URINARY CLUSTERIN CONCENTRATIONS – A POSSIBLE MARKER OF NEPHROPATHY? PILOT STUDY.

BACKGROUND
Clusterin is a glycoprotein which participates in a number of pathophysiological processes in the organism. Information about clusterin use in the diagnosis of nephropathy and the differential diagnosis of proteinuria has been published recently.


AIM
Search for correlations between urinary clusterin concentration and other renal function markers. Evaluation of urinary clusterin measurement use in the differential diagnosis of nephropathy.


METHODS
Urea, creatinine, IgG, transferin, Na, K in serum and 24-hour collected urine were measured in a sample of 82 individuals. Cystatin C in sera was also measured as were GMT, alpha-1 microglobulin, albumin, total protein in urine. In all probands urinary clusterin was assayed (ELISA).


RESULTS
Urinary clusterin values correlated with urinary total protein concentrations (r = 0.28; p = 0.018), total protein/creatinine index (r = 0.26; p = 0.02). No correlation was found between urine clusterin concentration and glomerular filtration rate, age, urine GMT/creatinine, alpha-1-microglobulin, urine albumin and albumin/creatinine ratio or Na, K fractional excretions. We found no urinary clusterin differences by sex of probands. No evidence of any relationship between urine clusterin and presence of defect of renal function, number of risk factors (chi(2) = 16.0; DF = 15; p = 0.38), albumin/creatinine index (chi(2) = 0.76; DF = 3; p = 0,86), total protein/creatinine (chi(2) = 6.5; DF = 3; p = 0.09), GMT/creatinine (chi(2) = 2.3; DF = 3; p = 0.51), high urinary alpha-1-microglobulin (chi(2) = 4.1; DF = 3; p = 0.25) or decreased of GFR (chi(2) = 1.3; DF = 3; p = 0.74).


CONCLUSIONS
A positive correlation exists between urinary clusterin and urinary total protein and total protein/ creatinine index. Urinary clusterin measurement with ELISA test does not offer any advantage over routinely used parameters for nephropathy diagnosis and the differential diagnosis of proteinuria type.


INTRODUCTION
The diff erential diagnosis of proteinuria is an essential component in the diagnosis of renal diseases.Proteinuria may be caused by increased capillary permeability to proteins, with excess tubule capacity for back resorption (glomerular proteinuria), disorders in back resorption of fi ltrated proteins (tubular proteinuria), extreme increase in some blood proteins (prerenal proteinuria) or infl ammatory and cytolytic processes in the urinary tract (postrenal proteinuria).The diagnostic examination of proteinuria is made by determining the urinary protein/creatinine index, urine electrophoresis, measurement of albumin and urinary albumin/creatinine index, calculation of selectivity proteinuria index, determination of α-1-microglobulin, light chain immunoglobulins and urinary paraproteins.
Recent information suggests the possible importance of other potential diagnostic urinary parameters.
A number of papers have been published on the possible use of urinary clusterin concentration analysis in the assessment of type proteinuria and renal function disorders [5][6] .

METHODS
The study tested 82 individuals after obtaining their informed consent.The probands had suspect nephropathy of various etiology (49 persons with suspect early diabetic nephropathy, 5 individuals with primary glomerulopathy, 28 subjects with nephropathy due to chronic pyelonephritis).
To evaluate the presence and severity of nephropathy, the following scheme of minimally three risk parameters was used: a) decreased GF value according to MDRD (Modifi cation of Diet in Renal Disease) 7 < 1.1 ml/s, b) enhanced urinary albumin/creatinine index > 2.5 g/mol in men and > 3.5 g/mol in women, c) enhanced urinary α-1-microglobulin > 10 mg/l, d) enhanced urinary GMT/ creatinine > 94.6 nkat/mmol, c) enhanced urinary protein/creatinine > 20 g/mol.
To compare the studied parameters, the group of probands was divided according to clusterin assessment into quartiles.The presence of other measured risk pa-Urinary clusterin concentrations -a possible marker of nephropathy?Pilot study.rameters were also calculated as their overall number and then statistically processed.
The data were processed statistically by means of the MedCalc program (Belgium).Values of individual parameters were expressed as means ± standard deviation and medians.Correlations of individual parameters were studied using a Spearman correlation coeffi cient (by data distribution).A value of p < 0.05 was considered statistically signifi cant.The data were evaluated for correlations between clusterin levels (measurable vs. non-measurable) and other studied parameters (over vs. below a limit of detection) and using a chi-squared test (χ 2 -test).

RESULTS
Most data showed an abnormal distribution.Only 28 probands (34 %) had higher clusterin values than the limit of detection (50 kU/l) (Tab 1).

DISCUSSION
Clusterin (70-80 kDa; synonym ApoJ) is a glycoprotein involved in many physiological and pathophysiological processes.Two clusterin forms exist: the fi rst (secretory, exogenous) occurs in biological fl uids; the second form (endogenous) is contained in the cytoplasm and nucleus 4 .Secretory clusterin originates from preclusterin (60 kDa), which is subsequently cleaved into two chains of mature protein (alpha, beta).This form has antiapoptotic and antiangiogenetic eff ects [2][3][4] .Endogenous clusterin originates by alternative cleaving of preclusterin; the fi nal molecule acts as a strong stimulator of apoptosis 4 .
Clusterin eff ects can be summarized as follows: secretory clusterin acts as a cytoprotective extracellular chaperon and its expression and synthesis are increased in individuals suff ering from various froms of stress and especially in patients with malignant tumors [1][2][3][4] .
Extracellular clusterin plays a number of roles, e.g. in the production of amyloid in Alzheimer's disease, binding abnormal prions, "heat shock proteins" and reduced degradation of many components of the extracellular matrix.Clusterin is also involved in immune processess regulation, lipid transport, sperm maturation and protection of cell membranes 4 .
The results of experimental studies indicate the pathophysiologic importance of clusterin particularly in the pathogenesis of glomerulonephritis, polycystic kidneys, renal tubular diseases, neurodegenerative disorders, atherosclerosis, myocardial infarction and malignant tumors 4 .
As the data are still sporadic and often contradictory, the aim of the present pilot study was to determine relations between urinary clusterin concentration and other parameters of renal functions in urine, as well as assess the use of clusterin measurement in the diff erential diagnosis of proteinuria.
With regard to the presence and diagnostic importance of urinary clusterin there is ambiguity and numerous discrepancies.Several years ago, the potential importance of serum clusterin was written in the diagnosis of renal disorders; many patients with renal disorders had lower urinary clusterin, which was considered as one cause of renal diseases (e.g.clusterin defi ciency leads to activation of complement cascade and thus to possible renal injury) [5][6][7][8][9][10][11] .On the other hand, there is experimental evidence that individuals with impaired renal parenchyma or toxic injury have higher urinary clusterin concentrations (together with other proteins) 6,10,12 .This is interesting apropos clusterin production by a stressed cell; enhanced urinary clusterin levels could be permanent and independent of diuresis changes or renal perfusion 4,6 .
Our fi ndings confi rme signifi cant correlations between urinary clusterin and total protein and total protein/creatinine index, but no unambiguous proof was found for We also failed to confi rm the assumption that clusterin determination more signifi cantly correlated with tubular proteinuria than glomerular proteinuria.It should be mentioned here that clusterin in the organism is enhanced nonspecifi cally under extreme cell stress.These reasons could be limiting for the generalized diagnostic eff ectiveness of urinary clusterin in the diagnosis of nephropathy.
It can be concluded that despite the proved existence of a positive correlation between urinary clusterin and urinary total protein, urinary clusterin measurement does not confi rm any evident advantage to routinely used parameters for nephropathy diagnosis and the diff erential diagnosis of proteinuria type.

Fig. 1 .
Fig. 1.Interactive dot diagram for evaluation of the diagnostic effi cacy of urine clusterin measurement

Table 2 .
Quartiles of urine clusterin and presence of nefropathy