95% CI , P=0.06). For cystatin C, the same logistic regression model showed that being HIV-seropositive (OR 3.66, 95% CI , P<0.0001) and being older (OR 2.26, 95% CI , P<0.0001) were significantly associated with higher cystatin C serum levels. As a result, being HIV-seropositive (OR 0.28, 95% CI , P<0.0001) or being older (OR 0.30, 95% CI , P<0.0001) were associated with an increased risk of a reduced egfr based on cystatin C International Medical Press

3 Cystatin C, renal function and HIV Cystatin C serum levels showed a positive correlation with higher HIV RNA (r=0.33, P<0.01) and an inverse correlation with CD4 + T-cell count (r=-0.29, P<0.01; Figure 1). In patients starting antiretroviral therapy (n=92), creatinine at baseline increased slightly from 0.85 to 0.89 mg/ dl at week 48 (P<0.001; Table 2). egfr calculated by MDRD decreased from 104 to 96 ml/min (P<0.001). Using Cockcroft Gault, egfr showed similar results (Table 2). In the same patients, baseline cystatin C serum levels decreased from median 0.92 to 0.77 mg/l at week 48 (P<0.001). Cystatin C-based egfr increased from 84 to 106 ml/min (P<0.001; Table 2). According to the MDRD formula, 23% of HIVseropositive individuals were classified as having renal insufficiency stage 2 (egfr ml/min) and none were in stage 3 (egfr ml/min), stage 4 (egfr ml/min) or stage 5 (egfr<14 ml/min) [14]. Among controls, stage 2 was observed in 45% of patients, stage 3 in 1% and stages 4 or 5 in none. A different pattern was observed using cystatin C, such that 36% of HIV-seropositive patients showed renal insufficiency stage 2, 5% showed stage 3 and none showed stages 4 or 5. In controls, stage 2 was observed in 10% of patients, stage 3 in 1% and stages 4 or 5 in none. The use of tenofovir had no effect on these findings. With MDRD, egfr decreased by 5 ml/min (range ml/min) in patients on tenofovir and by 3 ml/min (range ) in patients not on tenofovir (P=0.23). Using the Cockcroft Gault equation, egfr decreased by 5 ml/min (range ) in patients on tenofovir and by 3 ml/min (range ) in patients not on tenofovir (P=0.27). Patients with tenofovir (n=77) showed a median increase of cystatin C-based egfr of 15 ml/ min (range ) after 24 weeks of therapy compared with an increase of 23 ml/min (range ) in patients not treated with tenofovir (n=15, P=0.24). Discussion Among HIV-seropositive patients in this cross- sectional analysis, renal function assessed by formulas based on creatinine was markedly better than that estimated by cystatin C. This has been previously reported by four different papers, including one from our own group [4,15 17]. In all four publications, the possibility of underestimating renal impairment by using creatinine-based formulas compared with cystatin C was discussed. In the paper by Jaroszewicz et al. [16], cystatin C serum levels correlated positively with the level of HIV RNA. Odden et al. [4] reported an inverse correlation with the number of CD4 + T-cells [4]. In the present paper, we can confirm both observations. However, Figure 1. Association of cystatin C with HIV RNA or CD4 + T-cell count A Cystatin C, mg/l B Cystatin C, mg/l Spearman s ρ=0.329, P<0.01, n= HIV RNA, log 10 copies/ml Spearman s ρ=-0.287, P<0.01, n= ,200 1,400 CD4 + T-cell count, cells/µl Correlation between (A) serum cystatin C and HIV RNA and (B) serum cystatin C and CD4 + T-cell count in 261 HIV-seropositive patients naive to antiretroviral therapy. because of the cross-sectional nature of the previous studies the effect of HIV replication on cystatin C serum levels could not be separated from the effect of HIV and antiretroviral therapy on renal function. For this study, we included a longitudinal arm assessing the effect of antiretroviral therapy in previously untreated individuals on markers of renal function. Initiating antiretroviral therapy decreased serum cystatin C levels substantially, whereas serum creatinine levels were slightly increased. From our perspective, this suggests an interaction between HIV infection and cystatin C serum levels. Cystatin C is a cysteine protease inhibitor, for which serum levels have been shown to be altered by immunomodulating drugs such as prednisolone or cyclosporine A [18]. In addition, cystatin C level correlates with inflammation markers, such as C-reactive protein [19,20]. The positive correlation of cystatin C levels with HIV RNA and the inverse correlation with CD4 + T-cell count do suggest an increase of cystatin C Antiviral Therapy

4 S Mauss et al. This pdf is for personal use only. To obtain commercial reprints, Table 2. Effect of initiating antiretroviral therapy on serum creatinine, cystatin C and the respective calculated glomerular filtration rate Variable Baseline Week 12 Week 24 Week 48 Total, n Creatinine, mg/dl 0.85 ( ) 0.89 ( )* 0.89 ( )* 0.89 ( )* Cystatin C, mg/l 0.92 ( ) 0.84 ( )* 0.79 ( )* 0.77 ( )* egfr MDRD, ml/min 104 (68 180) 97 (69 149)* 99 (54 152)* 96 (64 135)* egfr CG, ml/min 117 (63 181) 110 (56 201)* 106 (63 183)* 108 (55 175)* egfr cystatin C, ml/min 84 (39 194) 96 (45 155)* 103 (52 179)* 106 (56 246)* HIV RNA<40 copies/ml, n (%) 0 (0) 52 (46) 76 (83) 70 (90) CD4 + T-cells, cells/µl 255 (17 833) 418 (81 1,445) 457 (93 1,445) 494 (161 1,406) Data shown are median (range) unless otherwise indicated. *P< CG, Cockcroft Gault; egfr, estimated glomerular filtration rate; MDRD, Modification of Diet in Renal Disease. by replicative HIV infection. This is further indicated by the decrease of cystatin C after suppression of HIV replication under antiretroviral therapy. Excluding or including tenofovir in the analysis did not change the findings. The data on the effect of tenofovir on renal function are conflicting, mostly showing little to modest changes of egfr, probably because of impairment of tubular function [21 23]. In conclusion, using cystatin C-based calculations of renal function might result in an overestimation of renal impairment, particularly in therapy-naive HIVseropositive patients. In patients with fully suppressed HIV replication, cystatin C levels are lower and could more accurately reflect renal function. This observation on cystatin C strongly suggests that new diagnostic markers, in particular markers associated with the immune system, have to be specifically validated for the use in HIV-seropositive patients. Transferring the use of those markers from studies in the general population without any further validation might lead to biased results. Disclosure statement The authors declare no competing interests. References 1. Mocroft A, Kirk O, Gatell J, et al. Chronic renal failure among HIV-1-infected patients. AIDS 2007; 21: Perrone RD, Madias NE, Levey AS. Serum creatinine as an index of renal function: new insights into old concepts. Clin Chem 1992; 38: Dharnidharka VR, Kwon C, Stevens G. Serum cystatin C is superior to serum creatinine as a marker of kidney function: a meta-analysis. Am J Kidney Dis 2002; 40: Odden MC, Scherzer R, Bacchetti P, et al. Cystatin C level as a marker of kidney function in human immunodeficiency virus infection. Arch Intern Med 2007; 167: Galteau MM, Guyon M, Gueguen R, Siest G. Determination of serum cystatin C: biological variation and reference values. Clin Chem Lab Med 2001; 39: Grubb A, Nyman U, Björk J, et al. 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A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Ann Intern Med 1999; 130: Levey AS, Greene T, Kusek JW, Beck GJ. A simplified equation to predict glomerular filtration rate from serum creatinine. J Am Soc Nephrol. 2000; 11:Abstract A Cockcroft DW, Gault MH. Prediction of creatinine clearance from serum creatinine. Nephron 1976; 16: Levey AS, Coresh J, Balk E, et al. National Kidney Foundation practice guidelines for chronic kidney disease: evaluation, classification, and stratification. Ann Intern Med 2003; 139: Mauss S, Berger F, Schmutz G. Antiretroviral therapy with tenofovir is associated with mild renal dysfunction. AIDS 2005; 19: Jaroszewicz J, Wiercinska-Drapalo A, Lapinski TW, Prokopowicz D, Rogalska M, Parfieniuk A. Does HAART improve renal function? An association between serum cystatin C concentration, HIV viral load and HAART duration. Antivir Ther 2006; 11: Jones CY, Jones CA, Wilson IB, et al. 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5 Cystatin C, renal function and HIV 20. Samouilidou EC, Grapsa E. Relationship of serum cystatin C with C-reactive protein and apolipoprotein A1 in patients on hemodialysis. Ren Fail 2008; 30: Fux CA, Simcock M, Wolbers M, et al. Tenofovir use is associated with a reduction in calculated glomerular filtration rates in the Swiss HIV Cohort Study. Antivir Ther 2007; 12: Cassetti I, Madruga JV, Suleiman JM, et al. The safety and efficacy of tenofovir DF in combination with lamivudine and efavirenz through 6 years in antiretroviral-naive HIV-1- infected patients. HIV Clin Trials 2007; 8: Nelson MR, Katlama C, Montaner JS, et al. The safety of tenofovir disoproxil fumarate for the treatment of HIV infection in adults: the first 4 years. AIDS 2007; 21: Accepted for publication 14 September 2008 Antiviral Therapy