The nuts and bolts of proteinuria (Proceedings)

Persistent proteinuria of renal origin is an important marker of chronic kidney disease (CKD) in dogs and cats. Unfortunately, due to the high incidence of false-positive results for proteinuria on the urine dipstick screening test and proteinuria associated with lower urinary tract inflammation, positive reactions for urine protein are quite common and therefore often disregarded. Ruling out false-positive proteinuria and identifying proteinuria of renal origin are necessary first steps when evaluating the results of tests for proteinuria. In the case of CKD, albumin is usually the primary component of renal proteinuria. In addition to being a diagnostic marker for CKD, the potential for renal proteinuria/albuminuria to be a mediator of CKD progression also exists. The recent development of species specific albumin ELISA technology that enables detection of low concentrations of canine and feline albuminuria has stimulated discussion about what level of proteinuria/albuminuria is normal and what levels may be associated with renal disease progression. For these reasons, detection and monitoring of renal proteinuria in dogs and cats has recently received renewed interest. Perhaps somewhat similar to our changing definition and treatment guidelines for systemic hypertension, the need to recognize, monitor, and potentially treat renal proteinuria, that not long ago may have been considered normal, is increasing.

Screening tests for proteinuria:

Proteinuria is routinely detected by semi-quantitative, screening methods, like the conventional dipstick colorimetric test (very common) and the sulfosalicylic acid (SSA) turbidimetric test (less common). The dipstick test is inexpensive and easy to use. This test primarily measures albumin, however both the sensitivity and specificity for albumin are relatively low with the dipstick methodology. False-negative results (decreased sensitivity) may occur in the setting of Bence Jones proteinuria, low concentrations of urine albumin, and/or dilute or acidic urine. The conventional dipstick test has a sensitivity level of > 30 mg/dl. False-positive results (decreased specificity) may be obtained if the urine is alkaline or highly concentrated or the dipstick is left in contact with the urine long enough to leach out the citrate buffer that is incorporated in the filter paper pad. False-positive results with the dipstick occur more frequently in cats compared with dogs but are common in both species.

The SSA test is performed by mixing equal quantities of urine supernatant and 3-5% SSA in a glass test tube and grading the turbidity that results from precipitation of protein on a 0 to 4+ scale. In addition to albumin, the SSA test can detect globulins and Bence Jones proteins. False-positive results may occur if the urine contains radiographic contrast agents, penicillin, cephalosporins, sulfisoxazole, or the urine preservative thymol. The protein content may also be overestimated with the SSA test if uncentrifuged, turbid urine is analyzed. False-negative results are less common in comparison with the conventional dipstick test due to the increased sensitivity of the SSA test for protein (> 5 mg/dl). Because of the relatively poor specificity of the conventional dipstick analysis, many reference laboratories will confirm a positive dipstick test result for proteinuria with the SSA test. Grading of both the color change on the dipstick test and the turbidity on the SSA test is subjective and therefore results can vary between individuals and laboratories.

Proteinuria detected by these semi-quantitative, screening methods has historically been interpreted in light of the urine specific gravity and urine sediment. For example, a positive dipstick reading of trace or 1+ proteinuria in hypersthenuric urine has often been attributed to urine concentration rather than abnormal proteinuria. In addition, a positive dipstick reading for protein in the presence of microscopic hematuria or pyuria was often attributed to urinary tract hemorrhage or inflammation. In both examples, the interpretation may not be correct. Given the limits of the conventional dipstick test sensitivity, any positive result for protein regardless of urine concentration may be abnormal (except in the case of false-positive results). Likewise, hematuria and pyuria have an inconsistent effect on urine albumin concentrations; not all dogs with hematuria and pyuria have albuminuria.

Localization of proteinuria:

When proteinuria is detected by screening tests, it is important to try to identify its source. Proteinuria may be caused by physiologic or pathologic conditions. Physiologic or benign proteinuria is often transient and abates when the underlying cause is corrected. Strenuous exercise, seizures, fever, exposure to extreme heat or cold, and stress are examples of conditions that may cause physiologic proteinuria. The mechanism of physiologic proteinuria is not completely understood; however, transient renal vasoconstriction, ischemia, and congestion may be involved.

Pathologic proteinuria may be caused by urinary or non-urinary abnormalities. Non-urinary disorders associated with proteinuria often involve the production of small-molecular-weight proteins (dysproteinemias) that are filtered by the glomeruli and subsequently overwhelm the reabsorptive capacity of the proximal tubule. An example of this "pre-renal" proteinuria is the production of immunoglobulin light chains (Bence Jones proteins) by neoplastic plasma cells. Genital tract inflammation (e.g., prostatitis or metritis) can also result in pathologic non-urinary proteinuria (post-renal proteinuria). Obtaining urine samples via cystocentesis reduces the potential for urine contamination with protein from the lower urinary tract.

Pathologic urinary proteinuria may be renal or non-renal in origin. Non-renal proteinuria most frequently occurs in association with lower urinary tract inflammation or hemorrhage (also referred to as post-renal proteinuria). Changes observed in the urine sediment are usually compatible with the underlying inflammation (e.g., pyuria, hematuria, bacteriuria, and increased numbers of transitional epithelial cells). On the other hand, renal proteinuria is most often caused by increased glomerular filtration of plasma proteins associated with intraglomerular hypertension or the presence of immune complexes, structural damage, or inflammation in the glomerular capillaries. Renal proteinuria may also be caused by decreased reabsorption of filtered plasma proteins due to tubulointerstitial disease. In some cases, tubulointerstitial proteinuria may be accompanied by normoglycemic glucosuria and increased excretion of electrolytes (e.g., acute tubular damage). Glomerular lesions usually result in higher magnitude proteinuria compared with proteinuria associated with tubulointerstitial lesions. Renal proteinuria caused by glomerular and tubular disease is most frequently accompanied by an inactive urine sediment, the exception being the presence of hyaline casts. In addition to glomerular and tubulointerstitial disease, renal proteinuria may be caused by inflammatory or infiltrative disorders of the kidney (e.g., neoplasia, and pyelonephritis) which are often accompanied by an active urine sediment.

Detection of albuminuria/microalbuminuria:

Albuminuria can be measured by point-of-care, semi-quantitative tests (e.g., the Heska E.R.D.-HealthScreen™ Urine test) and quantitative immunoassays at reference laboratories (Heska and Antech). Like proteinuria, albuminuria can be caused by pre- and post-renal disorders and therefore it is important to localize the source of albuminuria as discussed above. Microalbuminuria (MA) is defined as concentrations of albumin in the urine that are greater than normal but below the limit of detection using conventional dipstick urine protein screening methodology (i.e., ≤ 30 mg/dl). Urine albumin concentrations above 30 mg/dl are referred to as overt albuminuria and can often be detected using the routine screening tests as well as the urine protein/creatinine ratio (UP/C).

Indications for the use of MA tests include: 1) When conventional screening tests for proteinuria produce equivocal or conflicting results or false-positive results are suspected. 2) When conventional screening tests for proteinuria are negative in apparently healthy, older dogs and cats and a more sensitive screening test is desired. 3) When conventional screening tests for proteinuria are negative in apparently healthy, young dogs and cats with a familial risk for developing proteinuric renal disease and a more sensitive screening test is desired. 4) When conventional screening tests for proteinuria are negative in dogs and cats with chronic illnesses that are associated with proteinuria renal disease and a more sensitive screening test is desired. 5) When a previous MA test(s) was positive and monitoring for persistence and/or progression of the MA is desired.

Comparison of screening tests, up/c, and quantitative elisa for detection of canine and feline albuminuria:

Compared to the ELISA, positive dipstick and SSA results (≥ trace positive reaction) in canine urine samples have only moderate specificity and poor positive predictive value. These values improve when stronger positive dipstick and SSA results (≥ 2+ positive) are compared to the ELISA with specificity and positive predictive values > 90%. In cat urine, both the dipstick and SSA test have poor specificity and positive predictive value regardless of the magnitude of the positive reaction. Urine protein/creatinine ratio results have high specificity for canine and feline albuminuria (> 99%), but relatively low sensitivity (< 30%).

Quantitation of proteinuria:

If the results of the screening tests suggest the presence of renal proteinuria/albuminuria, urine protein excretion should be quantified. This helps to evaluate the severity of renal lesions and to assess the response to treatment or the progression of disease. Methods used to quantitate proteinuria include the UP/C and immunoassays for albuminuria that are expressed as either urine albumin/creatinine ratios or in mg/dl in urine samples that have been diluted to a standard urine specific gravity (e.g., 1.010). Albumin ≥ 30 mg/dl in urine that has been diluted to a specific gravity of 1.010 will usually result in UP/C's above the normal range in cats and dogs. Urine that contains enough albumin to register > a medium reaction on the ERD test will also often have a UP/C above the normal range. The UP/C and urine albumin/creatinine ratios from spot urine samples have been shown to accurately reflect the quantity of protein/albumin excreted in the urine over a 24-hour period. Because of the difficulty of 24-hour urine collection, this methodology has greatly facilitated the diagnosis of proteinuric renal disease in veterinary medicine. Most studies have shown that normal urine protein excretion in dogs and cats is ≤ 10-30 mg/kg/24 hours and that normal UP/C's are ≤ 0.2 – 0.3. Initially recommended normal values for canine UP/C's of < 1.0 were likely conservative and have more recently been lowered. Today, UP/C's < 0.5 and < 0.4 are considered to be normal for dogs and cats, respectively. Persistent proteinuria that results in UP/C's > that 0.4 and 0.5 in cats and dogs, respectively, where pre- and post-renal proteinuria have been ruled out, are consistent with either glomerular or tubulointerstitial CKD. Urine protein/creatinine ratios > 2.0 are strongly suggestive of glomerular disease. It is possible that the definition of normal will continue to change with additional research. For example, even the ultra-low level, single nephron proteinuria that can arise secondary to intraglomerular hypertension in hypertrophied nephrons in CKD, is abnormal in the face of what may be considered normal whole-body or whole-kidney proteinuria.

Monitoring renal proteinuria:

Transient renal proteinuria/albuminuria is likely of little consequence and does not warrant treatment. On the other hand, persistent proteinuria/albuminuria indicates the presence of CKD. Persistent proteinuria/albuminuria can be defined as positive test results on ≥ 3 occasions, ≥ two weeks apart. Because persistent proteinuria/albuminuria can be constant or increase or decrease in magnitude over time, monitoring should utilize quantitative methods to determine disease trends and/or response to treatment. Changes in the magnitude of proteinuria should always be interpreted in light of the patient's serum creatinine concentration since proteinuria may decrease in progressive renal disease as the number of functional nephrons decrease. Decreasing proteinuria in the face of a stable serum creatinine suggests improving renal function, whereas decreasing proteinuria in the face of an increasing serum creatinine suggests disease progression.

Implications of proteinuria/albuminuria:

In addition to the classic complications of moderate to heavy proteinuria (hypoalbuminemia, edema, ascites, hypercholesterolemia, hypertension, and hypercoagulability), there is increasing evidence in dogs and cats that proteinuria is associated with glomerular and tubulointerstitial damage and progressive nephron loss. In cats with naturally occurring CKD, relatively mild proteinuria (UP/C's > 0.43 and ≥ 0.2) appeared to be negative predictors of survival. Proteinuria has also been associated with decreased survival time in cats with systemic hypertension and the development of azotemia in geriatric cats. In dogs with naturally occurring CKD, the relative risk of uremic crises and mortality was approximately three times greater in dogs with UP/C's > 1.0 compared with dogs with UP/C's < 1.0. In this study the risk of an adverse outcome was approximately 1.5 times greater for every 1 unit increase in UP/C and the decline in renal function was greater in dogs with higher UP/C's.