Urinalysis: with an emphasis on proteinuria (Proceedings)

The urinalysis (UA) is an often overlooked and underutilized test in veterinary medicine. A complete UA includes three components: 1) physical assessment, 2) chemical assessment, and 3) microscopic examination of the sediment. When performed properly, the UA can provide important information regarding renal function and urinary tract disease. The UA can also aid in the diagnosis and treatment of extra-urinary tract disorders such as diabetes mellitus and hepatic and hemolytic disorders.

Cystocentesis is the ideal way to obtain a urine sample for UA. This method minimizes contamination from the lower urinary tract that can occur when urine is obtained by urethral catheterization or mid-stream voiding. Whenever possible, 10-12 ml of urine should be collected to allow part of the sample to be set aside for additional analysis (e.g., culture and sensitivity, protein/creatinine ratio, or enzyme/creatinine ratio).

The following steps are recommended to perform a complete UA:

1) Save a portion of the sample for potential additional analyses.

2) Warm the sample to room temperature if it has been refrigerated.

3) Thoroughly mix the sample so that any particulate matter that has settled due to gravity is included in the urinalysis.

4) Transfer a standard volume of urine (6-10 ml) to a clear, transparent conical centrifuge tube. (Normal values for the sediment examination are determined by centrifuging a standard volume of urine. For example, if < 5 WBCs/hpf is normal when 8 ml of urine is centrifuged, 2-4 WBCs/hpf could be abnormal if a lesser volume of urine is centrifuged.) Alternatively, the urine sediment pellet can be re-suspended with a standard 10 or 20% of the original volume of urine that was centrifuged.

5) Evaluate the color and turbidity of the sample in the centrifuge tube.

6) Perform specific gravity analysis with a refractometer. This analysis may be completed on uncentrifuged urine if the sample is clear. If the urine sample is turbid the specific gravity should be performed on the urine supernatant (see below).

7) Perform reagent strip analyses (the nitrate, urobilinogen, urine specific gravity, and leukocyte tests that may be present on human reagent strips are not reliable for canine and feline urine). It's best to use a pipette to apply urine individually to each test strip pad. Be careful not to allow urine to run from one test strip pad to the next which can result in cross contamination.

8) Centrifuge the sample for 3-5 minutes @ 2000 – 3000 rpm. These variables should be standardized.

9) Remove all but a standard volume (0.25 – 0.5 ml) or percent of the original volume (see #4 above) with a pipette or by decanting.

10) Re-suspend the sediment in the remaining supernatant by gently tapping the tube with your finger. Overzealous agitation may break cells and casts.

11) Transfer a drop of the re-suspended sediment to a microscope slide and cover it with a cover slip.

12) Decrease the microscope light intensity by lowering the condenser and partially closing the diaphragm.

13) Scan the entire sample via the low power objective and then record the average number of elements (e.g., cells, crystals, casts, bacteria) per several high-power fields.

14) Air-dried urine sediment slides may be stained for additional microscopic evaluation.

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, the urine sediment is active (pyuria, hematuria, and/or bacteriuria), 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. For example, when 298 canine and feline urine samples were analyzed by both a conventional urine protein dipstick method (Multistix® Reagent Strips, Bayer Corporation, Elkhart, IN) and a canine or feline albumin specific quantitative ELISA (Heska Corporation, Ft. Collins, CO), there were disparate results. The sensitivity for the conventional urine protein dipstick for albuminuria in canine and feline urine was 54% and 60%, respectively and the urine protein dipstick specificity for canine and feline albuminuria was 69% and 31%, respectively. If urine samples with an alkaline pH (≥ 7.5) and/or hematuria (≥ 10 RBC/hpf), pyuria (≥ 5 WBC/hpf), or bacteriuria were excluded, the dipstick specificity for canine and feline albuminuria increased to 84% and 55%, respectively. These data demonstrate that conventional urine protein dipsticks have a high percentage of false-negative and false-positive results for detection of albuminuria in canine and feline urine when compared with an albumin-specific ELISA. Urine protein dipstick false-positive results in both species can be decreased by excluding alkaline urine and urine with hematuria, pyuria, and/or bacteriuria from analysis.

The SSA test is performed by mixing equal quantities of urine supernatant and 5% SSA in a glass test tube and grading the turbidity that results from precipitation of protein on a 0 to 4+ scale (Figure 3). 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 are thought to be involved. Decreased physical activity may also affect urine protein excretion in dogs; one study showed that urinary protein loss was higher in dogs confined to cages than in dogs with normal activity levels.

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. 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 vascular 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., Fanconi syndrome and 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, pyelonephritis, and leptospirosis) which are often accompanied by an active urine sediment.

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.