CJASN ePress. Published on January 13, 2015 as doi: 10.2215/CJN.03190314 Article

Chronic Dietary Exposure to Aristolochic Acid and Kidney Function in Native Farmers from a Croatian Endemic Area and Bosnian Immigrants

 || Bojan Jelakovic,* Ivana Vukovic Lela,* Sandra Karanovic,* Zivka Dika,* Jelena Kos,* Kathleen Dickman, Maja Sekoranja,ˇ ‡ Tamara Poljicanin,ˇ § Maja Misˇic, ¶¶ Vedran Premuzi c,* Mirta Abramovic, ¶ Vesna Matijevic,** | | Marica Miletic Medved,†† Ante Cvitkovic, †† Karen Edwards,‡‡ Mirjana Fucek,ˇ §§ Ninoslav Leko, Tomislav Teskera, || Mario Laganovic,* Dubravka Cvorisˇ ˇcec, §§ and Arthur P. Grollman Departments of *Nephrology, Hypertension, Abstract Dialysis and Background and objectives Improvements in agricultural practices in have reduced exposure to Transplantation and §§ consumption of aristolochic acid-contaminated flour and development of endemic (Balkan) nephropathy. Clinical Laboratory Therefore, it was hypothesized that Bosnian immigrants who settled in an endemic area in Croatia 15–30 years Diagnostics, School of Medicine, University ago would be at lower risk of developing endemic nephropathy because of reduced exposure to aristolochic of Zagreb, University acid. To test this hypothesis, past and present exposure to aristolochic acid, proximal tubule damage as a hall- Hospital Center mark of endemic nephropathy, and prevalence of CKD in Bosnian immigrants were analyzed. Zagreb, Zagreb, Croatia; ‡Faculty for Natural Sciences, Design, setting, participants, & measurements In this cross-sectional observational study from 2005 to 2010, University of Zagreb, 2161 farmers were divided into groups: indigenous inhabitants from endemic nephropathy and nonendemic Zagreb, Croatia; nephropathy villages and Bosnian immigrants; a-1 microglobulin-to-creatinine ratio .31.5 mg/g and §National Institute for , 2 Public Health, Zagreb, eGFR 60 ml/min per 1.73 m were considered to be abnormal. | Croatia; General fi Hospital, “Josip Results CKD and proximal tubule damage prevalence was signi cantly lower in Bosnian immigrants than Benceviˇ c”, Department inhabitants of endemic nephropathy villages (6.9% versus 16.6%; P,0.001; 1.3% versus 7.3%; P=0.003, respec- of Internal Medicine, tively); 20 years ago, Bosnian immigrants observed fewer clematitis in cultivated fields (41.9% versus Dialysis Unit, ¶¶ 67.8%) and fewer seeds among wheat seeds (6.1% versus 35.6%) and ate more purchased than homemade bread Cytology Unit, Slavonski Brod, Croatia; compared with Croatian farmers from endemic nephropathy villages (38.5% versus 14.8%, P,0.001). Both ¶ fi Outpatient Clinic Croatian farmers and Bosnian immigrants observe signi cantly fewer Aristolochia growing in their Bebrina, Bebrina, fields compared with 15–30 years ago. Prior aristolochic acid exposure was associated with proximal tubule Croatia; **Outpatient fi Clinic Slavonski, Kobasˇ, damage (odds ratio, 1.64; 95% con dence interval, 1.04 to 2.58; P=0.02), whereas present exposure was not †† (odds ratio, 1.31; 95% confidence interval, 0.75 to 2.30; P=0.33). Furthermore, immigrant status was an inde- Croatia; Institute for fi Public Health County pendent negative predictor of proximal tubule damage (odds ratio, 0.40; 95% con dence interval, 0.19 to 0.86; Brodsko-Posavska, P=0.02). Slavonski Brod, Croatia; ‡‡Department of Conclusions Bosnian immigrants and autochthonous Croats residing in endemic areas are exposed significantly Epidemiology, Genetic Epidemiology Research less to ingestion of aristolochic acid than in the past. The prevalence of endemic nephropathy and its associated Institute, School of urothelial cancers is predicted to decrease over time. Medicine, University of ccc–ccc California, Irvine, Irvine, Clin J Am Soc Nephrol 10: , 2015. doi: 10.2215/CJN.03190314 || CA; and Department of Pharmacological Sciences, State Introduction EN/UTUC ultimately was shown to be an environmental University of New York at Stony Brook, Stony Endemic (Balkan) nephropathy (EN) is a chronic form of aristolochic acid nephropathy (12,14). Brook, New York tubulointerstitial nephropathy associated with otherwise The consumption of Aristolochia herbs in the practice rare upper tract urothelial carcinoma (UTUC) reported of traditional Chinese medicine is a major cause of Correspondence: exclusively in rural areas along the larger tributaries of aristolochic acid nephropathy worldwide (15–17). In con- Dr. Ivana Vukovic the Danube river in Bosnia, , Croatia, , trast, EN involves long-term, low-dose ingestion of the Lela, School of and that affects genetically predisposed individ- toxin through home-baked bread prepared from flour Medicine, University – of Zagreb, Salata 3, uals exposed to aristolochic acid in the diet (1 9). EN has contaminated with seeds of Aristolochia clematitis (14,18,19). 10000 Zagreb, notbeenreportedinindividualsresiding,15 years in A study performed on Ukrainian immigrants (Ukraine Croatia. Email: an endemic area; therefore, it is believed that the intake has no patients with EN) who settled in Croatia in the ivemedex@yahoo. of an environmental toxin over this period of time is re- early 20th century provided the first solid evidence that com quired for the disease to develop. Although a number of lifestyle plays an important role in this environmental agents has been considered as potential causes (10–13), disease (7). Over time, the risks of developing EN among www.cjasn.org Vol 10 February, 2015 Copyright © 2015 by the American Society of Nephrology 1 2 Clinical Journal of the American Society of Nephrology

Ukrainian settlers in endemic areas became similar to those nine endemic and three nonendemic villages. The overall of native Croats, whereas those who settled in nonendemic participation rates in the endemic and nonendemic villages areas proved not to be at increased risk for EN (7). were 67.2% and 73.7%, respectively. In recent decades, improved harvesting and milling tech- Of 2822 enrolled farmers, 2161 were eligible for addi- nologies have essentially eliminated the contamination of tional analyses (Figure 1). Participants from EN villages wheat grain with Aristolochia seeds, thus decreasing exposure were classified using the consensus diagnostic criteria to aristolochic acid in home-baked bread and consequently, (23) into four groups: affected participants (EN diseased), reducing the prevalence of EN in many but not all endemic participants suspected of having EN, high-risk partici- areas (19–22). Recent emigration of residents from nonendemic pants for EN, and other participants (Figure 1, Table 1). areas of Bosnia, where presumably, they were not previously Because we wanted to test whether diminished exposure exposed to aristolochic acid, into the Croatian endemic area to aristolochic acid that took place a few decades ago re- after improvements in agricultural practices provided an op- flects in today’s unaltered kidney function, we were par- portunity to test the hypothesis that environmental exposure ticularly focused on a subgroup of BoENs that settled after to this toxin is reduced, which would be reflected in unaltered agricultural changes took place and lived in the EN area kidney function of immigrants. Here, we investigate the prev- for .15 years, which is considered a long enough period to alence of CKD and proximal tubule damage (PTD) in Croatian ingest cumulative toxic doses of aristolochic acid if expo- endemic areas, comparing the subgroup of Bosnian immi- sure was present. grants (BoENs) with long-term residents. Study interviewers recruited adult participants by going door to door in the target villages. Informed consent was obtained from each participant who completed an exten- Materials and Methods sive survey and provided a spot urine and fasting blood This cross-sectional observational study conducted in sample. All participants underwent ultrasound evaluation Croatia from 2005 to 2010 included 2822 inhabitants from during the field work. Renal ultrasound examination was

Figure 1. | A study flow diagram. Proximal tubule damage (PTD) defined as a-1 microglobulin level corrected for urine creatinine .31.5 mg/g. EN, endemic nephropathy. 1Specific gravity ,1.002 or .1.030. 2ESRD caused by other kidney diseases (one patient with nephrolithiasis, one patient with adult polycystic kidney disease, one patient with planocellular cancer, two patients with diabetic nephropathy, two patients with nephroangiosclerosis, one patient with rheumatoid arthritis, and two patients with chronic pyelonephritis). Clin J Am Soc Nephrol 10: ccc–ccc, February, 2015 Chronic Dietary Exposure to Aristolochic Acid, Jelakovic et al. 3

Table 1. Criteria for diagnosis of endemic nephropathy and classification of villagers from endemic areas

Affected Patients Suspected of Diagnostic Criteria At High Risk Others (Diseased) Having EN

(a) Residence in an endemic ++++++ 2 household.15 yr/positive family history (b) Tubular proteinuria + + + 22 2 2 (c) Anemia + 22+ 22 2 (d) Decreased eGFR + 22+ 22 2 (e) UTUC 2 + 22+ 22

Tubular proteinuria indicates a-1 microglobulin level corrected for urine creatinine .31.5 mg/g and a-1 microglobulin level corrected for urine albumine to urine creatinine ratio$0.91. Anemia indicates hemoglobin,12.0 g/dl for men and women ages .50 years old and ,11.0 g/dl for women ages ,50 years old. Decreased eGFR indicates eGFR,60 ml/min per 1.73 m2. Affected/diseased indicates a+b +c+d or a+b+c. Suspected of having endemic nephropathy (EN) indicates a+b, a+c+d, or a+e. At high risk indicates a. Others indicates other villagers without any relation to EN. UTUC, upper tract urothelial cancer (23). ACR indicates albumin-to-creatinine ratio.

done using a Siemens Sonoline SI 250 ultrasound machine wheat fields. After seeing photographs of the specificweeds, with a sector probe of 3.5 MHz by study personnel (i.e., participants also were asked to provide the traditional name three physicians: N.L, T.T., and M.L.) who were blinded to of the weed used in the region. Recognition of the weed was participants’ origins and clinical and laboratory data. considered positive if the traditional name of the weed All study personnel were trained to collect survey and matched the Latin binominal name. Participants also were clinical information in a standardized manner. Weight and asked to identify seeds from the plants. Aristolochic acid ex- height were measured, and body mass index (BMI) was posure was considered positive if the Aristolochia was calculated. BP was measured on consecutive visits; each time, observed in the farming fields and/or seeds of Aristolochia three measurements were obtained for the nondominant were among wheat seeds. arm with an Omron M6 device as per European Society of Fasting blood and urine samples were collected on the Hypertension/European Cardiology Society guidelines (24). day of interview and stored on dry ice for transfer to the Hypertension was defined as BP$140/90 mmHg and/or University Hospital Center Zagreb. Urinary a-1 microglobulin the use of antihypertensive drugs. Diabetes was defined as and albumin concentrations were determined using latex- fasting blood glucose .126 mg/dl and/or the use of antidi- enhanced immunonephelometry on a Behring Nephelometer abetic drugs. CKD was defined as an eGFR,60 ml/min per II (Dade Behring, Marburg, Germany). Hemoglobin was 1.73 m2; CKD stages were classified according to the recent determined with a Cell Dyn 1800 Hematology Analyzer Kidney Disease Improving Global Outcomes guidelines (25). (Abbott, Santa Clara, CA), and glucose was determined PTD was assessed according to a-1 microglobulin level cor- by ultraviolet photometry with hexokinase (AU 2700; rected for urine creatinine (a-1CR), and PTD was considered Olympus, Tokyo, Japan). Serum and urine creatinine concen- if a-1CR values were .31.5 mg/g. trations were measured by continuous photometry with alkyl A survey was used to collect demographic data along picrate (AU 2700; Olympus). Kidney function was assessed by with information regarding medical and family histories, estimation of the GFR using the CKD Epidemiology Collab- dietary practices, and environmental exposures, with an oration formula (26). emphasis on possible exposure to aristolochic acid. Specific This study was approved by the Ethical Boards of the questions were asked about wheat farming and grain-milling School of Medicine, University of Zagreb, the Croatian Na- practices, observations of A. clematitis plants, sources of wheat tional Institute of Public Health, and General Hospital and flour, and bread consumption to establish a history of Dr. Josip Benceviˇ c. aristolochic acid exposure. Importantly, traditional farming Statistical analyses were performed using STATISTICA and lifestyle practices began to change several decades ago, software, version 10. Variance homogeneity was tested by and many of these are no longer maintained. Thus, at the time Lindeman’s test before the analysis of correlation and between- of the survey, questions focused on both current and tradi- group differences. Normality of distribution was tested by tional practices 20–30 years ago. Visual cues were used to using the Kolmogorov–Smirnov test. None of the variables increase the sensitivity of recall about A. clematitis.Specifically, came from normally distributed populations. Differences 20325-cm cards were developed showing common weeds between groups for independent variables were analyzed growing in the region, including Ambrosia artemisiifolia, using the Kruskal–Wallis test. Differences in the prevalence Agropyron repens, A. clematitis, Brassica napus, Cirsium lanceolatum, of individual conditions were tested using the chi-squared Convolvulus arvensis, Echinochloa crus-galli, Equisetum arvense, test. Logistic regression models were used to evaluate the Galium aparine, Lolium spp., Mentha arvensis, Papaver rhoeas, association between prior and present aristolochic acid expo- Sorghum halepense,andSorghum saccharatumpers.Duringthe sure, residence in an EN village, positive household history survey interview, participants were shown photographs of for EN, and immigrant status with PTD (a-1CR.31.5 mg/g). local plants and asked to identify and name the plant and if For the multivariate logistic model, all indigenous residents they recalled seeing them grow in meadows, gardens, and and all immigrants were included in the analysis. Odds ratios 4 Clinical Journal of the American Society of Nephrology

and 95% confidence intervals were estimated for each of the area between 15 and 30 years ago, a period when agricul- independent variables with adjustment for variables that can tural practices affecting consumption of wheat grain had influencePTD(age,sex,BMI,historyofarterialhypertension, begun to change. Exposure to aristolochic acid among the and diabetes mellitus). Statistically significant differences were various groups was estimated using parameters similar to defined as a P value ,0.05 in all analyses. those in a related study (19). Twenty years ago, BoENs had fewer wheat fields than indigenous Croatian farmers Results (P,0.001), and their fields were less frequently flooded— Table 2 summarizes the demographic and clinical char- a factor related to the growth of Aristolochia (P,0.01) (Ta- acteristics of the study participants. Baseline characteristics ble 5). Immigrants and farmers from the nonendemic area were similar in terms of age, sex, BMI, BP, and prevalence observed fewer Aristolochia plants in their fields (P,0.001) of hypertension and diabetes. Immigrants resided in the as well as fewer Aristolochia seeds among wheat seeds endemic area for significantly shorter periods of time than (P,0.001) than farmers from endemic villages. Interestingly, other study participants (P,0.001). The prevalence of CKD there were no differences between BoENs and Croatian (percentage) was higher in endemic villages compared farmers from nonendemic areas with respect to the same with nonendemic villages and BoENs (16.6 versus 9.1 ver- observations. At the time, immigrants baked bread using sus 6.9; P,0.001). Distribution of CKD stages (Table 3) their own flourlessoftenthannativeCroatianfarmers shows that CKD stage $3a was significantly higher in (P=0.001). Presently, observations of Aristolochia plants in EN villages (P=0.001), whereas no difference between farming fields as well as Aristolochia seeds among wheat BoENs and residents of control nonendemic villages was seeds are comparable between the three groups (P.0.05 observed (P,0.99). for all), and participants from EN villages less frequently As shown in Table 4, values for urine albumine to urine observe Aristolochia plants in their farming fields and creatinine ratio, hemoglobin level, and urine specificgrav- Aristolochia seeds among wheat than they did in the past ity were comparable among all three subgroups (all with (67.8% versus 40.0%; 35.6% versus 8.3%; P,0.001 for both). P.0.05). eGFR was higher among BoENs compared with Farmers from all groups reported that the amount of bread the two indigenous groups (P=0.003), whereas the shortest ingested today and 20 years ago is comparable (P=0.30). kidneys were found in participants living in endemic vil- However, native Croatian farmers bake bread using home- lages (P,0.001). More farmers with biomarkers of kidney made flour less often than previously, choosing to purchase function above the cutoff for abnormal values resided in bread in stores (P=0.04). endemic villages compared with farmers in nonendemic History of prior aristolochic acid exposure (.30 years villages and BoENs. However, there were no differences ago), residence in an EN village, and family household between BoENs and autochthonous residents of nonen- history of EN were positively associated with PTD (Table demic villages. 6). On the contrary, present aristolochic acid exposure and According to the diagnostic criteria for EN (Table1), time period of exposure 30–15 years ago were not related among autochthonous EN villagers, there were 30 affected to PTD. Furthermore, immigrant status was identified to and 119 suspected participants, whereas 535 participants be an independent negative predictor of PTD, regardless were at high risk for EN (Figure 1). Importantly, EN was of time periods of exposure (odds ratio, 0.40; 95% confi- not diagnosed in immigrants who settled in the endemic dence interval, 0.19 to 0.86; P=0.02).

Table 2. Baseline clinical and biologic data of enrolled subjects

Endemic Villages Control Nonendemic Bosnian Immigrantsa Clinical and Biological Data P value (n=1687) Villages (n=372) (n=102)

Age, yr 50 (18–91) 53 (18–90)b 52 (20–86) 0.02 Sex (men), % 40.4 40.9 37.3 0.80 Living in village, yr 41 (24–55) 45 (26–60) 18 (15–21)b,c ,0.001 Family history of 37.2 1.1b 2.0b ,0.001 EN (positive), % Height, cm 168 (161–175) 167 (160–175) 167 (160–174) 0.40 Weight, kg 76 (66–88) 79 (68–89) 74 (64–87) 0.09 Body mass index, kg/m2 26.87 (23.71–30.74) 27.66 (24.30–31.25) 26.59 (23.81–29.91) 0.07 Systolic BP, mmHg 135 (120–154) 138 (123.5–159.8) 137 (126–156) 0.07 Diastolic BP, mmHg 81 (75–90) 81 (75–92) 82 (76–90) 0.77 Hypertension, % 41.2 44.6 43.8 0.48 Diabetes, % 6.1 9.2 7.9 0.09 CKD, % 16.6 9.1 6.9 ,0.001

aBosnian immigrants who settled in the EN area 15–30 years ago. Values are expressed as medians and interquartile ranges (25th–75th; minimum to maximum for age) or percentage if indicated. bSignificant difference (P,0.05) with endemic villages group. cSignificant difference (P,0.05) with control nonendemic villages group. Clin J Am Soc Nephrol 10: ccc–ccc, February, 2015 Chronic Dietary Exposure to Aristolochic Acid, Jelakovic et al. 5

Table 3. Frequency of CKD stages in endemic and nonendemic villages and Bosnian immigrants who settled EN villages 15–30 years ago

Endemic Villages Control Nonendemic Villages Bosnian Immigrants CKD Stage (%; n=1687) (%; n=372) (%; n=102)

1 1.3 1.9 1.0 2 4.2 5.9 4.9 3a 9.4 7.5 4.9 3b 4.6 1.3 1.0 4 1.4 0.0 1.0 5 1.2 0.3 0.0

Table 4. Markers of kidney function in autochthonous villagers and Bosnian immigrants who settled EN villages 15–30 years ago

Endemic Villages Control Nonendemic Bosnian Laboratory Parameter P value (n=1687) Villages (n=372) Immigrants (n=102)

a-1CR, mg/g 5.98 (3.75–10.53) 7.39 (4.74–12.47)a 7.04 (4.66–11.17) ,0.001 a-1CR.31.5 mg/g, N (%) 82 (7.3) 10 (0.3)a 1(1.3)a 0.003 ACR, mg/g 5.29 (3.51–10.68) 5.68 (3.87–10.61) 5.58 (3.95–9.64) 0.30 ACR.30 mg/g, N (%) 189 (15.8) 38 (11.4)a 7(6.7)a 0.05 Serum creatinine, mg/dl 1.09 (0.97–1.25) 1.05a (0.94–1.21) 0.98 (0.9–1.12)a,b ,0.001 eGFR, ml/min per 1.73 m2 80 (67–94) 81 (70–92) 88 (73–100)a,b 0.003 eGFR,60 ml/min 280 (16.6) 34 (9.1)a 7(6.9)a ,0.001 per 1.73 m2, N (%) Hemoglobin, g/dl 13.7 (12.9–14.7) 14 (13–14.8) 13.8 (13–14.7) 0.13 Urine specific gravity 1.015 (1.010–1.021) 1.015 (1.010–1.020) 1.016 (1.011–1.022) 0.22 Left kidney length, mm 109 (100–117) 113 (108–119)a 112 (103–120) ,0.001 Right kidney length, mm 109 (101–117) 114 (106–120)a 114 (102–118) ,0.001

Values are expressed as medians and interquartile ranges (25th–75th). a-1CR, a-1 microglobulin level corrected for urine creatinine; ACR, urine albumine to urine creatinine ratio. aSignificant difference (P,0.05) with endemic villages group. bSignificant difference (P,0.05) with control nonendemic villages group.

Discussion We failed to detect any participants with EN among The main finding of this survey is that the prevalence of BoENs. Additionally, the prevalence of CKD and PTD in CKD and tubular proteinuria in Bosnians who immigrated BoENs is the same as that observed in residents of non- to Croatian endemic areas is lower compared with native endemic villages. Diagnosis of UTUC was made in eight residents, whereas no differences were detected in these farmers from endemic villages, and all of them were parameters between BoENs and the nonendemic Croatian diagnosed and classified as patients with EN, whereas rural population. Because of changes in lifestyle and agricul- none of the suspected or high-risk participants for EN had tural practices, exposure to aristolochic acid in the diet is UTUC. Additionally, UTUC was not diagnosed in any reduced compared with several decades ago; thus, a gradual farmers from nonendemic villages or among BoENs. These decrease in the prevalence of EN is predicted. The prevalence data support the hypothesis that exposure of this popula- of CKD is higher in Croats residing in the endemic area tion to aristolochic acid is significantly diminished. Using compared with BoENs and Croats residing in control villages. logistic regression analyses, we observed that immigrant Furthermore, the high prevalence of CKD in endemic areas status is an independent protective variable for PTD. In (16.6%) is greater than that reported in the majority of other contrast, positive family/household history for EN, resi- geographic regions worldwide (27–35). Importantly, it is dence in an EN village, and past aristolochic acid exposure essential to follow such patients not only with the aim of were significantly positive predictors. Present exposure to postponing progression to ESRD but also, because as many aristolochic acid was not associated with PTD, confirming as 50% of patients with EN are likely to eventually develop our hypothesis. In the group of BoENs who were excluded UTUC (1,4,17,36–38). In nonendemic villages, 9.1% of vil- from this analysis, because they settled in the Croatian lagers had CKD stage $3, similar to estimates in other endemic area .30 years ago, two farmers were diagnosed countries (27,29,31–33,35). The higher prevalence of CKD as EN affected, and three farmers were diagnosed as EN in endemic areas is attributed to prior exposure to aristolochic suspected. This observation is in line with our hypothesis, acid (14). Presently, the prevalence of both affected and sus- because these farmers moved to the endemic area before pected patients is lower than previously reported, and a shift improvement in agricultural practices occurred. Indeed, to older ages is observed (1,22,39,40). theseindividualsshowedthesameriskfordeveloping lnclJunlo h mrcnSceyo Nephrology of Society American the of Journal Clinical 6

Table 5. Exposure to aristolochic acid on the basis of differences in farming practices and dietary habits presently and 15–30 years ago

Past (15–30 years ago) N (%) Present N (%)

Question/Answer Bosnian Bosnian EN Non-EN P value EN Non-EN P value Immigrant Immigrant

Did/do you have farming fields? Yes 886 (91.2) 190 (88.8) 46 (73)a,b ,0.001 1119 (81.1) 214 (77.3) 63 (78.8) 0.32 Did/do you grow wheat in your farming fields? Yes 838 (95.1) 180 (94.7) 36 (76.6)a,b ,0.001 789 (71) 156 (73.6) 29 (45.3)a,b ,0.001 Were/are farming fields flooded? Yes 388 (45.4) 97 (51.3) 10 (24.4)a,b ,0.01 277 (27.5) 74 (35.6)a 19 (34.5) 0.05 Did/do you observe Aristolochia plants in your fields? Yes 581 (67.8) 98 (51.9)a 18 (41.9)a ,0.001 418 (40) 78 (37.9) 26 (41.9) 0.79 Did/do you observe Aristolochia seeds among wheat seeds? Yes 285 (35.6) 25 (14.5)a 2 (6.1)a ,0.001 68 (8.3) 10 (6) 2 (4.7) 0.45 Did/do you bake bread? Yes 916 (92.6) 206 (95.4) 50 (74.6)a,b 0.001 553 (42.5) 105 (39) 43 (55.1)a,b 0.04 If yes, where did/do you get the flour? Grind wheat at home 114 (12.4) 16 (7.8) 9 (18) ,0.001 14 (2.6) 2 (2) 2 (4.8) 0.001 Miller 770 (84.1) 176 (86.3) 28 (56) 410 (77.4) 63 (62.4) 23 (54.8) Store 32 (3.5) 12 (5.9) 13 (26) 106 (20) 36 (35.6) 17 (40.5) Did/do you buy bread rather than bake? Yes 147 (14.8) 20 (9.3) 25 (38.5)a,b ,0.001 1191 (86.2) 231 (83.1) 62 (75.6)a,b 0.02 Did you eat more or less bread 20 years ago compared with today? More than now 628 (64.3) 140 (65.1) 36 (57.1) 0.30 Less than now 79 (8.1) 19 (8.8) 10 (5.9) About the same 269 (27.6) 56 (26) 17 (27) aSignificant difference (P,0.05) with endemic villages group. bSignificant difference (P,0.05) with control nonendemic villages group. Clin J Am Soc Nephrol 10: ccc–ccc, February, 2015 Chronic Dietary Exposure to Aristolochic Acid, Jelakovic et al. 7

Table 6. Independent predictors of proximal tubule damage

Independent Predictor OR (95% CI) P value

Living in EN village (yes) 3.97 (1.78 to 8.85) 0.001 Family/household history for EN (positive) 2.74 (1.77 to 4.24) ,0.001 Past aristolochic acid exposure (yes) 1.64 (1.04 to 2.58) 0.03 Time of exposure 30–15 yr ago (yes) 1.59 (0.46 to 5.42) 0.57 Present aristolochic acid exposure (yes) 1.31 (0.75 to 2.30) 0.33 Immigrant status (yes) 0.40 (0.19 to 0.86) 0.02

Proximal tubule damage defined as a-1 microglobulin level corrected for urine creatinine .31.5 mg/g. Covariates in the multivariate logistic regression models included age, sex, body mass index, hypertension, and diabetes. OR, odds ratio; 95% CI, 95% confidence interval.

EN as autochthonous Croats and the same risk as Ukrai- in other endemic areas. Recall bias could be questioned. nians who immigrated to this area previously. Thus, our However, we did not ask participants to recognize only the results are consistent with those of an earlier study inves- A. clematitis but also, several other common weeds as well. tigating EN in Ukrainian immigrants (7). Theissueofparticipationbiascouldbealsoraised.Neverthe- According to a report from 1985, the prevalence of less, we recruited participants on the door-to-door basis, and patients with EN and those suspected of having EN among participation rate was good and without differences among Ukrainians who settled in endemic villages was the same as groups. Also, we determined a-1CR and urine albumine to the prevalence in autochthonous Croatian villagers (10.5% urine creatinine ratio in a single spot urine sample. However, versus 12.1%; P.0.05), whereas EN was not reported in recent studies suggest that similar measurements of a-1CR Ukrainians who settled in nonendemic villages (7). In our are a reliable marker for EN (23,25,39–45) and other chronic survey, we observed a lower prevalence of EN in autoch- tubulointerstitial diseases, such as cadmium nephropathy thonous Croats than Ceovic et al. (7) found 40 years ago (46–49). Most authors believe that a-1 microglobulin has ad- (1.8% versus 12.1%). Both Ukrainian immigrants and vantages over b-2 microglobulin. Stefanovic et al. (43) found BoENs settled in the same endemic area; therefore, the that both a-1 and b-2 microglobulin are significant predictors two studies seem comparable. In the past, the presence for differentiation of EN from healthy participants and other of aristolochic acid in the environment coupled with agri- kidney diseases. Interestingly, analyses odds ratios and P val- cultural practices in the endemic area led to similar risk ues were much more in favor of a-1 than b-2 microglobulin. factors for EN among Ukrainian immigrants and autoch- Other works evaluated diagnostic criteria for the diagnosis of thonous farmers. Presently, advances in agriculture prac- EN and concluded that a-1 microglobulinuria significantly tices contribute to decreased contamination of flour with discriminated EN from other kidney diseases (50,51). Finally, aristolochic acid and therefore, decreased risks of expo- in our cohort with .2000 enrolled farmers, we obtained the sure. This conclusion is confirmed with our observations same results (42). Accordingly, a-1CR was included in the of BoENs who settled in this area after those changes oc- latest consensus EN document prepared by leading experts in curred. In the last three decades, farmers began using the field (23). CKD was diagnosed using a single measure- modern combines, which improved the separation of ment of eGFR; however, in most epidemiologic studies of this weed seeds; importantly, wheat grain now is processed kind, only a single measurement is obtained. in large common mills. Thus, despite the continued pres- In conclusion, our results are fully consistent with the ence of A. clematitis in farming fields, the risk of contam- hypothesis that chronic dietary exposure to aristolochic acid is ination of wheat grain with aristolochic acid is unlikely to the cause of EN (9,12,14,18,19). We also confirm the relation- occur. Furthermore, villagers rarely prepare their own ship between reduced exposure to aristolochic acid in endemic flour or bake their own bread as in the past, because the areas and less PTD. Additionally, we confirm that present majority of farmers now purchase bread in bakeries or exposure to aristolochic acid is reduced. Although we predict stores. Results from this survey conducted in a general additional decreases in the prevalence of EN, the prevalence of rural population are in concordance with data obtained CKD remains high in endemic areas, underscoring the need from patients with EN undergoing dialysis or surgery for screening and strict follow-up of suspected patients. for aristolochic acid–induced UTUC (12,14,19). Finally, our research highlights the important role of en- This study has several important strengths. It is the first vironment and lifestyle on renal disease. It is also an ex- report on the overall prevalence of CKD in an endemic area ample of how human behavior, knowledge, and advances and the largest epidemiologic study to evaluate early renal in technology can positively influence the natural course damage specific to EN. Additionally, we report that exposure of an environmental disease. of residents of the endemic area to aristolochic acid has significantly decreased in the last two to three decades. Acknowledgments Our study also has certain limitations inherent to a cross- This research was supported by the Ministry of Science of the sectional observational study design. The survey was con- Republic of Croatia (108-0000000-0329), Croatian Foundation for Sci- ducted in a single endemic focus. Thus, although our results ence Grant 04/38, and National Institute of Environmental Health are statistically significant, similar studies should be performed Sciences Grant ES-04068. 8 Clinical Journal of the American Society of Nephrology

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