Antibodies to TammHorsfall protein in endemic nephropathy
Bojan Jelakovi
1,
Jasminka Benkovi
2,
Nada
ike
1,
Du
ko Kuzmani
1,
Dubravka
vori
ec2,
Stjepan
eovi
3,
Tomislav Ron
evi
1 and
eljko Krznari
1
1 Department of Internal Medicine,
2 Clinical Institute of Laboratory Diagnosis, Zagreb University School of Medicine and Clinical Hospital Center, Zagreb,
3 General Hospital Dr Josip Ben
evi
', Slavonski Brod, Croatia
Correspondence and offprint requests to:
Bojan Jelakovi
MD PhD, Department of Internal Medicine, Division of Nephrology and Arterial Hypertension, Zagreb University Hospital Center, School of Medicine, University of Zagreb, 10000 Zagreb, Ki
pati
eva 12, Croatia.
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Abstract
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Background. The aim of the study was to investigate the possible role of antibodies to TammHorsfall protein (anti-THP) in the early diagnosis of endemic nephropathy (EN).
Methods. Anti-THP (IgA, IgM, IgG classes) antibodies were determined by direct ELISA in a random sample of 159 subjects from the endemic village of Kani
a who were divided into four groups according to the WHO criteria, i.e., `diseased', `suspect', `at risk', and `others'. These groups were compared to subjects from the non-endemic village of Klakar and healthy subjects from Zagreb.
Results. No differences for all the classes of antibody were observed among the groups of subjects from the endemic village of Kani
a (P>0.05) or between these subjects and those from the non-endemic village of Klakar (P>0.05). The values of IgM anti-THP antibodies exceeded those of the IgA and IgG classes in all groups of subjects (P<0.05). The values for all three classes of antibodies were higher in the rural than the urban population (P<0.05).
Conclusion. Determination of anti-THP antibodies was not found to be useful in the early diagnosis of endemic nephropathy. The results suggest that most of the anti-THP antibodies are `natural' and/or cross reactive. The highest values observed in the rural population could probably be explained by exposure to some ubiquitous antigen or more likely they are consequences of fever.
Keywords: antibodies; endemic nephropathy; TammHorsfall protein
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Introduction
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Endemic nephropathy (EN) is a chronic tubulointerstitial nephritis of unknown aetiology that occurs in rural areas along the basins of big rivers in Bulgaria, Bosnia, and Herzegovina, Croatia, Romania and Yugoslavia [18]. EN has an insidious onset and progresses slowly to chronic renal failure. In Croatia, where approximately 10000 people are at risk and where the prevalence in the endemic area is about 4% [1], great attention has been paid to the early diagnosis and follow-up of EN. During the past decade, we focused on the urinary enzymes, beta-2 microglobulin and alpha-1 microglobulin [912]. Increased urinary excretion of TammHorsfall protein (THP) was also observed in patients with EN, as well as in subjects `at risk' and in those suspected to have EN [13,14]. THP is of utmost interest in EN since it may be a marker for the early stage of the disease and also an aetiological agent and promoter of renal failure progression. THP is a glycoprotein synthesized in epithelial cells of the ascending limb of the loop of Henle and in the proximal part of the distal tubule [15]. It has been considered as a `hidden antigen' that is exposed to the immune system only after pathophysiological alterations which then result in formations of antibodies to THP (anti-THP). Anti-THP antibodies have been determined in various kidney diseases, but its role in their pathogenesis or in diagnosis has not yet been established [1619]. We determined anti-THP antibodies in persons from the endemic village of Kani
a over 3 consecutive years with the purpose of investigating whether anti-THP antibodies could be of diagnostic value in EN.
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Subjects and methods
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Antibodies to TammHorsfall protein were determined in 159 inhibitants of the endemic village of Kani
a. Random subjects making up over 25% of the population of the endemic village were investigated. The subjects were divided according to WHO criteria into four groups: `diseased', `suspect for EN', `at risk' and `others' [20]. The diagnosis was based on the following parameters: positive proteinuria,sulphosalicylic acid and/or low-molecular-weight test (P), anaemia (A), serum creatinine above 132.6 µmol/l (C), and positive family history of EN (FH). Subjects with P, A and C, or P, A and FH were considered to be afflicted with EN (`diseased'). Those who had P and A, or P and FH were considered `suspect for EN', while persons with only FH were considered to be `at risk' for EN. Inhabitants from the endemic village who had none of these criteria were classified as `others'. Eight subjects were classified as `diseased', 15 as `suspect for EN', 54 as those `at risk' and 82 as `others'. All groups from the endemic village completely met previously defined criteria which, together with the number of subjects included in the study, indicates that the obtained results and conclusions are generally valid for the whole population. One control group, included subjects from the non-endemic village of Klakar in the same geographical area of Croatia (Brod Posavina), while the other control group consisted of 30 subjects from Zagreb who had never suffered from any renal disease. In addition, acute urinary tract infection was ruled out by history and clinical inspection.
THP was isolated from the urine of healthy adult males and females by multiple precipitation with 0.58 mmol/l NaCl as described earlier [21]. The values of anti-THP antibodies were determined by direct enzyme immunoassay (ELISA) according to the procedure proposed by Fasth et al. [22] and modified by Benkovi
et al. [16]. Microtitre plates (Behring, Marburg, Germany) were coated with THP (10 µg/ml in PBS buffer, pH 7.4) for 1 h at 37°C. Upon emptying the wells, human serum albumin (1 g/l in PBS buffer, Behring, Marburg, Germany) was added and incubation for 1 h at 22°C was performed in order to block the free binding sites. Thereafter the plates were washed three times with PBS-Tween 20 and twice with distilled water. Then, serum samples were diluted: 1:5 with PBS-Tween 20 for the determination of IgA and IgG class antibodies and 1:10 for IgM and 100 µl were added per well. After incubation for 1 h at 22°C, the plates were washed three times with PBS-Tween 20. Secondary antibodies labelled with alkaline phosphatase (anti-human IgG, IgA and IgM, Sigma, St Louis, USA) were added. IgA, IgG and IgM class antibodies were diluted with PBS-Tween 20 at ratios of 1:4000, 1:5000 and 1:25000 respectively. After incubation for 1 h at 22°C and washing with PBS-Tween 20 three times, 100 µl substrate, p-nitrophenylphosphate, was added per well (1 mg/ml in diethanolamine buffer, pH 9.8, Sigma, St Louis, USA). The plates were incubated for 1.5 h in the dark at 22°C, and colour development was stopped by the addition of 3 mol/l NaOH, 200 µl per well (Kemika, Zagreb). Absorbance was measured with a Behring ELISA analyser at the wavelength of 405 nm. The background absorbance (THP-coated wells) and the value of unspecific binding (THP-coated wells with secondary antibodies) were determined for each microtitre plate. All values were determined on three occasions and their means were calculated. Prior to statistical analysis and interpretation of results obtained by ELISA, the stability and reliability of the method were tested.
Final confirmation of the method's reliability was obtained by testing variability within and between some plates. This was done using the ANOVA method by abandoning the starting hypothesis that variability between the plates exceeded the one within the plate, under the condition that the same random sample was on each plate.
The data were statistically processed on a PC using Statistics for Windows program. As the result distribution was not normal for all antibody classes, the significance of the differences was determined by non-parametric methods, i.e. by MannWhitney U test and KruskallWallis test.
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Results
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Basic clinical features of subjects from Kani
a and Klakar are shown in Table 1
and laboratory findings are presented in Table 2
.
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Table 1. Basic clinical features of subjects from the endemic village of Kani a and from the non-endemic village of Klakar
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Table 2. Laboratory findings of subjects from the endemic village of Kani a and from the non-endemic village of Klakar
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There were no differences between the values obtained for nonspecific binding of secondary antibodies over the 3 years for any of the antibody classes (IgG: 0.3±0.2 vs 0.3±0.3 vs 0.4±0.2; P>0.05, IgA: 0.5±0.3 vs 0.3±0.3 vs 0.4±0.2; P>0.05, IgM: 0.3±0.2 vs 0.3±0.3 vs 0.4±0.2; P>0.05) or between antibody classes (P>0.05). Based on these two results, we conclude that the method is reproducible and reliable. This was confirmed by the fact that no correlation between the final result and the values of non-specific binding for any of the antibody classes was found (P>0.05).
The variability of the anti-THP antibodies in the serum over the 3 years was higher than the variability during a single year. This result probably reflects real variations through time. Individual variations of IgG class antibody titres in four sick, four suspect, and four control subjects are shown in Figure 1
. However, as THP is a large molecule with a number of epitopes, low specificity also cannot be completely ruled out, which is quite consistent with our conclusion.

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Fig. 1. Individual variations of IgG class anti-THP antibody titres obtained during 3 consecutive years in four sick persons (P), four suspects (S) and four control subjects (C) from the endemic village of Kani a.
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Diseased subjects and those suspected for EN were older than those classified as at risk and others (P<0.05). Diseased subjects had significantly higher blood pressure (P<0.05) than those categorized as `at risk' and others, while values of blood pressure in other subgroups were similar. There were no differences between inhabitants of Kani
a as a whole and Klakar in age or blood pressure (P>0.05). Diseased and suspected for EN groups had significantly higher values of proteinuria than others and at risk persons, as well as persons from Klakar (P<0.05). There were no differences in proteinuria between inhabitants of Klakar and at risk persons and others from Kani
a (P>0.05). Serum creatinine values were significantly higher in the diseased (P<0.01). The suspected for EN group had higher values of serum creatinine than at risk persons, others, and inhabitants of Klakar, but the difference was not statistically significant (P>0.05). Haemoglobin values were significantly lower in the diseased and suspected for EN groups, than in the at risk, others, and Klakar groups (P<0.05).
The values of antibodies to TammHorsfall protein measured in subjects from the endemic village of Kani
a over 3 consecutive years are shown in Table 3
. No statistically significant differences were observed for any class of anti-THP antibodies between subgroups (P>0.05). There were also no differences in the values of any class of anti-THP antibodies observed over three consecutive years in any of the subgroups from Kani
a (P>0.05). Values of antibodies determined in subjects from the non-endemic village of Klakar and healthy subjects from Zagreb are presented in Table 4
. There were no differences in any class of anti-THP antibodies between the subgroups of Kani
a and inhabitants from Klakar (P>0.05). However, the values of all three classes of anti-THP antibodies were significantly lower in healthy subjects from Zagreb than from inhabitants of the endemic village of Kani
a and the non-endemic village of Klakar (P<0.01).
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Table 3. Values of antibodies to TammHorsfall protein in subjects from the endemic village of Kani a observed in 3 consecutive years
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Table 4. Values of antibodies to TammHorsfall protein in subjects from the non-endemic village of Klakar and in healthy persons from Zagreb
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Values for IgM class of anti-THP antibodies were significantly higher than the IgA and IgG classes in all subgroups of subjects (P<0.05).
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Discussion
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The clinical course of EN is characterized by an insidious onset and slow progression to chronic renal failure [8]. Patients might be without any obvious sign of renal disease until the final phase when uraemic syndrome is evident. Intermittent macroscopic haematuria, loin pain, and profuse urination occur only in very few patients. Blood pressure remains normal and tends to rise in ESRD, which was also confirmed by our results. The disease becomes manifest at the age of 4060 years or when patients had lived more than 20 years in the endemic area. The diseased and suspects for EN groups were also older than those who were at risk of EN and other inhabitants of the endemic village of Kani
a (P<0.05). Tubular proteinuria is one of the hallmarks of EN [8,9,23,24]. Not only the diseased, but even those suspected for EN excrete more tubular proteins than other subjects. One of the signs of EN at the early stage is normochromic, normocytic anaemia. In the diseased and suspected for EN, anaemia is more pronounced than expected for the observed renal lesion [8]. The values of haemoglobin in the diseased and suspected for EN groups were significantly lower than the values observed in others from Kani
a and from the values obtained in inhabitants of the non-endemic village of Klakar. It is obvious that the subjects in our groups truly fulfilled the criteria defined before [20], thus representing subgroups from the endemic area.
The early sub-clinical course and great prevalence in endemic areas point toward the necessity of early diagnosis. Investigations on low-molecular-weight proteins, i.e. beta-2 microglobulin, alpha-1 microglobulin [9,11,12] and enzymes originating from tubular cells, i.e., alanine aminopeptidase, N-acetyl-beta-D-glucosaminidasae, lactate-dehydrogenase and lysozyme [10,14] showed that they were very sensitive indicators of tubular damage. Nevertheless, these parameters are not adequately specific to be considered as markers for the early phase of EN. Attention was also focused on the distal tubule. Urinary THP was analysed and correlation between elevated values of THP excretion and proximal tubular proteinuria was observed [25]. Moreover, increased THP excretion was found in patients who were at risk for EN. One can speculate that an undefined aetiological agent of EN damaged the cells of the distal part of the nephron, resulting in increased urinary excretion and concurrent interstitial deposition of THP. Interstitial deposits of THP have been observed in other tubulointerstitial renal diseases [26]. Animal models and studies in vitro have shown that THP influences cellular and humoral immunity [15,27] and, therefore, THP might be an agent that contributes and perpetuates interstitial inflammation. Some authors have proposed anti-THP antibodies as a marker for induced immune response to THP in patients with acute pyleonephritis and vesicoureteral reflux [18,19,22,28]. Fasth et al. [19] and Sandberg and Fasth [28] have found increased levels of IgA and IgG anti-THP antibodies in patients with acute pyelonephritis. We reported [16] on elevated IgM anti-THP antibodies directed toward native THP molecule, while IgA class was a dominant immunoglobulin in the reaction directed to the THP subunits obtained in vitro [29]. Recently we reported on a rise of IgA anti-THP antibodies in patients with nephrolithiasis who were treated with extracorporeal shock-wave lithotripsy [30]. The observed rise could also be explained by the stimulation of the immune system with THP.
In subjects from the endemic area, the values of IgM anti-THP antibodies were significantly higher than values of IgG and IgA classes (P<0.05). There were no differences between particular subgroups of inhabitants of the endemic village of Kani
a (P>0.05) and therefore we conclude that the determination of anti-THP antibodies is of no value for the diagnosis of EN. The results obtained in the non-endemic village of Klakar, where the values of anti-THP antibodies did not differ from those obtained in the endemic area, are in line with this conclusion (P>0.05). The same pattern was observed during the three consecutive years of our investigation. The values of all three classes of anti-THP antibodies observed in rural population, in the endemic village of Kani
a as well as in the non-endemic village of Klakar were significantly higher than the values found in subjects from an urban centre (P<0.05). The values of anti-THP antibodies observed in the rural population were higher compared to healthy persons from Zagreb, but they were also significantly higher than the values found in urban patients with acute pyelonephritis [16,29] or nephrolithiasis [30]. This could be explained by the presence of so-called native antibodies or binding of cross-reactive antibodies and it might reflect polyclonal activation induced by some ubiquitous antigen that is permanently present only in the rural environment. It is speculative to propose that this antigen is a part of an agent that could, in genetically predisposed [31] persons, initiate a chain of pathophysiological events leading to tubulointerstitial inflammation, scarring and eventually chronic renal failure. Should the observation of a significant difference of anti-THP antibodies between rural and urban populations be confirmed in a larger population sample with other demographic characteristics, differing for instance in age and gender, this would prompt a reappraisal of all observations on anti-THP antibodies.
The delayed onset of EN implicates that direct toxicity of a putative agent is not the most important mechanism for this type of chronic tubulointerstitial nephritis. More likely, this aetiological agent could damage kidney tissue, unmask some sequestrated antigens (like THP), and activate the immune system and/or it could act by increasing gene expression, important for the progression of renal disease (i.e. ACE, angiotensinogen, TGF-beta). Parallels that could be drawn between EN and Chinese herb nephropathy [32] are very interesting and intriguing since Chinese herbs contain aristolochic acid. Aristolochia clematitis is very abundant in the rural area of this part of Croatia and it was one of the first agents investigated [7,8]. Perhaps it is time to turn back to this plant.
In conclusion, we can say that determination of anti-THP antibodies is of no use in the early diagnosis of EN. Further attempts are needed to find a specific marker for this disease. This should be accompanied by investigations on the pathogenesis of this unusual chronic tubulointerstitial nephritis.
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Received for publication: 19.11.98
Accepted in revised form: 22. 6.99