Quantitative Assessment of glomerular permeability: Proteinuria

a.      Quantitative Assessment of glomerular permeability: Proteinuria.

The normal urinary total protein excretion is <150 mg/day and about 60% of this is albumin (<30 mg/day) and some smaller proteins, together with proteins secreted by tubules, of which Tamm-Horsfall glycoprotein (THG) is the predominant (70 mg/day).

The appearance of significant amount of protein in the urine suggests renal disease. Commonly proteinuria is classified as either tubular or glomerular depending on the pattern of proteinuria observed. A third is overflow proteinuria where excess low molecular weight proteins are excessively produced.

It is recognized that 24 hour urine is definitive means of demonstrating presence of proteinuria although early morning urine or random samples are also used. Since the creatinine excretion in urine is fairly constant throughout 24 hour, measurement of protein/creatinine (or albumin/creatinine) ratio allows correction for variation in urinary concentration. So, spot urine protein/creatinine ratio (or ACR) has been used to rule out necessity for 24 hour collections. EMU sample is preferred since it correlates will with 24 hr protein excretion and excludes the orthostatic proteinuria. Multiplying PCR measured in mg/mmol by 10 gives daily protein excretion in mg/24 hr. 10 is assumed since daily excretion of creatinine depends on muscle mass and average of 10 mmol creatinine per day is assumed.

i.                    Measurement of total protein:

These includes (1) original lowry method, (2) turbidimetry after mixing with TCA or sulfoslicylic acid, (3) dye binding with comassie brilliant blue and pyrogallol red molybdate.  For measurement of individual protein in urine, the most common approach today employs a light-scattering immunoassay with either turbidimetric or nephelometric detection of immunoaggregate formation.

Dry chemistry systems have been developed for quantitation of albumin in urine. For example in one such device the urine albumin flows laterally along a porus matrix through an area containing gold particle labeled antibodies to albumin. In the presence of albumin, these antibody molecules are neutralized and pass through a portion of the matrix containing immobilized albumin to a detection zone, where they appear as a pink coloration. The excess labeled antibody is captured by the immobilized albumin.

ii.                  Urinary albumin and microabluminuria screening:

Microalbuminuria (30-300 gm/day) is defined as an increase in urinary excretion of albumin above the reference interval for healthy non diabetic subjects but at a concentration that is not generally detectable by crude clinical tests, such as dipsticks.  It is now a clinical indicator of deteriorating renal function in diabetic subjects to predict the risk of renal and cardiovascular disease.

The diagnosis of microabluminuria requires presence of increased albumin excretion (either increased ACR or increase albumin excretion rate) in at least two out of three urine sample collected in the absence of infection or an acute conditions like intercurrent illness. Screening should commence 5 years after diagnosis in patients with type 1 diabetes and at diagnosis in patients with type 2 diabetes without proteinuria. Screening is not indicated in patients with established proteinuria. An ACR <23 mg/g (<2.5mg/mmol) in male or <32 mg/g (<3.5 mg/mol) in female requires no further investigation until next annual review. Patients with ACR above or equal to this cut-off should have urine samples sent to laboratory on two further occasions (ideally within 1 month) for albumin estimation. Patients demonstrating increased ACR in one or both of these further samples have microabluminuria. Other conditions of albuminuria should be considered e.g. menstrual contamination, vaginal discharge, uncontrolled hypertension, UTI, uncontrolled diabetes, heart failure, intercurrent illness and strenuous exercise.

Once microabluminuria has been established an angiotensin converting enzyme (ACE) inhibitor should be prescribed as this has antiproteinuria effect and it will also reduce rate of fall in GFR. 

Microalbuminuria is also associated with risk of CVD as it indicates generalized vascular endothelial dysfunction and currently it is being increasingly used in cardiovascular risk assessment clinics.

i.                    Bence jones proteinuria:

Presence of light chains (Bence Jones proteins, 22 kDa) in the urine is an important indication of the presence of myeloma. Classical heat test and Bradshaw test (urine is layered over conc nitric acid which forms round ppt), electrophoresis supplemented by immunofixation is the most reliable approach.

ii.                  Tubular proteinuria:

Tubular integrity and its functional status can be measured by measuring urinary concentrations of low molecular weight proteins using immunoassay technology. These are freely filtered at glomerulus and then reabsorbed and catabolized within the proximal tubule. Increased quantities in urine reflect failure of tubular reabsorptive mechanisms. The most commonly measured proteins are retinol binding protein (RBP) and α₁-microglobulin.  α₁-microglobulin (31 kDa) is also referred as protein HC because of its human complex-forming capacity with IgA. It is synthesized by liver and freely filtered at glomeruli. RBP (22 kDa) is also synthesized by liver and is found in plasma as complex with prealbumin; the protein is the carrier protein for vitamin A. Urinary RBP is more sensitive than α₁-microglobulin to detect tubular damage but the higher concentration and excellent stability of the α₁-microglobulin in human urine ex vivo facilitate its use as a marker of tubular damage. THG located in thick ascending limb of loop of Henle, has also been used as marker of more distal tubular damage.

Tubular damage can be demonstrated by identifying intracellular components into the urinary tract and their measurement which reflects functional integrity of tubule. The enzyme N-acetyl-β-D-glucosaminidase (NAG) is an example. Measurement of α-glutatione S-transferase (for proximal tubular damage) and π-glutatione S-transferase (for distal tubular damage) are also been proposed.

iii.                Characterization of proteinuria:

As glomerular damage increases the permeability of membrane increases with an increasing proportion of HMW proteins appearing in urine. The relative clearance of a number of proteins has been measured to assess the selectivity of membrane and provide an assessment of glomerular damage.