The term “proteinuria” is generally used to indicate the presence of abnormal quantities of proteinin the urine (>20 mg/dl/day in the dog). In healthy subjects, the amount of protein in the urine is mainly constituted by a mucoprotein known as Tamm-Horsfall protein, produced by the epithelial cells lining the loop of Henle, the distal tubule and the collecting ducts.
Proteinuria can represent the main symptom of renal disease, meant as both glomerular and tubular dysfunction. For a better understanding of the pathological mechanisms underlying proteinuria it is essential to make some reference to pathophysiology.
The nephron is the anatomical and functional unit of the kidney (Fig. 1): it starts at the renal corpuscle, it continues in the proximal convoluted tubule and then entersthe renal medulla, with a long “U” shaped loop known as the loop of Henle, to then return within the renal cortex with the distal convoluted tubule, which then flows into the collecting duct that carriesurine to the renalpelvis. The renal corpuscle (Fig. 2), responsible forplasmatic ultrafiltration, consists of a vascular pole, where the afferent and efferent arterioles enter and exit, and a urinary pole, which opens into the proximal convoluted tubule. The afferent arteriole generates a tuft of capillaries, which make up the glomerulus. The capillary tuft is delimited by "Bowman's capsule", consisting of an external parietal layer and of an internal visceral layer; both layers delimit the urinary space, in which the liquid, filtered through the wall of the capillaries and the visceral layer, is collected.Theexternal parietal layer consists of a simple pavement epithelium supported by a basal lamina and by a thin layer of reticular fibres. The internal layer is formed by specialized epithelial cells, known as "podocytes," characterized by a cell body with different primary prolongations, from which numerous secondary prolongations, known as "pedicels", extend. The interdigitation of pedicels forms the filtration slits, approximately 25 nm wide. The pedicels are in close contact with the basal lamina, which derives from both the endothelial cells of the capillaries and from the podocytes, and fundamentally makes up the glomerular filtration barrier (Fig. 3). Electron microscopy allows the identificationwithin the basal membrane of a central electrodense layer (lamina densa) flanked by two electrolucent layers (lamina rara), rich in heparan sulfate, a polyanionic molecule (Fig. 4).
The glomerular filtration barrier acts in fact as a protein sieve, the function of which depends on:
- protein molecular weight: it allows the passage of low molecular weight proteins (MW ≤ 66,000 D)
- protein electric charge: it blocks the passage of negatively-charged proteins (e.g., albumin)
- protein shape; sherical proteins (e.g., immunoglobulins) pass more easily than cylindrical ones (e.g., albumin)
- hydrostatic pressure and GRF (glomerular filtration rate): an increased hydrostatic pressure can facilitate the passage of proteins; albumins in particular, despite their negative charge, can transit because the increased hydrostatic pressure wins over the electric repulsion mechanism generated by heparan sulfate.
The resorption of low molecular weight proteins present in the ultrafiltrate occurs within the proximal convoluted tubule, where protein hydrolysis and the subsequent recovery of amino acids takes place, thanks to pinocytosis and lysosome fusion (Fig. 5).
As proteinuria may also result from extra-renal causes, before using proteinuria as a marker of kidney disease a proper diagnostic workup is necessary.
From an academic point of view, depending on the site of origin proteinuria may be classified as (Fig. 6):
- pre-glomerular
- glomerular
- post-glomerular
PRE-GLOMERULAR proteinuria, independent of renal lesions, is divided into:
- FUNCTIONAL PROTEINURA
- GLOMERULAR PROTEIN OVERLOAD PROTEINURIA
Functional proteinuria is recognized for its mild and reversible nature. Conditions associated with this finding are fever, strenuous physical exercise, seizures and stress, all linked to an increased protein catabolism.
Glomerular protein overload proteinuria is caused by an abnormal content of plasma proteins, which, passing through the wall of glomerular capillaries in view of their low MW, subsequently leak into the urine. Haemoglobinuria, myoglobinuria, lysozymuria, Bence-Jones proteinuria and iatrogenic protein administration are all possible causes of this condition.
Haemoglobinuria occurs during intravascular haemolysis. The haemoglobin released from red blood cells is complexed by haptoglobin, a moderate positive acute phase protein, which does not cross the glomerular filtration barrier in view of its MW (80,000 D). The haptoglobin-haemoglobin complex is subsequently engulfed by macrophages and hepatocytes, and converted into bilirubin. As the concentration of haptoglobin is 1000 times lower than the concentration of haemoglobin (mg/dl versus g/dl), in the presence of significant haemolysis there is a rapid saturation of available haptoglobin. In view of this, the non-complexed haemoglobin (MW 17,000 D in monomeric form, 34,000 D in dimeric form) freely passes through the glomerular filtration barrier and is absorbed by the proximal convoluted tubule, up to a threshold of maximum resorption. The unabsorbed amount is the excreted with the urine (haemoglobinuria). Myoglobinuria develops following muscle necrosis. Lysozymuria has been documented in the course of acute leukaemias, primarily with monocytic differentiation: monocytes present high levels of lysozyme (MW 17,000 D) in the cytoplasm; the increased cell turnover occurring during acute leukaemia results in an increased concentrations of this enzyme in the serum, which similarly to haemoglobin is absorbed up to a maximum tubular resorption threshold; the excess is then detected in the urine. Bence-Jones proteinuria is characterized by the presence of monoclonal free light chains in the urine (MW 27,000 D), caused by immunoglobulin-secreting lymphoproliferative disorders (e.g., multiple myeloma, Waldenström’sMacroglobulinaemia, lymphoma).
GLOMERULAR PROTEINURIA, attributable to renal structural or functional lesions, is a high molecular weight (MW> 66,000 D) persistent proteinuria. It may be selective or non-selective: the first type, attributed to an early and reversible glomerular lesion with loss of the anionic filter function, allows the passage of proteins with MW up to 80,000 D; the second type is indicative of advanced basal membrane lesions with loss of the molecular sieve function, and consequent passage of proteinswith MW even in excess of 80,000 D.
Glomerular proteinuria may be of variable degree, ranging from mild (microalbuminuria) to severe (nephrotic syndrome). The term "microalbuminuria" is correctly used when the presence of albumin in the urine of dogs and cats ranges from 1 to 30 mg/dl. In humans, it is an important predictive sign of kidney disease in diabetic and/or hypertensive individuals. In the past, the term nephrotic syndrome was used in the presence of marked proteinuria, hyperlipidaemia (high cholesterol), hypoalbuminaemia, ascites or oedema. Currently, nephrotic syndrome is defined as a proteinuria greater than 3.5 g in 24 hours (in human medicine).
POST-GLOMERULAR PROTEINURIA is divided into:
- TUBULAR PROTEINURIA
- POST-RENAL PROTEINURIA
Tubular proteinuria is a mild, typically persistent, low MW proteinuria (<66,000 D), which is sometimes associated with normoglycaemic glycosuria and with possible electrolyte abnormalities. It may also include non-significant quantities of albumin. It may be the result of tubulardamage [e.g., acute renal failure, Fanconi syndrome, chronic kidney disease (CKD)] or instead of tubular protein overload (comparable to glomerular protein overload).
Post-renal proteinuria identifies those proteins that have leaked into the urine from the renal pelvis onwards. It is divided into urinary and extra-urinary system proteinuria. Urinary system proteinuria derives from haemorrhagic and/or exudative processes involving the walls of the urinary excretory system, following inflammatory (e.g., pyelonephritis, cystitis), traumatic (e.g., bladder rupture) or neoplastic (e.g., bladder carcinoma) events. Conversely, extra-urinary system proteinuria originates from haemorrhagic and/or exudative processes involving the genital apparatus, following inflammatory (e.g., vaginitis), traumatic (e.g., rupture of penile urethra) or neoplastic (e.g., prostate carcinoma) events.
Having now understood the pathophysiological mechanisms underlying proteinuria, the laboratory tests available for dosing urinary proteins are divided into:
Semi-quantitative methods
- Colorimetric test with test strips
- Turbidity test with sulfosalicylic acid (SSA) or trichloroacetic acid (TCA)
Quantitative methods
- Coomassie blue and Ponceau S method
- Colorimetric method with pyrogallol
- Microalbuminuria assay
Qualitative methods
- Urinary electrophoresis
The semi-quantitative methods provide an estimate of proteins in the urine.
Colorimetric tests with test strips must be carried outon a sample of well-mixed fresh urine, maintained at room temperature and not centrifuged. If the test is not performed within half an hour, refrigeration of the sample is recommended(stable for up to 24-48h at 2-8 °C), bringing the sample back to room temperature before performing the test. Recently, new test tubes have been introduced in the market, distinguishable thanks to the characteristic variegated yellow-red coloured cap (Fig. 7): thanks to a preservative adhering to the test tube wall, urine may be preserved for up to 72 h at room temperature before performing the chemical and physical examination of the sediment and the urine chemical tests. In order to use these test tubes, it is essential to collect 7.5 ml of urine, corresponding to the minimum level indicated on the label.
The test strips have a pad containing an acid pH colorimetric indicator (tetrabromophenol blue) (Fig. 8); negatively charged proteins bind to the pad and change its colour, the intensity of which gives an estimate of protein concentration. The method involves a comparison between the colour variations of the reagent and the standard reference colours provided by the manufacturer. This method presents undeniable advantages as it is easy to perform, it gives immediate results and the costs are contained. As for the disadvantages, the subjectivity of the test is the first to be mentioned. Pigmenturia, an alkaline pH, the presence of quaternary ammonium salts or of chlorhexidine may interfere with the reading. In case of urine with high specific gravity (SG) (typically in cats), since the strips are for human use the pad can be rapidly saturated, with colorimetric variations present even in the absence of proteinuria (false negative result). Moreover, given that negatively-charged proteins are the ones that principally bind to the pad, the test has a higher sensitivity for albumins. The minimum albumin detection threshold in the urine is equal to 20 mg/dl, whereas for γ-globulins it is greater than 1000 mg/dl.
Turbidimetric tests are based on the denaturation of proteins. Proteins denaturated by sulfosalicylic acid (SSA) ortrichloroacetic acid (TCA) tend to precipitate, and thus increase the turbidity of the sample. The results may be expressed using a turbidity scale (from 1+ to 4+). As an example, in order to perform an SAA turbidimetric assay a 3-5% solution of sulfosalicylic acid is prepared and urine is then centrifuged at 1,500 rpm for 5 minutes to separate the supernatant. Ten (10) drops of the supernatant are then dispensed into a conical container and an equal volume of sulfosalicylic acid is added. After mixing the obtained solution, the degree of turbidity of the sample is to be interpreted as follows:
- No turbidity: negative (<10 mg/dl)
- Traces: weak precipitate visible on a black background (10 mg/dl)
- 1+: mild degree of turbidity (10-50 mg/dl)
- 2+: moderate turbidity (50-200 mg/dl)
- 3+: intense turbidity (200-500 mg/dl
- 4+: flocculations (> 500 mg/dl).
This test is also easy and immediate, as well as economical. According to some textbooks, the test could also be used to suspect the presence of Bence-Jones proteinuria: in the past, a negative colorimetric test performed using reactive test strips together with a positive turbidity assay was considered indicative of Bence-Jones proteinuria.
As for the disadvantages, it should be noted that the presence of penicillin, tolbutamide and crystals may generate a false increase of proteinuria. On the contrary, when the SSA assay is performed an alkaline pH may cause a false reduction of proteinuria. Finally, it is important to highlight that being TCA sensitive to temperature, the test must be performed at temperatures ranging between 20° and 25° C.
Unlike semi-quantitative methods, quantitative methods give an accurate and objective measurement of proteinuria. These tests are carried out on fresh or frozen urine.
Proteinuria is assayed by means of a spectrophotometric method, based on a colorimetric reaction: photometry measures the light being absorbed (absorbance) by the solution, which contains the substance the concentration of which is being assayed. The following formula is used:
A= log I0/It A = absorbance, I0= incident light, It = transmitted light
As an example,pyrogallol red combined with molybdate forms a red complex with a maximum absorbance of 470 nm. When the pyrogallol-red molybdate complex binds with the amino groups of proteins, it generates a blue-purple complex with a maximum absorbance of 600 nm. The absorbance of this complex is directly proportional to the urinary protein concentration in the sample.
The inestimable advantage of the quantitative method is that it allows to calculate the U:P/C ratio (urine protein:creatinine ratio), which provides a reliable measurement for both the diagnosis and the monitoring of proteinuria.
ELISA tests for the determination of urinary albumin (microalbuminuria) in the dog and cat are available on the market. These tests are very sensitive (false positive results), but have a low specificity: this implies a high number of false positive results. In fact, also in humans there is evidence of microalbuminuria in the presence of chronic inflammatory processes not primarily localized in the kidneys, but rather secondary to neoplasms, sclerosis, IBD (inflammatory bowel disease) and spondylitis. One study has in fact shown that tumour necrosis factor alpha (α-TNF) alters glomerular permeability, causing microalbuminuria, even in the absence of renal disease. The result is that the poor specificity of the test makes it less applicable in clinical practice.
Unlike the previous methods, qualitative methods are not used to understand how many proteins are present in the sample, but rather which proteins are present. These methods should be performed at the end of a diagnostic approach that has already verified/confirmed the presence of proteinuria.
An example of a qualitative method is the agarose gel electrophoresis of urinary proteins in a neutral buffer solution of sodium dodecyl sulfate (SDS-AGE), which allows the separation of urinary proteins based on their molecular weight.
SDS-AGE should be carried out on a sample of urine as such or centrifuged (1,500 rpm for 5 minutes), collected by cystocentesis. It is performed on fresh urine (stable for up to 24-36 h at refrigeration temperature) or on fresh frozen urine, brought back to room temperature before running the test. A volume equal to 1 ml of urine or of supernatant is sufficient for the test. The method involves the comparison of each electrophoretic run with a standard run, in which proteins with a known molecular weight are made to migrate through the gel (molecular weight control): in Fig. 9, run No. 1 consists respectively of 4 electrophoretic bands which identify, bottom-up, immunoglobulins (MW 150,000 D), albumin (MW 66,000 D), triose phosphate isomerase (26,600 D) and finally lysozyme (14,300 D). To understand the origin of proteinuria, the molecular weight control is compared to the run of the patient’s sample being examined. As an example, in run No. 2 a tubular proteinuria is identified, in No. 3 albuminuria, in No. 4 mixed proteinuria and finally in run No. 5 a non-selective glomerular proteinuria.
The main advantage of SDS-AGE is that it is a non-invasive test for the early detection of renal damage; moreover, in the approach to the nephropathic patient it allows to assess the progression of the disease over time as well as the possible response to therapy.
specifically, scientific publications have shown that SDS-AGE allows the identification of subjects with severe tubulointerstitial lesions, as the MW of urinary proteins inversely correlates with the severity of the tubulointerstitial damage (high specificity). With regard to the finding of glomerular proteinuria, although the method provides high sensitivity in detecting it, it is not however capable of giving precise information on the type of glomerular disorder present, as there are no specific electrophoretic patterns for predicting any possible histological lesion. In view of what has been said, renal biopsy remains the "gold standard" for discriminating among the different glomerular disorders.
Suggested reading
- Langston C. 2004, Microalbuminuria in cats. J Am Anim Hosp Assoc. 2004 Jul-Aug;40(4):251-4.
- Lees GE, Brown SA, Elliot J, et al. Assessment and management of proteinuria in dogs and cats: ACVIM Forum Consensum Statement (small animals). J Vet Intern Med. 2005;19:377–385.
- Osborne CA, Finco DR. Nefrologia e urologia del cane e del gatto – UTET 1999.
- Pressler BM, Vaden SL, Jensen WA, et al. Prevalence of microalbuminuria in dogs evaluated at a referral veterinary hospital. J Vet Intern Med. 2001;15:300.
- Stockham SL, Scott MA. Fundamentals of Veterinary Clinical Pathology, 1st edition. Blackwell 2002.
- Willard MD, Tveden H. Small Animal Clinical Diagnosis by laboratory Methods, 4th edition. Saunders 2004.
- Zini E, Bonfanti U, Zatelli A. Diagnostic relevance of qualitative proteinuria evaluated by use of sodium dodecyl sulfate-agarose renal histologic findings in dogs. Am J Vet Res. 2004;65:964–971.