Tubular Proteinuria and Glomerular Filtration 6 Years after Puumala Hantavirus-Induced Acute Interstitial NephritisMiettinen M.H.a · Mäkelä S.M.b, d · Ala-Houhala I.O.b, d · Huhtala H.S.c · Kööbi T.e · Vaheri A.I.f · Pasternack A.I.b · Pörsti I.H.b, d · Mustonen J.T.b, d
aDepartment of Internal Medicine, Central Hospital of Jyväskylä, Jyväskylä, bMedical School, cTampere School of Public Health, University of Tampere,Departments of dInternal Medicine and eClinical Physiology, Tampere University Hospital, Tampere, and fDepartment of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland Corresponding Author
Background/Aims: We previously found increased urinary protein excretion, glomerular filtration rate (GFR) and blood pressure in a retrospective analysis of patients with previous nephropathia epidemica (NE). Here, we evaluated the long-term outcome after NE in a prospectively recruited patient group. Methods: Proteinuria, GFR and ambulatory 24-hour blood pressure were assessed 4–7 years (mean 6) after acute NE in 37 patients, and these values were compared to those from 38 seronegative controls. Results: Six years after NE, the prevalence of elevated urinary α1-microglobulin excretion was higher in the patients than controls (9/35 vs. 1/38; p = 0.005). The patients also had higher urinary protein excretion (0.17 ± 0.05 vs. 0.14 ± 0.04 g/day; p = 0.006), GFR (119 ± 19 vs. 109 ± 14 ml/min/1.73 m2; p = 0.016) and mean systolic (123 ± 11 vs. 117 ± 9 mm Hg; p = 0.012), nighttime systolic (109 ± 11 vs. 100 ± 9 mm Hg; p = 0.001) and nighttime diastolic blood pressure (70 ± 7 vs. 66 ± 7 mm Hg; p = 0.035) than the controls. Conclusions: These results confirm our previous findings of a higher prevalence of tubular proteinuria and increased urinary protein excretion, GFR and systolic blood pressure 6 years after acute NE.
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Nephropathia epidemica (NE) is a mild type of hemorrhagic fever with renal syndrome. It is caused by Puumala virus, a member of the hantavirus genus in the Bunyaviridae family that is carried by bank voles (Myodes glareolus) [1, 2]. NE is characterized by sudden onset of fever, headache, backache and abdominal pain [3,4,5]. Proteinuria, hematuria and oliguria followed by polyuria are common clinical findings, while acute tubulointerstitial nephritis is the typical histopathologic lesion on renal biopsy [3, 6]. The clinical picture of the disease varies in severity, and complete recovery is the usual outcome [3,4,5].
Previously, we performed a retrospective analysis of 46 patients 5 years after acute NE and found higher urinary protein excretion and increased glomerular filtration rate (GFR), filtration fraction (FF) and systolic blood pressure in the patients than in healthy control subjects . Recently, we were able to re-examine 36 patients from the same group 5 years later, i.e. 10 years after acute NE. At that stage the differences between the patients and controls with regard to proteinuria, GFR, FF and blood pressure were no longer present, which supports the view that the long-term prognosis of NE is favorable .
In the present study, we evaluated the reliability of the results of our previous retrospective investigation  and examined urinary protein excretion, renal function and ambulatory blood pressure (ABP) in a new prospectively recruited group of patients 4–7 years (mean 6) after acute NE. The results were compared with those of the same seronegative controls as in our previous study , in order to avoid the influence of possible variations between different control populations.
The patient group consisted of 43 consecutive patients hospitalized due to serologically verified NE at Tampere University Hospital , Finland, during the years 1997–1999. They were prospectively collected for this longitudinal study. Six of these 43 patients were excluded from the present study; 4 of them had hypertension before acute NE, 1 patient had diabetic nephropathy and 1 had mesangial glomerulonephritis. Thirty-seven patients (29 males and 8 females) aged 29–70 years (mean 49) were examined as outpatients 4–7 years (median 6) after acute NE in 2004. One patient had suffered from Henoch-Schöenlein purpura in childhood and 1 patient had been treated for renal tuberculosis in the 1960s. Neither of them showed any findings of chronic renal disease following these conditions. Prior to the episode of acute NE, the other chronic diseases in 7 of the patients were hypothyroidism (in 3 patients) and celiac disease, coronary heart disease, bronchial asthma and ankylosing spondylarthritis (each in 1 patient). The patient group was compared with the same group of 38 healthy, Puumala virus-seronegative controls (22 males and 16 females), aged from 27 to 64 years (mean 44), that was used in our previous study . One of the controls had hypothyroidism. All 4 subjects with hypothyroidism were euthyreotic with thyroxin substitution.
All subjects gave informed consent before participation, and the study was approved by the Ethics Committee of Tampere University Hospital.
Blood specimens were obtained in the morning after a minimum 12-hour fast. In the studies that took place from 1997 to 1999, serum creatinine, urea, triglycerides, blood glucose and total, high-density and low-density lipoprotein cholesterol were determined by Vitros (Johnson & Johnson, Rochester, N.Y., USA), and the blood cell count was evaluated by Technicon H3 (Bayer Diagnostics, Elkhart, Ind., USA). In 2004, the same laboratory parameters were analyzed by Cobas Integra 700/800 and Bayer Advia 120, respectively. Serum creatinine concentrations showed 10% lower values in 2004 than during the years 1997–1999 due to the above change in the determination method. Fasting blood glucose was determined from whole blood until the year 2003, after which the analysis was performed in plasma, thus producing 12% higher values. In this study, the results of serum creatinine concentrations from the years 1997–1999 were multiplied by the coefficient 0.9, and the results of fasting plasma glucose before the year 2003 were multiplied by the coefficient 1.12. The limits for impaired fasting glucose (IFG) were 6.1–6.9 mmol/l, according to the guideline of the European Diabetes Epidemiology Group .
Nighttime, daytime and 24-hour urinary protein excretion was measured by the pyrogallol red molybdate method (Olli C, Kone Instruments, Helsinki, Finland) in the 1990s and by Cobas Integra 700/800 in 2004. The overnight urinary excretion of α1-microglobulin, albumin and immunoglobulin G (IgG) was measured by nephelometry (Behring Nephelometer II Analyzer, Behringwerke AG, Marburg, Germany). Values for overnight urinary excretion of α1-microglobulin ≥7 μg/min, albumin ≥11 μg/min and IgG ≥5 μg/min were considered abnormal, based on the reference values for healthy subjects from our laboratory. The spot samples of morning urine were collected after a minimum of 12 h of fasting and analyzed for osmolality, erythrocytes, leukocytes, albumin, nitrite, glucose, pH and ketones. Urine osmolality was measured by the Advanced Cryomatic™ Osmometer (Advanced Instruments Inc., Needham Heights, Mass., USA). Urine for the measurement of proteinuria was not collected for 3 patients.
Electrocardiograms (ECGs) were evaluated according to the Minnesota codes. Left ventricular mass was evaluated by the sum of the height of the S wave in lead V1 and the R wave in lead V5 of a 12-lead resting ECG. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in square meters).
GFR was determined by a single-injection method as the plasma clearance of 51Cr-EDTA after a light meal and expressed in values normalized for body surface area. Effective renal plasma flow (ERPF) was estimated by the clearance of 131I-hippurate. The FF was calculated as the quotient of GFR and ERPF. Based on studies in healthy subjects, increased GFR was defined as a GFR ≥130 ml/min/1.73 m2 .
The 24-hour ABP was measured with a fully automatic recorder. The cutoff point for hypertension was a 24-hour mean ABP ≥133/82 mm Hg, according to the results of a large population study . One control subject and 10 patients either refused ABP measurement or had missing measurements due to technical failure.
To describe the data, means and standard deviations (SDs) are given for normally distributed variables and medians and ranges for skew-distributed continuous variables. Groups were compared using independent-sample t tests for normally distributed variables and Mann-Whitney U tests for skewed variables. Categorical data were analyzed by χ2 test or by Fisher’s exact test. Correlations were analyzed by Pearson’s correlation coefficient. Skew-distributed urinary excretion of protein was log transformed before analysis. Binary logistic regression analysis was carried out to determine whether the differences in proteinuria, GFR and mean systolic, nighttime systolic or nighttime diastolic blood pressure between the groups could be ascribed to the differences in age and gender distribution between the groups. Odds ratios (ORs) and their 95% confidence intervals (CIs) were given. All tests were two-sided, and analyses were performed with SPSS for Windows, version 13 (SPSS Inc., Chicago, Ill., USA).
The characteristics of the study population and basic laboratory values are shown in table 1. The patients were on average 5 years older than the controls, and their fasting blood glucose and serum low-density lipoprotein cholesterol levels were slightly higher than in controls. Medication for type 2 diabetes had been initiated for 1 male patient after acute NE, but he showed no evidence of albuminuria during this study. One female patient had fasting blood glucose of 7.1 mmol/l and her hemoglobin A1 concentration was 6.4%, pointing to type 2 diabetes. Recurrent microscopic hematuria, in the absence of proteinuria, was also detected after NE. Her clinical, laboratory and radiological examinations showed normal blood pressure, renal function and renal ultrasound. Moreover, there were no specific findings on cystoscopy, while urinary cytology showed class II cells with atypical degenerative changes. Seven out of 37 patients (19%) had IFG, while none of the 38 controls had abnormal fasting blood glucose (p = 0.005, Fisher’s exact test). Altogether, 9 of the 37 patients (24%) had IFG or type 2 diabetes.
|Table 1. Clinical and basic laboratory findings in patients 6 years after acute NE and in controls|
One patient had right bundle branch block on the ECG, but no pathological Q waves or ST-T wave alterations were recorded. The sum of the S wave in lead V1 and R wave in lead V5 did not differ between the patients and controls (24 and 23 mm, respectively; p = 0.482, independent-samples t test).
Table 2 and figure 1 show proteinuria (fig. 1a) and renal function (fig. 1b) in patients 6 years after acute NE compared with seronegative controls. The urinary protein excretion was higher in patients than in controls, and after adjusting for age and sex, the difference still remained statistically highly significant. A positive correlation between proteinuria and GFR was detected in the patients (r = 0.495, p = 0.003, Pearson’s correlation) but not in the controls (r = 0.255, p = 0.123), while proteinuria did not correlate with age or systolic or diastolic blood pressure in either group (data not shown). In addition, the prevalence of elevated urinary excretion of α1-microglobulin was higher among the patients than the controls (9/35 vs. 1/38; p = 0.005, Fisher’s exact test), but there was no difference between the groups in the overnight excretion of albumin or IgG (data not shown).
|Table 2. Proteinuria, renal function and ABP in patients 6 years after acute NE and in controls|
|Fig. 1. Urinary protein excretion (a), GFR (b) and mean ambulatory systolic blood pressure (SBP) (c) in controls (n = 38) and prospectively recruited patients (n = 36) 6 years after acute NE. Variables are depicted as means (█), standard errors of the mean (boxes) and SD (whiskers). * p < 0.05; ** p < 0.01.|
The mean GFR was higher in patients than in controls, but there was no difference in ERPF or FF between the groups (table 2, fig. 1b). After adjusting for age and sex, the difference in GFR remained statistically significant (OR 1.047, 95% CI 1.01–1.09; p = 0.010). Increased GFR (≥130 ml/min/1.73 m2) was found in 10 of the 37 patients (27%) compared to 4 of the 38 controls (11%) (p = 0.067, χ2 test). GFR correlated inversely with age in the patient group (r = –0.512, p = 0.001, Pearson’s correlation) but not in controls (r = 0.008, p = 0.963). Furthermore, GFR showed no correlation with blood pressure or fasting blood glucose in either group (data not shown). The mean GFR was 118 ± 15 ml/min/1.73 m2 in patients with diabetes or IFG and 119 ± 20 ml/min/1.73 m2 in patients with normal fasting blood glucose (p = 0.194, independent-samples t test). Furthermore, increased GFR was found in 3 of the 9 patients with diabetes or IFG compared to 7 of the 28 patients with normal fasting blood glucose (p = 0.679, Fisher’s exact test). The clinical severity of acute NE, as indicated by the highest serum creatinine concentration measured during the hospital stay, had no relation to proteinuria or GFR 6 years later (data not shown).
ABP monitoring was successfully completed in 27 patients and 37 controls. The patients had higher mean (fig. 1c) and nighttime systolic blood pressure and nighttime diastolic blood pressure than the controls (table 2).
After adjusting for age and sex, the difference between the groups with regard to mean and nighttime systolic blood pressure remained (OR 1.07, 95% CI 1.00–1.14, p = 0.038, and OR 1.08, 95% CI 1.01–1.14, p = 0.019, respectively). In the whole group of study subjects, neither the mean, daytime nor nighttime systolic and diastolic blood pressure correlated with age (data not shown). The highest serum creatinine measured during acute NE had no relation to the results of ABP measurement 6 years later (data not shown).
Antihypertensive medication had been initiated in 5 of the patients 1–6 years after acute NE, and all continued their medication during the examinations. The classes of the medications were β-adrenoceptor blockers, calcium channel blockers, angiotensin-converting enzyme inhibitors and angiotensin II receptor antagonists. None of the controls were on antihypertensive medication during the study. It is noteworthy that differences in mean systolic and nighttime systolic blood pressure between the groups remained significant even if the subjects using antihypertensive medication were excluded from the statistical analysis.
The long-term prognosis of Puumala hantavirus-induced acute tubulointerstitial nephritis is considered to be favorable. However, this longitudinal study with a prospectively recruited patient group confirmed our previous findings that 4–7 years after acute NE, patients have higher urinary protein excretion, GFR and ambulatory systolic blood pressure than healthy controls .
As also previously described, this study showed increased urinary protein excretion and higher prevalence of abnormal α1-microglobulin excretion in patients than in controls several years after NE [7, 13]. In a recent retrospective analysis of 30 Croatian patients, performed 3–6 years after acute hemorrhagic fever with renal syndrome, 6 of the 23 patients (26%) had increased excretion of β2-microglobulin, while proteinuria exceeding 150 mg/day was detected in 11 of the 23 patients (48%) . The pathophysiological mechanism of mild proteinuria several years after acute hemorrhagic fever with renal syndrome is obscure. However, in 1978, Lähdevirta et al.  from Finland published a study of renal biopsies from 9 patients 4–5 years after the episode of acute NE. The samples showed slight residual interstitial fibrosis and occasionally atrophic tubuli, tubular casts, increased numbers of hyalinised glomeruli and minor changes in other glomeruli. Two specimens also showed fibrotic scars in the cortex.
In this study, the patients who presented with higher GFR than the controls 6 years after acute NE were older than the controls, who were age-matched with the previous patient cohort . Usually GFR declines with age , and also in the present study, GFR correlated inversely with age in the patient group. This indicates that age cannot explain the difference in GFR between the groups. The mechanism underlying increased glomerular filtration after acute interstitial nephritis is not known at present. In early diabetic nephropathy, glomerular hyperfiltration is a typical feature, and similar findings have been reported in essential hypertension and polycystic kidney disease [17,18,19,20]. Furthermore, in a recent report on stage I hypertensive patients, glomerular hyperfiltration was shown to predict the development of microalbuminuria . In the present investigation, increased GFR in patients might have been related to the higher frequency of IFG in patients than in controls. However, the GFR of patients with diabetes or IFG compared with the patients with normal fasting blood glucose was essentially the same. Neither did we find any correlation between the GFR and fasting blood glucose levels in this study. Higher GFR could also have been a consequence of the higher systolic blood pressure in the patient group, although the fact that GFR showed no correlation with blood pressure is in conflict with this view.
Our patients had higher ambulatory systolic blood pressure than the controls 4–7 years after acute NE, as in our previous study . Furthermore, antihypertensive medication had been started in 5 patients during the follow-up, and all of them continued their medication during the study. If the subjects treated with antihypertensive compounds were excluded from the analyses, the outcome of the analysis of ABP results remained the same, i.e. the patients still showed increased systolic blood pressure. Furthermore, we did not find any significant correlation between age and blood pressure, and the difference in blood pressure between the groups remained after adjusting for age and sex.
We recently re-examined 36 patients from our previous retrospective analysis of a patient cohort 10 years after acute NE . The results showed no difference in proteinuria, GFR or FF between the patients and the controls. As in all long-term clinical follow-up studies, there were some dropouts. As the dropouts did not differ essentially from the study population, it seems safe to conclude that the 10-year prognosis of NE is favorable . Nevertheless, the possibility remains that NE may predispose some patients to the development of hypertension.
In conclusion, in a new prospectively recruited population of patients with prior NE, we were able to confirm our previous findings of higher prevalence of tubular proteinuria, increased GFR and higher systolic blood pressure 6 years after acute Puumala hantavirus-induced nephritis compared with a healthy control group. The underlying mechanisms of the tubular proteinuria and the increased glomerular filtration remain unclear.
The study was financially supported by the Medical Research Foundation of Central Finland Health Care District, the Pirkanmaa Regional Fund of the Finnish Cultural Foundation, the Medical Research Fund of Tampere University Hospital, the Finnish Kidney Foundation and the European Commission Project ‘Diagnosis and control of rodent-borne viral zoonoses in Europe’ (QLK2-CT-2002-01358).
The skilful technical assistance provided by Ms. Katriina Ylinikkilä is greatly appreciated.
Marja Miettinen, MD
Department of Internal Medicine, Central Hospital of Jyväskylä
FI–40620 Jyväskylä (Finland)
Tel. +358 14 269 1811, Fax +358 14 269 2711, E-Mail email@example.com
Received: March 4, 2008
Accepted: October 16, 2008
Published online: April 23, 2009
Number of Print Pages : 6
Number of Figures : 1, Number of Tables : 2, Number of References : 22
Nephron Clinical Practice
Vol. 112, No. 2, Year 2009 (Cover Date: May 2009)
Journal Editor: El Nahas M. (Sheffield)
ISSN: 1660-2110 (Print), eISSN: 1660-2110 (Online)
For additional information: http://www.karger.com/NEC