Dr. NK Hase brings a new masterclass pertaining to kidney disease- proteinuria. In this first part in the series, he discusses pathophysiology, types, and tests used to detect proteinuria.

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What is Proteinuria?

Increased excretion of proteins in the urine is called as proteinuria. Normal individuals excrete less than150 mg of total proteins per day. It consists of albumin less than 30 mg derived from serum, others mainly, Tamm-Horsfall/uromodulin secreted by thick ascending limb of Henle and early distal convoluted tubule, small molecular weight proteins that have filtered and escaped reabsorption or degradation by renal tubular cells and immunoglobulin IgA, and urokinase secreted by renal tubule. In adults, proteinuria is defined as protein excretion of more than 150 mg/day, in children more than 100 mg/m²BSA/day and in neonates more than 300 mg/m²BSA/day (BSA: Body surface area).

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What is the clinical importance of proteinuria?

Around 2400 years back, Hippocrates, the father of modern medicine had described association between “bubbles” on the surface of the urine and kidney disease. In the seventeenth century Frederick Dekkers demonstrated that urine samples shows coagulum after exposure to heat and acetic acid. In 1827 Richard Bright described the association between proteinuria, kidney disease, and left ventricular hypertrophy.

1. Persistent proteinuria and albuminuria is well established as a marker of structural damage to kidney.
2. Albuminuria is the earliest marker of glomerular injury occurring before reduction in glomerular filtration rate in diabetes, hypertension, obesity, and heart diseases.
3. Degree of proteinuria is responsible for pathogenesis of progression of CKD.
4. Proteinuria predicts CKD progression, cardiovascular and all cause mortality in general population.
5. There is evidence that reducing Proteinuria retards the progression of CKD.
6. The monitoring of urine protein and albumin helps in assessing response to therapy in diabetes and glomerular diseases.

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What is the pathophysiology of proteinuria?

The glomerular filtration barrier consists of three major layers:

• Endothelial cells
• Glomerular basement membrane
• Podocytes

This filtration barrier prevents the diffusion of proteins and macromolecules across the glomerular capillary wall depending on the basis of size, shape, and charge. Glomerular capillary wall is negatively charged; negatively charged protein is prevented from passing to tubular fluid to a greater extent than neutral or cationic proteins. Proteins with molecular weight less than 20000 Da can readily diffuse across the glomerular capillary wall (low molecular weight proteins: beta 2 micro globulins, retinol binding proteins, alpha1 globulins).

Albumin, the dominant high molecular weight protein in plasma, is negatively charged, approximately 67 kDa protein. Size and charge selectivity restricts the passage of albumin through the glomerular filtration barrier. The plasma proteins that are filtered at the glomerulus and present in the tubular luminal fluid are reabsorbed in the proximal tubule and catabolised by the proximal tubular cell.

Finally, urine contains little albumin derived from serum, small molecular weight proteins filtered and escaped reabsorption or degradation by renal tubular cells. The others are mainly Tamm-Horsfall/uromodulin secreted by thick ascending limb of Henle and early distal convoluted tubule, and immunoglobulin IgA, IgG and urokinase secreted by renal tubule.

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What are the factors that can increase proteinuria?

As protein is mainly handled by glomeruli and tubules, glomerular and tubular injury may result in proteinuria. The factors that can increase proteinuria are, loss of electrical charge of glomerular barrier, increased glomerular hydraulic pressure, altered permselectivity of the glomerular capillary wall, decreased tubular reabsorption and catabolism of protein, and increased plasma concentration of proteins normally filtered by the glomeruli.

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Types of proteinuria

Glomerular proteinuria

It is due to the damage to glomerular filtration barrier leading to increased filtration of plasma proteins that exceed tubular reabsorptive capacity. It is predominantly albuminuria. More than 2 g/day of proteinuria is likely to be due to glomerular origin. Proteinuria more than 3.5 g/day is called as as “nephrotic range proteinuria” almost always due to glomerular involvement.

Glomerular Proteinuria can be characterised as selective or non-selective based on the ratio of the clearance of IgG to that of albumin or transferrin in the urine (IgG is a γ globulin with MW 150 kD; transferrin is in the β1 band on protein electrophoresis and has MW 88 kD).

1. C IgG/C Albumin or C IgG/C Transferrin <0.1: Suggests highly selective proteinuria and indicates that it is primarily due to defective charge selectivity with intact size selectivity. This is characteristic of steroid responsive childhood minimal change disease (MCD). (C stands for clearance)
2. C IgG/C Albumin or C IgG/C Transferrin >0.2: Suggests non-selective proteinuria and indicates a significant component of abnormal size selectivity. The non-selective proteinuria is seen in FSGS and other non-MCD nephropathies.

Urine albumin to beta2 micro globulin ratio is >1000:1. Glomerular proteinuria may be accompanied by dysmorphic RBC or RBC cast and acanthocytes in urine.

Tubular proteinuria

It is due to inadequate reabsorption of low molecular weight proteins (beta2 microglobulin, retinol binding protein and polypeptides). This may be due to tubular or tubulointerstial injury. Tubular proteinuria may coexist with glomerular proteinuria. Typically, tubular proteinuria is <2g/day. It is not detected by dipstick. Urine albumin to beta2 miroglobulin ratio is <100:1. Urinary electrophoresis shows more globulins than albumin.

Overflow proteinuria

This is due to systemic overproduction of abnormal proteins of low molecular weight that exceeds capacity of tubular reabsorption. It is most commonly seen in multiple myeloma where abnormal immunoglobulins and light chains are produced in excess due to plasma cell proliferation. It may also be due to lysozyme (in acute myelomonocytic leukemia), myoglobin (in rhabdomyolysis), or free haemoglobin (in intravascular hemolysis). This light chain proteinuria is dipstick negative and sulfosalicylic test positive.

Post-renal Proteinuria (Tissue proteinuria)

Inflammation occurs in urinary tract by infection, stones, neoplasm, and may be associated with increased protein excretion. This protein is non-albumin predominantly IgA or IgG in small amounts.

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Methods of detection of proteins in urine

Currently two most commonly used test to detect proteinuria are:

• Standard urine dipstick
• Sulfosalicylic acid test

Other tests not commonly used nowadays are:

• Heat coagulation test
• Heller’s nitric acid test

Urinary dipstick test

This is a semi-quantitative, simple and cheap test for screening proteinuria. This dye-impregnated paper uses tetrabromophenol blue as pH indicator. Albumin in the urine binds to reagent on the strip changes its pH, which then results change in color from yellow to green. The spectrum of color change is directly depends on degree of proteinuria. The dipstick should be read in 60 seconds.

• Yellow: negative
• Trace: 15 to 20 mg/dl
• 1+: 30 to 100 mg/dl
• 2+: 100 to 300 mg/dl
• 3+: 300 to 1000 mg/dl
• 4+: >1000 mg/dl

1. The dipstick is very specific but not sensitive to low levels of albumin excretion. The lower limit of detection of albumin by standard dipstick is equal to 10 to 20 mg/dl. This is roughly equivalent to 300 to 500 mg of protein excretion in 24 hours. The patient with moderately increased albuminuria formerly called as 'micoalbuminuria' will not be detected by this strip.
2. Dipstick does not detect light chain proteins (Bence Jones Protein) and immunoglobulins in urine.
3. Dipstick gives false negative results in multiple myeloma.
4. False positive results can occur when urine is highly alkaline (pH >8), highly concentrated, presence of gross haematuria and after use of radio contrast use.
5. Urine should not be tested for proteins at least for 24 hours after radio contrast studies.

Albumin sensitive test strips

1. Albustix, Micral tests: These are the strips that are more sensitive to detect albumin concentration as low as 20 mg/dl. These are used to screen for microalbuminuria (30 to 300 mg/dl for moderately increased albuminuria) where standard urine dipstick is negative.
2. These are dye-impregnated strips with special immunoassays, and can detect albumin concentration as low as 20 mg/dl. Their sensitivity and specificity rage from 80 to 97% and 33 to 80% respectively.
3. Screening test should be followed by quantification of proteins.

Sulfosalicylic acid (SSA) test

SSA detects all proteins in urine at a sensitivity of 5 to 10 mg/dl. The SSA test requires centrifugation of urine, followed by addition of 2.5 ml of supernatant urine to 7.5ml of 3% of SSA. The presence of protein gives white precipitate/turbidity. The degree of turbidity is quantified as follows:

• 0 = Clear
• 1-10 mg/dl (opalescent trace slight turbidity)
• 15-30 mg/dl [turbidity through print can be read: (1+)]
• 40-100 mg/dl [white cloud without precipitate, heavy black line can be seen (2+)]
• 50-350 mg/dl [white cloud with fine precipitate, through heavy black line cannot be seen (3+)]
• >500 mg/dl: Flocculent precipitate (4+)

1. False positive results can be seen in patients on tolbutamide, penicillin, sulfonamide, and with gross haematuria.
2. Dipstick negative and SSA positive urine for protein suggest it may be light chain proteinuria (Bence Jones Proteinuria).
3. Once proteins are detected on dipstick or by SSA should be followed by quantification and complete urinalysis for haematuria and casts.

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Key practice points

1. The normal daily protein excretion is less than 150 mg/day (usually 40 to 80mg/day). Normal urine is composed of 40% Tamm-Horsfall protein, 40% albumin, 15% Immunoproteins and 5% other plasma proteins.(Albumin is less than 20 to 30 mg/day).
2. In adults, proteinuria is defined as excretion of protein more than 150 mg/day.
3. Proteinuria is marker of structural injury to renal parenchyma; it may be glomerular, tubular, tubulointerstitial or urinary tract.
4. Glomerular Proteinuria is due to increased glomerular capillary wall permeability to proteins and occurs in primary and secondary glomerulopathy. Proteinuria is variable, predominantly albuminuria, dipstick positive, more 2 g/24 hours usually due to glomerular involvement. >3.5 g/day is called nephrotic range. Proteinuria is always almost due to glomerular origin. It can be further classified into selective and non-selective proteinuria. Selective proteinuria is characterised by the presence of predominantly anionic proteins with a molecular weight of 50 to 80 kDa (e.g. albumin). In the case of non-selective proteinuria, larger proteins with a molecular weight >50 to 80 kDa are detected, in addition to albumin.
5. Tubular proteinuria occurs due to decreased tubular reabsorption of filtered low molecular weight proteins due to tubular cell damage- usually less than 2 g/day; dipstick negative with UACR UPCR ratio <0.4.
6. Overflow proteinuria is secondary to an increased production of low-molecular weight proteins, which are filtered by glomeruli and usually reabsorbed by the proximal tubular cells, such as immunoglobulin light chains (which are increased in monoclonal gammopathies), lysozyme (which is increased in some leukaemias), or myoglobin (which is increased in rhabdomyolysis). The degree of proteinuria is variable upto 20 g/day; dipstick negative; sulfosalicylic acid test positive.
7. Post-renal proteinuria is due to inflammation and neoplasmin in urinary tract- less than 1 g/day; dipstick negative, predominantly IgA & IgG.

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In the coming parts, Dr. NK Hase will discuss the different methods of quantification of proteins in urine, 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.