**2.1 Study population, baseline cohort**

Between 1974 and 1986 all women, born between 1911 and 1945, who lived in the city of Utrecht and surroundings, the Netherlands, were invited for a breast-cancer-screening program, with a participation rate of 68 to 72%. A total number of 38,994 women, aged 39 to 68 years old at intake, participated (the baseline cohort). Baseline measurements, performed between 1974 and 1986, included extensive questionnaires, a short medical examination, and the collection of a midstream morning urine sample. Data obtained through the questionnaires included age, marital status, smoking habits, parity, menopausal age, diet and drug use. During the medical examination weight and height were measured. Approximately 200 ml urine was stored in plastic polypropylene jars, without preserving agents, and stored at –20°C for future analyses. All women gave oral consent to use their data and urine samples for future scientific research.

#### **2.2 Study population, follow-up cohort**

From 1993 to 1997, 50,313 women living in Utrecht and surroundings who were scheduled for breast cancer screening during this period received an invitation by mail to join an additional study to assess the relation between nutrition and cancer and other chronic diseases, the Prospect-EPIC study (the follow-up cohort). A total of 17,357 women (participation rate 34.5%) agreed to take part . Participants were between 49 and 70 years old at enrolment. Information was collected on the basis of two self-administered questionnaires and a medical examination including blood pressure. Non-fasting blood samples were successfully drawn from 97.5% of the women, and stored under liquid nitrogen at –196°C. Approximately 88% of the women signed a detailed informed consent, enabling the researchers to use their blood samples for future analysis, and to obtain information on future morbidity and mortality.

To address the relation between *E. coli* bacteriuria and renal function development, we performed a full cohort analysis for women who participated in both the baseline cohort and the follow-up cohort. *E. coli* bacteriuria was diagnosed by a real-time Polymerase Chain Reaction in this urine sample. Participants were between 49 and 70 years old at enrolment. The mean duration of follow-up was 11.5 ± 1.7 years, ranging from 8.1 to 18.6 years from baseline until participation in the follow-up study. Forty-eight of 490 women (10%) were classified with *E. coli* bacteriuria at baseline. At study endpoint, the mean creatinine clearance for women with baseline bacteriuria was 87 ± 21 ml and without baseline bacteriuria 85 ± 18 ml per minute, respectively (Figure 1). *E. coli* bacteriuria at baseline was not associated with creatinine levels at follow-up, adjusted for age and weight and the

membrane) can cause scarring in the renal parenchyma of rats, with large foci of inflammation. This might be due to the activation of polymorphonuclear leukocytes by type 1 fimbriatedstrains, which leads to the release of tissue destroying enzymes. Mice models have shown that although neutrophils are important in bacterial clearance, they can also cause renal damage. In a clinical study, renal scarring was detected in 29 of 63 adult women ten to twenty years after hospitalization for pyelonephritis. In contrast, no study has convincingly shown that ASB can lead to a clinically relevant decline in renal function in otherwise healthy women. Several authors in the first half of the twentieth century have suggested a role of bacteriuria

Between 1974 and 1986 all women, born between 1911 and 1945, who lived in the city of Utrecht and surroundings, the Netherlands, were invited for a breast-cancer-screening program, with a participation rate of 68 to 72%. A total number of 38,994 women, aged 39 to 68 years old at intake, participated (the baseline cohort). Baseline measurements, performed between 1974 and 1986, included extensive questionnaires, a short medical examination, and the collection of a midstream morning urine sample. Data obtained through the questionnaires included age, marital status, smoking habits, parity, menopausal age, diet and drug use. During the medical examination weight and height were measured. Approximately 200 ml urine was stored in plastic polypropylene jars, without preserving agents, and stored at –20°C for future analyses. All women gave oral consent to use their

From 1993 to 1997, 50,313 women living in Utrecht and surroundings who were scheduled for breast cancer screening during this period received an invitation by mail to join an additional study to assess the relation between nutrition and cancer and other chronic diseases, the Prospect-EPIC study (the follow-up cohort). A total of 17,357 women (participation rate 34.5%) agreed to take part . Participants were between 49 and 70 years old at enrolment. Information was collected on the basis of two self-administered questionnaires and a medical examination including blood pressure. Non-fasting blood samples were successfully drawn from 97.5% of the women, and stored under liquid nitrogen at –196°C. Approximately 88% of the women signed a detailed informed consent, enabling the researchers to use their blood samples for

To address the relation between *E. coli* bacteriuria and renal function development, we performed a full cohort analysis for women who participated in both the baseline cohort and the follow-up cohort. *E. coli* bacteriuria was diagnosed by a real-time Polymerase Chain Reaction in this urine sample. Participants were between 49 and 70 years old at enrolment. The mean duration of follow-up was 11.5 ± 1.7 years, ranging from 8.1 to 18.6 years from baseline until participation in the follow-up study. Forty-eight of 490 women (10%) were classified with *E. coli* bacteriuria at baseline. At study endpoint, the mean creatinine clearance for women with baseline bacteriuria was 87 ± 21 ml and without baseline bacteriuria 85 ± 18 ml per minute, respectively (Figure 1). *E. coli* bacteriuria at baseline was not associated with creatinine levels at follow-up, adjusted for age and weight and the

future analysis, and to obtain information on future morbidity and mortality.

in the etiology of hypertension, but the pathogenesis is not understood.

**2. ASB and renal function decline in healthy women** 

data and urine samples for future scientific research.

**2.2 Study population, follow-up cohort** 

**2.1 Study population, baseline cohort** 

distribution in stages of renal function was not different for women with bacteriuria compared to women without bacteriuria.

Fig. 1. Differences in creatinine clearance between women WITHOUT DM with and without ASB. (Meiland R, Stolk RP, Geerlings SE, Peeters PH, Grobbee DE, Coenjaerts FE, Brouwer EC, Hoepelman AI. Association between *Escherichia coli* bacteriuria and renal function in women: long-term follow-up. Arch Intern Med. 2007 Feb 12;167(3):253-7.)

#### **2.3 Nested case-control study population**

To obtain follow-up information on end-stage renal failure, we obtained data from the Renal Replacement Registry Netherlands (RENINE) that were available May 2002. RENINE is a foundation in which all Dutch nephrologists participate and where patients are registered who at one time have used kidney replacing therapy (hemodialysis or renal transplantation), with a coverage rate throughout the years of nearly 100%. Data from the baseline cohort and RENINE were matched on (maiden and married) name combined with date of birth to select the cases. A group consisting of four times the number of cases was randomly selected from the baseline cohort to form the control group. Four women participated in the follow-up cohort and were also selected as one of the cases who received kidney replacing therapy during follow-up; one woman underwent kidney transplantation before blood withdrawal (and was excluded for the cohort analysis), three women developed end-stage renal failure thereafter (and were included in both analyses). After excluding four individuals with a missing urine sample 49 cases and 206 controls were included. Among the cases, the mean duration until the date of kidney replacing therapy was 13.8 ± 7.4 years, with a minimum and maximum duration of 1.6 and 25.5 years, respectively. In the control group, the mean follow-up (i.e. the time from participation in the baseline cohort until study-endpoint in May 2002) was 27.0 ± 0.2 years.

Asymptomatic Bacteriuria (ASB), Renal Function and Hypertension 381

Although more recent studies also found a correlation, only one prospective study has shown that bacteriuria is associated with the development of hypertension. In the above mentioned cohort study, a higher prevalence of hypertension in the bacteriuric group after 12 years of follow-up was found. However, the underlying mechanism of this finding is not clear. Hypertension is a lasting increase in blood pressure with a heterogeneous etiology consisting of both genetic and environmental factors. Patients share the inability to excrete sodium at a normal arterial pressure. If bacteriuria would lead to hypertension, the most attractive explanation would be that hypertension arises secondary to renal scarring caused by the (type 1 fimbriae of the) uropathogens. In the multivariate analysis, correction for creatinine did not change the results, but hypertension can occur before the reduction in creatinine clearance becomes apparent. An alternative explanation is that both bacteriuria and hypertension are found more frequently among individuals with comorbidity or that they share a same (currently unknown) cause. This is supported by the higher prevalence of bacteriuria among women who used antihypertensive drugs at baseline. Given the importance of hypertension

the nature of this correlation needs to be studied in future studies.

**4. ASB and renal function decline and hypertension in patients with DM** 

function, we also studied the influence of ASB on the development of hypertension.

The association between ASB and renal function decline (and hypertension) in patients with DM was investigated in a prospective study with women with DM type 1 (n=296) and type 2 (n=348). All patients were interviewed and their medical records were reviewed at baseline and at study closure to collect all relevant information. All patients were asked to provide 1 or 2 midstream urine specimens. The women were followed up for a mean (SD) duration of 6.1 (1.9) years. Women with DM type 1 were younger, but had a longer duration of DM, than women with DM type 2. At baseline, 201 women with DM type 2 (58%) were treated with insulin only, 97 (28%) with oral hypoglycemic medication only, 41 (12%) with a combination of both, and five women (2%) were on a diet only (data were incomplete for 4 women). Because the Cockcroft-Gault formula for the estimation of the creatinine clearance includes age, adjusting for age in a multivariate model is not possible. Therefore patients were stratified into 3 age strata to assess the impact of age on the association between ASB and the (relative increase in the) creatinine clearance (respectively 18 to 36, 37 to 55, and 56 to 75 years old). All analyses were performed on the entire study population and on women with DM type 1 and DM type 2 separately.The prevalence of ASB was 17% in the study population, lower in

Women with DM have an increased prevalence of ASB, but also an increased risk on symptomatic UTI's and developing complications of UTI's such as renal abscesses. It was also shown that at short term follow-up treatment of ASB in women with DM did not appear to reduce complications. *E. coli* is the leading uropathogen in non-diabetic as well as in diabetic patients. Ninety percent of *E. coli* possesses type 1 fimbriae, the adhesive organelles found at the outer bacterial membrane. We have shown in vitro that type 1 fimbriated *E. coli* have an increased adherence to uroepithelial cells voided by women with DM. Others demonstrated that UTI's with type 1-fimbriated *E. coli* can lead to scar formation in the renal parenchyma of infected rats.At present, conclusive and prospective data with a long follow-up period directly relating ASB (with *E. coli*) to long-term risk of renal failure in diabetic patients are lacking. Taken together, we hypothesized that ASB in women with DM could lead to a faster decline in renal function, and decided to enlarge our cohort of diabetic women and to prolong the follow-up period. Besides the effects on renal

No difference in duration until kidney replacing therapy was found between bacteriuric and non-bacteriuric individuals (14.6 versus 13.7 years, p = 0.80). Seven of 49 women who developed renal failure had *E. coli* bacteriuria at baseline, compared to 29 of 206 women in the control group (both 14%). The OR for the development of renal failure in the presence of *E. coli* bacteriuria, corrected for age, was 1.1 (95% CI 0.4–2.8, p = 0.86).

In a Swedish study the prevalence of ASB in women was 4%. After 15 years a reinvestigation was carried out, 40 cases (with ASB) and 40 age-matched healthy controls participated. Nobody had developed progressive renal disease. The age-dependent decrease after 15 years was the same in both groups.

The results of these longitudinal findings give strong support to the absence of an association between ASB and renal function decline in healthy women. As an explanation, Svanborg et al. found that certain *E. coli* strains stop expressing adherence factors like type 1 and P fimbriae once they have established bacteriuria. Therefore, these strains can remain present in the bladder without triggering an inflammatory response from the host and without side effects.

In conclusion, no relation between ASB and renal function decline has been demonstrated in healthy women. It has been recommended in American and European guidelines not to screen or to treat ASB in premenopausal non-pregnant women and older persons living in the community. The results of these studies confirm these recommendations.
