Background

Balkan endemic nephropathy (BEN) is of great clinical importance in restricted areas of Bulgaria, Serbia, Croatia, Bosnia, Herzegovina, and Romania, since the etiology of BEN is still unknown.

Methods

In urine samples from 48 patients (41 females and 7 males, aged 65.6 6.87 years) with BEN living in an endemic area of Vratza district, Bulgaria, neopterin concentrations were measured by high-pressure liquid chromatography (HPLC) and compared with other clinical and laboratory investigations, including creatinine, hemoglobin, and erythrocyte sedimentation rates (ESRs).

Results

Urinary neopterin concentrations were 263 128 (mean SD; range, 78 to 786 mol/mol creatinine), 24 (50%) of BEN patients presented with increased concentrations as compared to the established normal ranges. Average ESRs were increased (1 hour, 29.0 14.7 mm/hour) and hemoglobin was decreased (109.3 16.4 g/L). Hemoglobin correlated inversely with ESRs (rs = -0.787 and –0.780) and creatinine concentrations (r = -0.690, all P < 0.001), but not with neopterin concentrations. Neopterin concentrations also did not correlate with serum creatinine levels. There existed an age relationship of ESR, creatinine, and hemoglobin, but not of neopterin. Neopterin concentrations were slightly lower in five females with low titers of antibodies against local B1 hantavirus strain (P < 0.05).

Conclusion

The findings can support an immune-mediated inflammatory process in the pathogenesis of BEN only in a subgroup of patients.

METHODS

Five endemic villages from Vratza district (Gorno Peshtene, Tishevitza, Vesletz, Beli Izvor, and Pudria), all in Bulgaria, were screened for BEN. First-morning urine samples from 48 patients (41 women and seven men; age, 65.5 6.9 years; range, 52 to 79 years) with BEN living in the endemic area were collected. Specimens were kept at –20°C until measured.

Clinical investigations comprised blood tests [hemoglobin concentrations, red blood cells, leukocytes, and erythrocyte sedimentation rate (ESR), blood urea, creatinine, and uric acid] and urine analyses (pH, protein, glucose, bilirubin, urobilinogene, blood, nitrite, specific urine weight, ketons, and leukocytes). None of the patients showed symptomatology of any acute viral infection.

Neopterin and creatinine concentrations in urine samples were determined by high-pressure liquid chromatography (HPLC)¹². Briefly 100 mL of urine was diluted in 1 mL 15 mmol/L Soerensen potassium phosphate buffer, pH 6.4 containing 5 mmol/L ethylenediaminetetraacetic acid (EDTA). Neopterin and creatinine were separated by reversed-phase HPLC and expressed as mol neopterin per mol creatinine ratio to take into account variations of urine dilution¹². As stationary phase, a LiChroCART 125-4 column (RP-18 endcapped, grain size 5 m) (Merck, Darmstadt, Germany) was used, the mobile phase was a 15 mmol/L potassium phosphate buffer, pH 6.4 (Soerensen). Neopterin was quantified by its natural fluorescence (excitation at 353 nm wavelength; emission at 438 nm wavelength) and creatinine was simultaneously quantified by ultraviolet absorption at 235 nm wavelength in one single run¹². HPLC measurements were performed in subsequent runs within 1 day. Intra-assay variation of the HPLC method is 6%¹².

Preliminary virus analyses could be performed only in a subgroup of 34 individuals. By indirect immunofluorescence, antibodies against nephrotopic viruses with endemic areas of circulation [e.g., hantaviruses (hantavirus strains, standard Hantaan and F3955, local B1)] and West Nile flavivirus virus were analyzed.

Because not all laboratory results showed normal distribution, for comparisons of groups nonparametric Mann Whitney U-test was employed. Associations between neopterin concentrations, age, and other laboratory results of patients were calculated by Spearman rank correlation analysis.

RESULTS

Demographic data and neopterin concentrations in the individual urine samples are shown in Table 1. Increased neopterin concentrations were observed in 50% of the BEN patients, as compared to the 95th percentile of healthy controls¹²Table 1. Mean neopterin concentration was 263.1 mol/mol creatinine (see Table 2), which exceeds the upper limits of normal for men and women of all age groups. Elevated ESRs, as well as decreased hemoglobin concentrations, were also evident in the patients Table 2.

Table 1 - Demographic characteristics and neopterin concentrations in urine samples of Balkan endemic nephropathy (BEN) patients.

Full table

Table 2 - Neopterin concentrations and erythrocyte sedimentation rates (ESR) in 48 patients with Balkan endemic nephropathy (BEN).

Full table

There was no difference of neopterin concentrations between men and women, and there existed no correlations between neopterin and age and also not to other available laboratory parameters Table 3. Creatinine concentrations correlated positively with ESRs and inversely with hemoglobin levels. In addition, lower hemoglobin levels were associated with higher ESRs Table 2. When splitting the patients into two groups at the median creatinine concentration (=132 mol/L), the group with higher creatinine concentrations presented with lower hemoglobin (mean SD, 101.5 15.7 g/L; P < 0.001) and higher ESRs (ESR 1 hour, 33.8 16.0 mm/hour; P = 0.01) as compared with the group with lower creatinine (hemoglobin, 118.9 10.6 g/L; ESR 1 hour, 22.9 10.4 mm/hour). No such differences were found for age and urinary neopterin concentrations. Of the two patients on hemodialysis, one presented with the highest (=786 mol neopterin/mol creatinine), the other with the lowest neopterin concentration (=78 mol neopterin/mol creatinine) of all patients. Age correlated only weakly and positively with creatinine and ESRs and negatively with hemoglobin, and when patients were split in two groups at the median of their age (=65 years), there was no difference between any of the parameters (all P> 0.05).

Table 3 - Associations between age and laboratory characteristics of patients with Balkan endemic nephropathy (BEN).

Full table

There was no difference between any of the measured parameters in cases with and without BEN family history. BEN patients with diagnosed malignancy were older (73.0 3.67 years; N = 5) than those without such diagnosis (64.6 6.63 years, P < 0.01), but again, none of the laboratory parameters differed between the two groups of BEN patients. All the results of statistical analyses did not change when two patients on hemodialysis were excluded.

Preliminary data in a subgroup of 34 patients revealed comparatively low levels of antibodies against hantavirus strains (standard Hantaan and F3955, local B1) in nine patients (27%) Table 4. Besides, in two cases (two out of 34) (6%) anti-West Nile virus antibodies have been recorded Table 4. There was no association between the serologic findings and any of the laboratory measurements with the exception of neopterin: neopterin concentrations were lower in the 5 females seropositive for local hantavirus strain B1 (168.2 + 66.1 mol neopterin/mol creatinine) compared to residual seronegative BEN patients (279.3 + 144.3 mol neopterin/mol creatinine; U = 1.98, P < 0.05).

Table 4 - Antibodies against West Nile virus (WNV) and hantavirus strains in serum samples of 34 patients with Balkan endemic nephropathy (BEN) (indirect immunofluorescence test).

Full table

DISCUSSION

The monitoring of neopterin concentrations in body fluids is a sensitive way to detect Th1-type immune response^12,13 initiated by various causes. Measurements of neopterin concentrations in urine, serum or cerebrospinal fluid are employed as a laboratory diagnostic tool (e.g., to earlier detect immunologic complications in transplant recipients or to predict prognosis in HIV infection and malignancy)¹². Neopterin concentrations are also increased with high frequency during acute virus infections^12,14. Neopterin concentrations were found to be increased during episodes of viral infection, including acute hepatitis A and B¹⁵, Epstein-Barr virus (mononucleosis), cytomegalovirus^14,16, and measles¹⁷. In cytomegalovirus infections¹⁶, increased neopterin concentrations are observed in the majority of patients before antibody seroconversion becomes detectable. Usually neopterin concentrations normalize after seroconversion and when clinical symptomatology has ceased. However, elevated serum and urine neopterin concentrations levels cannot only be detected in acute and late stage of HIV infection, they are also found in more than 75% of patients with asymptomatic HIV-1 infection^18,19.

Renal impairment may increase blood neopterin concentrations¹⁸. Therefore, we decided to determine urinary neopterin concentrations expressed as the neopterin per creatinine ratio. Data of this study show that there was no relationship between serum creatinine and urine neopterin concentrations when expressed as mol neopterin/mol creatinine. The lacking association between creatinine and urinary neopterin concentrations is further strengthened by the observation that patients on hemodialysis (N = 2) presented, on the one hand, with the lowest and, on the other, with the highest neopterin concentrations of the study population.

Because, among other reasons, viral infection is discussed as a possible cause for BEN^8,9,10,11, it was of interest to examine neopterin concentrations in urine samples from BEN patients. However, only 50% of BEN patients presented with elevated neopterin concentrations compared to healthy controls. There was no difference between BEN patients with and without family history. Survival of BEN cases with family history was found longer when compared with BEN cases without family history²⁰. Again, these subgroups in our study did not differ regarding their neopterin concentrations. Only a few patients tested weakly positive for antibodies against hantavirus strains or anti-West Nile virus. Five females seropositive for local hantavirus strain B1 had lower neopterin concentrations than seronegative BEN patients. The number of patients tested was limited and the titers measured were low, still the result would fit to the earlier observation that higher neopterin concentrations, indicating cellular (=Th1-type) immune activation, are related to lower Th2-type markers such as IgE or antibody titers^21,22 in the sense of a cross-regulatory influence of Th1- versus Th2-type cytokines. Nevertheless, our observations would fit the notion that at least half of the patients examined in this study do not comprise patients suffering from acute virus infections.

Earlier studies show that malignant tumor diseases are frequently associated with increased neopterin concentrations²³. Thereby, higher neopterin concentrations are associated with shortened survival. Half of the BEN patients presented with increased neopterin concentrations, which is similar to the frequencies with elevated neopterin observed earlier in patients with solid cancer such as gynecologic neoplasias or cancer of the prostate. However, only a minority of our BEN patients presented with tumors, and their neopterin concentrations did not differ from patients without such diagnosis. A possible predictive value of neopterin concentrations still has to be demonstrated.

In several malignant and infectious diseases, an association between higher neopterin concentrations and lower hemoglobin concentrations was observed²⁴. This was not found in our study population. In addition, in chronic or protracted infections very often an association exists between neopterin concentrations and ESR^24,25,26, again this was not true in our individuals. With this respect, patients with BEN differ considerably from observations made in patients with infections or malignant diseases.

The lack of a strong increase of neopterin concentrations may reflect the very slow progression of BEN, the pauci-cellular infiltrates, marked interstitial fibrosis without prominent inflammation. The absence of elevated neopterin concentrations in patients with malignant tumors might be due to the characteristically superficial, epithelial type of tumors seen in BEN patients. Overall, the absence of increased neopterin in half of the patients might support the hypothesis of a toxic nephropathy with a genetic predisposition and may argue against an ongoing viral infection.

CONCLUSION

Approximately 50% of BEN patients present with increased urinary neopterin levels. The patients with elevated neopterin levels did not have a higher degree of renal insufficiency, and there were no differences in other variables studied between the patients with increased neopterin and those with normal values. The background of this elevation, whether it is infectious or of other origin, remains unresolved. Also, an association between higher neopterin and ESR or lower hemoglobin concentrations, which is commonly observed in infections and malignant processes, is obviously missing in patients with BEN.

References

References

Acknowledgments

This work was supported by the Austrian Federal Ministry of Social Affairs and Generations, and by INCO-COPERNICUS project ERB IC15-CT98-0318.