The significance of proteinuria should always be evaluated in light of the method of collection, urine specific gravity, and measurement technique. The presence of protein in a urine sample collected by catheterization or manual expression may contain blood which by definition contains RBC and blood proteins; the proteinuria in that case is iatrogenic. The amount of protein assessed by qualitative methods such as colormetric dip sticks becomes more significant as the urine decreases in urine specific gravity. A +1 protein in a urine sample with a 1.003 specific gravity is much more significant than a +1 protein reading in a urine sample with a 1.030 specific gravity.

Protein can be measured by semiquantitative methods including colormetric dip sticks, turbidometric methods or by quantitative methods. The semiquatitative methods of protein measurement may result in erroneous results including both false positive and false negative results. Quantitative methods are less likely to return false positive or false negative results.

top of page

Colormetric dip stick protein readings

The changes on the color pads range from negative to +4 with the following approximate concentration correlations:

The chemicals on the color pads are more sensitive to the detection of albumin than globulins, Bence Jones proteins (the light chains of immunoglobulin molecules) or other proteins. These other proteins can be detected by the dipstick but only when present in large quantities. The lower limit of sensitivity is ~ 20 mg/dl protein.

The following readings are considered normal

The dipstick may register a false negative if the urine is very dilute urine. A false negative means that a significant amount of protein is actually present but the large urine volume dilutes the protein so its concentration is less than 20 mg/dl which is the lowest concentration recorded by the dipstick. The presence of Bence Jones proteins may also result in a false negative (may not be measured as protein). False positive color changes may be recorded if the urine sample is contaminated with the antiseptic, chlorhexidine (Novalsan) or if the urine sample is markedly alkaline. The H2 blocker, ranitidine, may also cause false positive readings. The range of pH of dog and cat urine in both health and disease rarely results in a pH high enough to result in a false positive reading.

top of page

Turbidimetric methods for protein measurement

Turbidimetric tests are based upon the precipitation of protein by the addition of sulfosalicylic acid (SSA) resulting in relative degrees of turbidity of the urine sample. The sample on the far left is strongly positive (+4) for protein and the sample on the near left is negative. This test has a lower limit of sensitivity of ~ 5 mg/dl protein.

False positive readings may occur if the urine contains radiographic contrast agents or large amounts of antibiotics including penicillins, cephalosporins or sulfonamides. In treatment of higher bacterial infections, actinomyces and nocardia, the doses of penicillins (actinomyces) or sulfonamides (nocardia) used in treatment, often exceed the doses of the same drugs used to treat other diseases.

Most laboratories routinely perform SSA testing on all urine samples or at least those that are positive by dip stick.

top of page

A new protein detection test the E.R.D. screening test was recently (2002) released by the Heska Corporation. The test is an in-office immunoassay that detects low levels of albumin in the urine of dogs. It is specific for albumin, not other types of protein. This test detects microalbuminuria which is defined as a concentration of albumin greater than 1 mg/dl but less than 30 mg/dl which is approximately the lower limit of protein detection using a dipstick.

Microalbuminuria has been reported to be an early indicator of progressive renal disease in humans with hypertension and diabetic nephropathy. Studies are underway in dogs to determine if the ERD test can be used to detect dogs with early renal dysfunction. Some preliminary reports, published as ACVIM abstracts are posted at: http://www.heska.com/erdscreen/erd_studies.asp

In addition to this test detecting lower concentrations of albumin compared with dipsticks, studies performed by the manufacturer state that some dogs with trace positive protein using a dipstick are false positives in 66% (33 of 50 urine samples) as defined by a negative ERD test.

Persistent microalbuminuria indicates ongoing glomerular damage which may be due to primary renal disease or secondary to a variety of systemic diseases. This does not mean that all persistently microalbuminuric dogs will progress to end stage renal disease although the manufacturer states that persistently microalbuminuric dogs are “at risk” for developing end stage renal disease.

Blood contamination will interfere with the ERD test when the urine sample is visibly discolored. The affect of lower urinary tract inflammation on ERD results is variable. In one study about half of dogs with lower urinary tract inflammation were negative for microalbuminuria. Therefore a positive ERD test in the presence of lower urinary tract inflammation does not diagnose nor preclude the existence of underlying glomerular disease. Re-testing after successful treatment of the lower urinary tract disease is recommended.

Preliminary results suggest that corticosteroids may increase the severity of microalbuminuria measured by the ERD test.

"A screening test is now also available for cats. 1243 cats visiting 59 clinics were tested for microalbuminuria in their urine. 24.5% of cats tested positive for microalbuminuria with the E.R.D.-HealthScreen™ Feline Urine Test. A statistically significant correlation (P <0.0001) was found between increasing age and a microalbuminuria positive test result. Observed prevalence of microalbuminuria in apparently healthy cats and increased prevalence of microalbuminuria in cats with medical conditions support routine testing to assess the current health status of the patient and to alert the veterinarian to identify and treat potential causes of renal damage."
http://www.heska.com/erd/erd_datacat.asp

Quantitative tests

The dipstick readings are "semi quantitative" with the degree of positivity providing some estimate of the amount of protein contained in the urine sample. Quantitative tests (e.g. biurete method) use chemical methods to provide an actual measure of protein in the urine. Urine protein should be quantitated when glomerular proteinuria is suspected in order to provide an objective baseline against which to compare the effects of treatment and to follow over time in order to determine progression or regression of disease. Quantitative tests should also be performed if the results of semi quantitative methods are in question (e.g. you are concerned that dipstick or turbidometric methods are yielding false positive or false negative results). Quantitative tests require a timed collection of urine or can be accurately estimated by a protein/creatinine ratio.

Timed urine collection

To perform a timed urine collection, collect all the urine produced over a period of at least 24 hours. The longer the collection period, the less significant are errors in which part of the urine produced is lost to collection. See the section on creatinine clearance for details of timed urine collections. Measure the volume of urine produced and determine what the 24 hour urine production would be. Send a sample of the collected urine to a laboratory for a chemical determination of protein concentration. This will be reported in units of mg/dl or mg/ml. Multiply the 24 hour urine volume by the protein concentration to determine total quantity of protein lost in the urine over 24 hours.

urine protein (mg/dl) X urine volume/24hours (dl) = mg of protein/24hours
one deciliter (dl) = 100 ml
Normal ~ < 22mg/kg/day

Example:

50 kg dog
24 hour urine production = 1,500 ml
protein concentration of 10 mg/dl
15 dl x 10 mg/dl = 150 mg
150 mg/ 50 kg = 3 mg/kg/24 hours

top of page

Protein/creatinine ratio (UPC ratio)

The protein/creatinine ratio (UPC ratio) is not affected by urine concentration or volume and correlates well with 24 hour urine protein excretion. Because the correlation of UPC ratios to timed 24 hour protein losses is good, there is little reason to perform timed urine collection to determine magnitude of proteinuria. UPC ratios are determined from a single urine sample. The urine can be collected by any collection method although potentially traumatic methods such as catheterization and manual expression can create iatrogenic hematuria and the blood proteins will result in a high upc ratio. There is little information be gained from performing a UPC ratio on grossly bloody urine. Remember that blood proteins are measured in units of g/dl whereas urine protein is usually measured in mg/dl.

UPC results

• < 0.5 normal
• 0.5 - 1.0 questionable significance
• > 1.0 abnormal

Formulas and graphs are published in text books that will allow you to convert UPC to a daily urine protein loss in units of mg/day. These formulas are specific to the technique used to measure urine protein. UPC ratio's do not identify the underlying disease process or location of lesion (pre- or postglomerular, glomerular or tubular).

top of page

Sources of urine protein

The blood entering the afferent arteriole into the capillary loop of the glomerulus contains plasma protein at an approximate concentration of 6 g/dl and an albumin concentration of approximately 3 g/dl. Albumin is small enough that some of it will leave the capillary lumen and enter glomerular filtrate. Because a large amount of glomerular filtrate is made (as much as 100 liters in large dogs), even though the concentration of albumin in glomerular filtrate is low (~2 mg/dl) the actual amount of albumin in glomerular filtrate is large.

The epithelial cells lining the renal tubules will reabsorb most of the albumin from glomerular filtrate before it is excreted as urine. Additionally the tubular epithelial cells secrete Tamm Horsfall mucoproteins into tubular fluid.

As the urine is eliminated through the urethra, the genital tract adds proteins to the urine. Immunoglobins (IgA) which are locally produced in the urinary tract are also added to the urine.  Therefore what is measured in the urine as protein may originate from all of the above sources.

Types of proteinuria

• functional (transient)
• over flow (preglomerular)
• glomerular
• tubular
• secretory (post glomerular)

A transient increase in glomerular permeability can occur as a consequence of heavy physical activity (exercise, seizures), fever, or congestive heart failure resulting in proteinuria in the absence of renal disease. The proteinuria is reversible if the under lying disease/circumstance which caused the increased permeability is reversible. If one of the previous circumstances/diseases exists, urine protein should be re evaluated several times to determine if it is transient or permanent. This type of proteinuria is also called "functional" proteinuria.

Over flow (preglomerular) proteinuria is the presence of protein in the urine that originated from a small protein which is increased in blood. Substances which may be increased in the blood and which are small enough proteins to pass the glomerular filtration barrier and enter the urine include hemoglobin, myoglobin, or Bence Jones proteins. Bence Jones proteins are the light chains of immunoglobin molecules which are produced by abnormal plasma cells in patients with multiple myeloma (plasma cell tumor). Bence Jones proteins will not be measured by the semiquantitative methods of protein determination but will be measured by quantitative methods. To determine if proteinuria is overflow, look at the patient's blood. Is the plasma red indicating the presence of free hemoglobin from intravascular hemolysis or free myoglobin released from muscle damage? Is the plasma globulin markedly increased as it will be in patients with multiple myeloma? If the protein in the urine is hemoglobin or myoglobin the urine sample will be red colored.

A small amount of albumin will normally pass the glomerular filtration barrier and enter tubular fluid. In health, tubular epithelial cells will reabsorb most of the filtered albumin and return it to the blood. If the tubules are diseased they may not reclaim the filtered albumin resulting in its loss in urine. Other evidence of tubular malfunction includes failure to reabsorb filtered glucose leading to glucosuria, isosthenuric urine specific gravity indicating inability to remove water from tubular filtrate and the presence of casts.

Glomerular proteinuria is generally caused by either immune complex glomerulopathy or amyloidosis.

Secretory (post glomerular) proteinuria originates from hemorrhage, inflammation, or infection of the kidneys, lower urinary tract or genital tract. When ever RBC or WBC are observed in the urine sediment, protein will also be present as blood contains both RBC and blood proteins and WBC are usually accompanied by proteins (immunoglobulins).

top of page

Assessing the significance of proteinuria

To summarize, when a urine sample contains protein you should evaluate the significance of that protein as follows:

• it may be normal, if a small amount is present in a concentrated urine sample
• rule out false positives (dipstick or turbidometric)
• determine if persistent
• look at blood for possible causes of protein overflow and evaluate urine color (hemoglobin or myoglobin will color the urine red)
• rule out secretory proteinuria by a sediment exam
• determine if tubular by looking for other signs of tubular dysfunction
• if everything above is not true, then the protein is glomerular in origin