Quantitative Evaluation of Proteinuria by Estimation of the Protein/Creatinine Ratio in a Random Urine Sample

Abstract

The aim of this study was to evaluate the severity of proteinuria using the protein/creatinine ratio in a random urine sample. In 45 patients (male 28, female 17; mean age 50.68 ± 18.26 years) with proteinuria of various causes, we measured the 24-hour protein excretion per 1.73 m² of body surface and, during the same day, the protein/creatinine ratio in three different urine samples (8 am, 12 pm, 4 pm). The 24 h proteinuria was defined as mild (<1 g), moderate (1–3.4 g), and severe (>3.4 g) in 7, 27, and 11 patients, respectively. The sensitivity for protein/creatinine ratio compared to the 24 h proteinuria as a method of reference was 86–100% in the mild, 78–100% in the moderate, and 73–82% in the severe proteinuria, whereas the specificity was 84–100%, 78–83%, and 100% respectively. The patients with better renal function had significantly higher proteinuria levels. There was a similarity in the 24 h proteinuria and the protein/creatinine ratio measurements in all renal function and level-of-proteinuria groups. The protein/creatinine ratio of the morning and midday samples had a very good association with the 24 h sample, whereas it was not associated significantly with the evening sample (4 pm). In conclusion, the degree of 24 h proteinuria levels can be evaluated by calculating the protein/creatinine ratio in a random urine sample collected at any time from morning until midday. Protein/creatinine ratio is independent of the severity of proteinuria or renal function, and it can replace in clinical practice the cumbersome 24 h urine collections.

Keywords:

• protein-to-creatinine ratio
• proteinuria estimation
• methods
• 24 h proteinuria
• chronic kidney disease

INTRODUCTION

Twenty-four hundred years ago, Hippocrates noted the association between “bubbles on the surface of the urine” and kidney disease.^[1] Proteinuria (PR) evaluation is important for the differential diagnosis, follow-up, assessment of prognosis, and response to therapy of primary renal diseases or systemic disorders with renal involvement. Quantitation of urinary protein excretion is used in a large number of diseases and a wide range of patient groups. The method most commonly used to measure urinary protein relies on 24-hour urine collections, which are time-consuming, cumbersome, and often inaccurate. A simplified validated determination of proteinuria is obtained by measuring urine protein/creatinine concentration in a spot urine sample.^[2,3] According to the literature, the random urine protein/creatinine ratio (Pr/Cr) provides an accurate assessment of quantitative protein excretion and avoids errors and difficulties associated with 24-hour urine collection.^[4] In various studies over the years, a significant correlation between the Pr/Cr and the 24-hour protein excretion per 1.73 m² of body surface (PR/24 h/1.73 m²) has been demonstrated in several conditions, including diabetes mellitus, pregnancy, and lupus nephritis.^[5–7] In a classic study by Ruggenenti et al., Pr/Cr was found to be more accurate than the 24-hour urine protein measurement.^[8]

Moreover, a circadian rhythm of proteinuria is demonstrated in patients with glomerular disease, and, consequently, it may be important to define the optimal period of the day to obtain a random urine sample.^[9]

The aim of the present study was to evaluate the severity of proteinuria by estimation of the Pr/Cr in a random urine sample in order to define the impact of renal function in the accuracy of proteinuria assessment and to evaluate the optimal period of day for the urine sample uptake.

MATERIALS AND METHODS

Forty-five patients (28 male, 17 female, mean age 50.68 ±18.26 years) with proteinuria of various causes were studied. The primary causes of proteinuria are listed in Table 1. In the patients of the study, we measured the 24 h proteinuria (PR/24 h/1.73 m²) and, during the same day, the protein/creatinine ratio (Pr/Cr) in samples from three different times (8 am, 12 pm, and 4 pm). The study patients were divided in three groups according to the severity of proteinuria (see Table 2). The PR/24 h/1.73 m² was defined as mild (<1 g), moderate (1–3.4 g), or severe (>3.4 g) in 7, 27, and 11 patients, respectively. The studied patients were also divided in three groups according to their renal function [creatinine clearance (ClCr)]: >70 mL/min (n = 8), 10–69 mL/min (n = 16), and <10 mL/min (n = 22) (see Table 3). Creatinine clearance was estimated using the Cockroft–Gault formula. Turbidimetric method was used for the quantitation of proteinuria in the timed and random urine samples,^[10] and the enzymatic method was used for the measurement of creatinine concentration in the random urine sample (RUS).^[11]

Table 1  Primary causes of proteinuria

Glomerulonephritis	21 pts
• Vasculitis	5 pts
• SLE	4 pts
• IgA nephropathy	6 pts
• FSGS	3 pts
• Membranous nephropathy	1 pt
• Minimal change disease	2 pts
Diabetic nephropathy	9 pts
Amyloidosis	3 pts
Multiple myeloma	2 pts
Unknown origin	10 pts
Total	45 pts

Table 2  Mean levels of PR/24 h 1.73 m² and P/Cr in three different urine samples according to the level of 24 h proteinuria

Severity of proteinuria	24 h proteinuria levels (g)	PR/24 h × 1.73 m^2 (g)	Pr/Cr (8 am) (mg/mg)	Pr/Cr (12 pm) (mg/mg)	Pr/Cr (4 pm) (mg/mg)
Severe	>3.5	6.15 ± 4.63	5.95 ± 4.52	5.91 ± 4.50	4.97 ± 4.63
Moderate	1–3.4	1.88 ± 0.57	1.87 ± 0.59	1.86 ± 0.59	1.56 ± 0.51
Mild	<1	0.68 ± 0.24	0.72 ± 0.27	0.68 ± 0.24	0.64 ± 0.22

Table 3  Mean levels of PR/24 h × 1.73 m² and Pr/Cr in three different urine samples according to renal function [creatinine clearance (ClCr)]

ClCr (ml/min)	PR/24 h × 1.73 m^2 (g)	Pr/Cr (8 am) (mg/mg)	Pr/Cr (12 pm) (mg/mg)	Pr/Cr (4 pm) (mg/mg)
>70	3.60 ± 6.64	3.51 ± 6.40	3.44 ± 6.43	3.05 ± 5.86
10–69	2.90 ± 2.03	2.77 ± 1.99	2.69 ± 1.94	2.38 ± 2.15
<10	2.46 ± 1.62	2.48 ± 1.59	2.52 ± 1.58	2.20 ± 1.52

Statistical Analysis

SPSS for Windows 16.0 (SPSS Inc, Chicago, Illinois, USA) was used in analyzing the data. Normality of distributions was checked and found to be fairly Gausian. We therefore conducted parametric tests in the statistical analysis of the study. We estimated the Pr/Cr sensitivity and specificity according to the severity of proteinuria, the levels of renal function, and the time of the sampling for the RUS. Using a multivariate analysis for PR/24 h/1.73 m² and factors of sex, renal function, and method of estimation of urine protein (24 h, 8 am, 12 pm, 4 pm), we investigated the impact of the stratification according to the PR/24 h/1.73 m² levels.

We also used a multiple linear regression model for renal function with a dependent variable of the PR/24 h/1.73 m² and independent variable of the Pr/Cr in the different times in order to show which of the three samples obtained during the day can predict with better accuracy the PR/24 h/1.73 m². Results are expressed as mean ± SD, and a p value < 0.05 was considered statistically significant.

RESULTS

Using the PR/24 h/1.73 m² as method of reference, Pr/Cr sensitivity was found 86–100% in the mild, 78–100% in the moderate, and 73–82% in the severe PR/24 h/1.73 m², whereas the specificity was 84–100%, 78–83%, and 100%, respectively. These results confirm the accuracy and reliability of Pr/Cr in the assessment of proteinuria. We found no differences on concordance of PR/24 h/1.73 m² and P/Cr between male and female patients (p = NS).

As seen in Table 2, the Pr/Cr ratio was equally accurate in all levels of proteinuria (p = NS). The samples taken in the morning and at midday were not significantly different from the PR/24 h/1.73 m² samples (p = NS) for any level of proteinuria. The samples taken at 4 pm were not significantly associated with the 24 h urine samples at any proteinuria level (p = 0.021).

In the various groups of patients divided according to their renal function (see Table 3) (ClCr: >70 mL/min, 10–69 mL/min, and <10 mL/ min), there was no significant differences between the PR/24 h/1.73 m² and the Pr/Cr taken at 8 am and at midday (p = NS), nor was there any association with the evening Pr/Cr measurement (p = 0.018). The levels of Pr/Cr were independent of the renal function of the studied patients (p = NS), which means that the Pr/Cr ratio is equally accurate in any levels of renal function (p = NS).

DISCUSSION

Proteinuria is one of the most important and accurate indicators of renal injury. The qualitative and semi-quantitative proteinuria measurement in random solitary urine samples has amplified the ability of the health providers to detect and follow up primary kidney diseases and systemic conditions associated with proteinuria.^[2] The method is easy and cheap, and, in many cases, it has been substituted for the protein measurements from 24 h urine samples, which may be cumbersome and inaccurate. Protein/creatinine ratio—in general—accurate corresponds to 24 h proteinuria.^[2] In view of the circadian rhythm of proteinuria^[4] and the potential differences between sexes, we conducted this study in order to measure the accuracy of urine samples in different hours of the day.

In this study, we found no differences between sexes and a very good concordance between 24 h proteinuria and P/Cr ratio in samples taken at 8 am and 12 pm. Another point to consider is the possibility of inaccuracies in the results from the RUS P/Cr measurements due to reductions in urine creatinine excretion in the process of chronic kidney disease. PR and Pr/Cr were associated in the different levels of renal function.

Moreover, it seems that there is a significant discrepancy between the protein/creatinine ratio values given before and after midday. The outpatient clinics are very often programmed for afternoon hours. Taking the circadian proteinuria fluctuation into consideration, a possible change in the hours of the programmed appointments may cause significant differences in the expected proteinuria results. This may lead to confusing results concerning the activity of a primary or secondary renal disease and the effectiveness of a given therapy. The results of the present study may help the therapeutic community to critically assess these dynamic changes and take the appropriate logistical steps in order to avoid potentially harmful methodological errors.

The results of the study indicate that P/Cr is an accurate estimate of quantitative protein excretion. P/Cr in spot urine samples is a precise indicator of proteinuria and a reliable predictor of progression of disease in patients with chronic nephropathies, and it represents a simple and inexpensive procedure in establishing severity of renal disease and prognosis. Finally, we concluded that the degree of PR can be evaluated by calculating the P/Cr in a random urine sample collected at any time from morning until midday.

ACKNOWLEDGMENTS

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.