Dr. NK Hase in the second part of the series on proteinuria writes on protein quantification methods, comparing and listing the advantages and disadvantages as well as pointers to remember while selecting patients for each of these tests.

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Why are quantification of proteins in urine important?

Semiquantative spot urine protein estimates are concentration dependent. Highly concentrated urine may show abnormal results ++, +, even when the absolute daily protein excretion is normal. Highly dilute urine may show normal or only modestly-elevated results for proteins (trace or +) concentration even when significant elevated amount of proteins are excreted.

Proteinuria may vary from day to day, timing of the day, posture and activity. The degree of the proteinuria is prognostically important in glomerular diseases. Higher the proteinuria, more rapid the progression to kidney failure. Quantification of proteinuria is important to monitor response to therapy.

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24-hour urinary protein or albumin excretion

The 24-hour urine collection and estimation of protein excretion is still considered as the 'gold standard' method for quantification of urinary protein and urinary albumin. The patient should be given written instructions about collection and preservation. First morning void should be discarded and second day morning sample should be included.

Adequacy of the collection can be verified by quantifying the 24 hours urinary creatinine in the same sample. It should be roughly 20 to 25 mg/kg in males and 15 to 20 mg/kg/day. If creatinine excretion is less than expected, error in sample collection should be suspected.

The main advantage of this timed urine collection for 24 hours is, it take into account protein fluctuations during the day resulting from changes in body composition, activity, body positions, emotional state, protein consumption and blood pressure.

Disadvantages

1. It is time consuming, cumbersome, inconvenient and more expensive to the patient.
2. There are always chances of under or over collection even in well-motivated patients.
3. 24 hours urine collection is difficult to carry out in children, elderly, intectually or physically handicapped and patients with incontinence.

A 24 hours urinary protein is preferred in patients whom urine creatinine excretion is less reliable, changing renal function is high and muscle mass low.

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Urinary protein/creatinine ratio (UPCR) & urinary albumin/creatinine ratio (UACR)

The spot (random) urine protein to creatinine ratio (P/C ratio) is an alternative, fast and simple method of detecting and estimating the quantitative assessment of proteinuria. The ratio of protein to creatinine is independent of urinary concentration and dilution.

Most commonly, the urine protein-to-creatinine ratio (UPCR) in a spot first- or second-morning urine sample after avoiding exercise is used to estimate 24-hour proteinuria.

The urine protein concentration in a spot sample is measured in mg/dL and is divided by the urine creatinine concentration, also measured in mg/dl. The protein to creatinine ratio in a normal person is less than 0.2 and is roughly equivalent to an excretion of less than 0.2 g per day/1.73m²..A ratio of 3 roughly translates to 24 hours protein excretion of 3 g.

If SI units are used, the value for the UPCR (or UACR) in units of mg of protein per g of creatinine is divided by 8.8. As an example, a UACR of 30 mg/g creatinine is equivalent to 3.4 mg/mmol creatinine. The accuracy of the total UPCR is related to the assumption that daily creatinine excretion is only slightly more than 1000 mg per 24 hours per1.73 m².

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What are the limitations of UPCR?

Although the UPCR correlates well with 24 hours proteins excretion on a population level, its usefulness in prediction, the true 24 hours proteins excretion in any given individual is debatable.

The UPCR and UACR are heavily influenced by the urinary creatinine concentration (the denominator of the ratio) and therefore by the total daily creatinine production. Urine protein excretion can vary through the day and day to day.

The accuracy of the ratio is diminished if creatinine excretion is either markedly higher or lower than the average population value of 1000 mg/day.

Individuals with large muscle mass in whom creatinine excretion may be much higher than 1000 mg/day. The UPCR and UACR will underestimate proteinuria.

In a cachectic patient with small muscle mass, creatinine excretion may be much lower than 1000 mg/day. The UPCR and UACR will overestimate proteinuria.

Lack of correlation between spot urine P/C ratio and protein concentration evaluated in 24-hour urine collection- if daily protein excretion exceeds 6 g/day and GFR <15 ml/min/1.73m². The spot urine P/C ratio result depends on ethnicity (lower values are observed in the African-Americans, because they excrete more creatinine to urine compared to Caucasians with similar height and weight).

1. Poor or lack of correlation between spot urine P/C ratio and protein concentration in 24-hour urine collection is in patients with minimal change disease or membranous nephropathy with nephrotic-range proteinuria.
2. The spot urine P/C ratio may be expressed in different units, i.e. in mg/mg, mg/g, mg/mmol, and it is often reported simply as a numerical value (without units, especially when it is reported in mg/mg). The use of different units can lead to difficulties in interpreting the results for both the physician and the patient.
3. The spot urine P/C ratio stronger correlated with protein concentration in 24-hour urine collection if the proteinuria ranged from 0.3 to 3.5 g/day (300 to 3449 mg/day) compared to patients with nephrotic syndrome >3.5 g/day (>3500 mg/day); no correlation was observed when the protein excretion exceeds 6 g/day.
4. The spot urine P/C ratio may be overestimated at GFR levels 10 mL/1.73m²

The diversity of methods used to assess protein and creatinine concentration make the comparison of spot urine P/C ratio results between laboratories impossible. Therefore, for the diagnosis of proteinuria, a physician using the P/C ratio should use results obtained in a single laboratory using the same analytical conditions.

Verification of the P/C ratio by means of protein concentration in 24-hour urine collection is needed before important diagnostic and therapeutic decisions e.g. before biopsy or before the change of immunosuppressive agents.

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What is utility of simultaneous measurement of UACR and UPCR?

Simultaneous measurement of UACR and UPCR allows for the calculation of the urine albumin/total protein ratio (UAPR). The UAPR aids in diagnosis of tubular proteinuria, a cut-off of<0.40 was 88%sensitive and 99% specific for diagnosis of tubular proteinuria. Total proteinuria more than albuminuria suggests tubular proteinuria.

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What is the relationship between UACR and UPCR?

Urine albumin-to-creatinine ratio (ACR) and protein-to-creatinine ratio (PCR) are used to measure urine protein. Majority of recent guidelines recommend quantifications of albuminuria rather than total proteinuria for screening of early kidney damage in diabetes, hypertension and cardiovascular diseases. UACR also has been incorporated into equations to predict risk of kidney failure. Finally, ACR is more sensitive than protein to-creatinine ratio (PCR) in detecting the onset of diabetic nephropathy.

Albumin is the predominant urinary protein in the majority of kidney diseases, and it is possible to accurately measure urine albumin at levels in the physiologic range. Despite this many laboratories estimate only proteinuria rather than albuminuria. KDIGO guidelines divides albuminuria into 3 categories A1, A2, A3.

Measure	Normal to mildly increased (A1)	Moderately increased (A2)- Microalbuminuria	Severely increased (A3)- Overt proteinuria
Protein reagent strip	Negative to trace	Trace to +	+ or greater
Protein excretion rate (PER: mg/24 hours)	<150 mg/24 hours	150 to 500	>500
Albumin excretion rate (AER: mg/24hours)	<30	30 to 300	>300
PCR: mg/g          mg/mmol	<150 <15	150 to 500 15 to 50	>500 >50
ACR: mg/g          mg/mmol	<30 <3	30 to 300 3 to 30	>300 >30

Albuminuria and Proteinuria can be measured using excretion rates in timed urine collections, ratio of concentrations to creatinine concentration in spot urine samples, and using reagent strips in spot urine samples. Relationship among measurement methods within a category are not exact. For example, the relationships between AER and ACR and between PER and PCR are based on the assumption that average creatinine excretion rate is approximately 1.0 g/d or 10 mmol/d

The conversions are rounded for pragmatic reasons (for an exact conversion from mg/g of creatinine to mg/mmol of creatinine, multiply by 0.113). Creatinine excretion varies with age, sex, race and diet; therefore the relationship among these categories is approximate only.

• ACR <10 mg/g (<1 mg/mmol) is considered normal
• ACR 10 to 30 mg/g (1 to 3 mg/mmol) is considered 'high normal'
• ACR >2200 mg/g (>220 mg/mmol)which is approximately equivalent to PER>3500 mg/24 hours or PCR (proteinto-creatinine ratio) >3500 mg/g [>350 mg/mmol] is called 'nephrotic range'

The relationship between urine reagent strip results and other measures depends on urine concentration. Therefore quantification is required.

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What is eAER?

Time urine collection either 24 hours or overnight for 8 hours is considered as a gold standard method for measurement of albuminuria. Considering several limitations of the UACR because of variability in creatinine excretion several investigators have developed an 'estimated albumin excretion rate' to predict 24 hours albumin excretion by using factors which affect creatinine generation.

eAER was derived in a large study of 1693 patients with kidney disease using age, sex, race and UACR and then validated in two large cohorts of patients with CKD and diabetes. The estimated albumin excretion rate (or eAER) can be calculated by multiplying the spot UACR by the expected 24-hour creatinine generation (which can be calculated using a variety of equations- online calculators are available).

The eAER was significantly more accurate and less biased than the ACR in predicting the measured 24-hour albumin excretion. Accuracy may be further increased if the eAER is calculated using values from a timed urine collection of 12 hours or more. Using the eAER rather than the ACR to estimate albumin excretion may be particularly important in patients who have very large or very small muscle mass (e.g. young athletic men and older adult women, respectively).

Estimated protein excretion rate (ePER) can be calculated by multiplying UPCR by 24 hours creatinine generation (online calculators are available).

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What is the difference between proteinuria and albuminuria?

Proteinuria means total proteins (albumin, globulins, low molecular weight proteins, immunoglobulins) and albuminuria means only albumin excretion. Proteinuria and albuminuria are not strictly interchangeable terms. Albuminuria is a marker of glomerular injury. It is derived from circulation. Proteinuria is marker of structural injury to renal parenchyma; it may be glomerular, tubular, tubulointerstitial or urinary tract.

• The normal mean albumin excretion rate (AER) is 5 to 10 mg/day
• AER more than 30 mg/day is considered as abnormal
• AER between 30 to 300 mg/day or 20 to 200 mcg/min is called moderately increased albuminuria; previously this was called as microalbuminuria or subclinical proteinuria; this is not detected by dipstick (dipstick negative)
• AER level greater than 300 mg/day is called severely increased albuminuria; previously this was called as macroalbuminuria or overt proteinuria or clinical proteinuria; this is detected by dipstick

Albuminuria is the earliest marker of kidney involvement in diabetes occurring before a reduction in glomerular filtration rate.

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What are the issues with measuring urine albumin?

Abnormal excretion of small amount of albumin in urine called as microalbuminuria currently designated by KDIGO as moderately increased albuminuria is an early marker of kidney injury in diabetes, hypertension and is a biomarker of endothelial dysfunction.

Standard dipstick cannot detect moderately increased albuminuria (microalbuminuria). Therefore, in dipstick-negative diabetic and hypertensive patients, urine albumin should be estimated by using albumin creatinine ratio (UACR) in early morning, overnight first voided urine sample to detect early glomerular injury.

• UACR value less than 30 mg/g is considered as normal
• 30 to 300 mg/g indicate moderately increased albuminuria (microalbuminuria)
• Values more than 300 mg/g is considered as severely increased albuminuria (macroalbuminuria or overt proteinuria) and is detected by standard routine dipstick

UACR is a more sensitive screening test than UPCR. Both have similar limitations. UPCR is less expensive and used in patients with established proteinuria more than 300 mg/day.

The National Kidney Foundation (NKF-DOQI) and Kidney Disease Improving Global Outcome (KDIGO), recommends annual screening for proteinuria in diabetes by UACR in early morning overnight sample. If it comes positive, it is confirmed with two additional first void specimens collected over the next 3 to 6 months. Two of the three samples positive for microalbuminuria are considered as persistent microalbuminuria requiring treatment.

Before testing, albuminuria diabetes should be well controlled. Hyperglycaemia, ketosis can lead to transient increase in albuminuria. Treat urinary tract infections, and advise patients to avoid vigorous exercise and smoking 24 hours before testing for albuminuria.

Direct measurement of albuminuria with traditional immunological-based laboratory methods such as immunonephelometry, immunoturbidimetry, and radioimmunoassay can produce results that vary considerably. Therefore, there is the need for standardisation of methods and protocol used for testing urinary albumin.

High performance liquid chromatography based measurement is able to assess all intact urinary albumins including immune non-reactive albumin. HPLC is a more sensitive and specific method for early detection of albuminuria. This may become the method of choice in the future.

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In the coming parts, Dr. NK Hase will discuss the protein evaluation methods, treatment approach to proteinuria and albumiuria, and case discussions on the topic.

Disclaimer- The views and opinions expressed in this article are those of the author's and do not necessarily reflect the official policy or position of M3 India.

The author Dr. NK Hase is a Director clinical Nephrology & Transplant working at Jupiter Hospital, Thane and former Professor & Head of Department of Nephrology Seth GS Medical College and KEM Hospital, Mumbai.