Proteinuria in cats

© August, JR (2006) Consultations in Feline Internal Medicine, Vol 5.  Elsevier Saunders

Kidney diseases that lead to renal insufficiency and failure are common in aging cats. Good epidemiological studies are lacking to determine the true prevalence of chronic renal insufficiency (CRI) and chronic renal failure (CRF), but estimates suggest that as many as one in three cats over the age of 12 may be affected. The importance of proteinuria as a prognostic indicator and therapeutic target in feline kidney disease only recently has been recognised.

Diagnosis

1. Qualitative testing

The most common screening tests for proteinuria involve some form of colorimetric dipstick test. These tests are semi-quantitative and depend  on the ability of amino groups of proteins to combine with indicator dyes (e.g. tetrabromophenol blue), which then change color. The degree of color change depends on the number of free amino groups in the protein. Because albumin has more free amino groups than hemoglobin or globulins, the reagent pads usually are two or three times more sensitive to albumin in the urine than to other proteins. The individual tests vary in their lowest limits of detection, but usually produce a positive reaction only if protein is present at a concentration above 0.3 g/L. False-positive results are common in dipstick tests, particularly in alkaline urine, and positive results must be interpreted in light of urine specific gravity.

An alternative screening test is the sulfosalicylic acid turbidimetric test. This involves addition of an equal volume of 3 - 5% sulfosalicylic acid to the urine sample and subjective assessment of the turbidity of the sample (0 to 4+). Highly alkaline urine may give false-negative results, but the likelihood of false-positive results is much lower with this screening test than the standard dipstick tests.

The final semi-quantitative screening test that is widely available is the Early Renal Damage test (ERD, Heska Ltd). This is a sensitive immunological test that employs antibodies specific for feline albumin. Because of a lack of immunological cross-reactivity, tests that have been developed for use in other species cannot be used to detect feline albumin. The test kit provides a method of diluting the urine sample to a specific gravity of 1.010 to control for urine concentration and therefore urine volume. The cut-off between negative and weakly positive has been set for an albumin concentration of 1 mg/ dL, a value some 30 times lower than most standard dipstick tests (although samples may be diluted several fold before testing). Therefore samples with the severity of proteinuria that traditionally has been considered clinically significant (i.e. those typically associated with primary glomerular disease) are likely to give readings in the high to very high positive range. The advantage of this test (and others like it) is its ability to detect low concentrations of protein that would not be detected by standard non-immunological methodologies. The term microalbuminuria has been coined to describe the low concentration of urinary protein detected by these immunological methods.

2. Quantitative testing

The gold standard for assessing urinary protein loss is to collect all the urine passed by the animal for a 24-hour period, measure its volume and protein concentration accurately, and calculate the amount of protein lost ( in mg of protein in urine per kg body wt) in the 24-hour period. The excretion of albumin over a 24-hour period can be measured in a similar method. This technique is used only in research settings.

Measurement of the urine protein to creatinine ratio (UPC) is used in veterinary practise as an alternative to measurement of 24-hour urine protein excretion. This method can be done on a single random (spot) urine sample. It is best performed after a period of confinement without access to a litter tray so that the volume of urine upon which it is based is as large as possible. Laboratories offering this test measure creatinine and protein by quantitative analyses, express the concentration of both analytes in g/L, and calculate the ratio of analyte concentrations. Such an approach has been shown to give results in cats that correlate with 24-hour urine protein excretion measured under research conditions. Standard practise chemistry analysers cannot be used to make measurements of urine protein and creatinine. These machines are calibrated for plasma in which the concentration of protein is 500 to 10000 times higher than in urine. Therefore, cross-contamination of urine with protein from previously analysed plasma samples is a significant problem. Furthermore, creatinine concentrations are 25 to 100 times higher in urine than in plasma, which makes it necessary to dilute urine samples considerably before the creatinine concentration falls within the working range of the machine.

A similar approach can be adopted for quantifying albumin concentration in feline urine. Feline-specific ELISA assays have been produced and may be used to provide a quantitative evaluation of the albumin concentration in urine. At present, we are aware of only one commercially available assay (Heska Corp). Traditionally, in human medicine, albumin has been quantified in mg / L, so the ratio has been expressed in units of mg albumin per g of creatinine. The cut-points used typically for human urine are 30 mg/g dividing between normal and microalbuminuric patients and 300 mg/g divided between microalbuminuria and grossly proteinuric (also termed macroalbuminuric). Preliminary studies suggest that similar cut-off points are appropriate for cats. However, of these definitions are accepted, some older, apparently healthy cats will be classified as microalbuminuric.

Aetiology

The glomerular filter consists of the fenestrated endothelium of the glomerular capillary, the basement membrane, and the visceral epithelium. Of these three structures, the endothelium prevents passage of cells into the glomerular filter, the basement membrane provides structural stability of the filter, anchoring cells in the correct position, whereas the transmembrane proteins of the podocytes (visceral epithelium), which form the slit diaphragm, act as the primary charge and size selective filtration barrier. Albumin appears in the ultrafiltrate at low concentrations, despite its presence in the plasma at high concentrations (about 0.05 mM). This is because, with a molecular weight of 69,000, it is close to the limit for filtration in terms of size. In addition, albumin is predominantly negatively charged and is repelled by the negatively charged proteins within the slit diaphragm. Nevertheless, recent data suggest a significant transglomerular flux of albumin so that more of this protein appears in the glomerular filtrate of normal kidneys than was once recognised.

Small proteins of molecular weight less than 7,000 are unimpeded in passage across the glomerulus, but most of this filtered protein is removed from the glomerular filtrate as it passes through the proximal convoluted tubule. Protein is removed from filtrate by pinocytosis; tubular cells break down the reabsorbed protein into its constitutive amino acids. Albumin that passes across the glomerular filter is dealt with inefficiently by such a process, and therefore usually is the major urinary protein detected in normal healthy animals. For proteins with a molecular weight between 7,000 and 70,000, an increasing impediment to filtration occurs in both size-dependent and charge-dependent. Loss of protein in the urine in normal healthy cats usually does not exceed 30 mg/kg/day. In addition, to small amounts of albumin, urine contains small amounts of protein secreted by the tubules (Tamm-Horsfall protein) and proteins that are derived from the lower urinary and genital tracts.

Classification of proteinuria

The origin of protein appearing in the urine can be classified as prerenal, renal, or postrenal. Prerenal proteinuria implies that significantly increased concentrations of protein are being presented to the kidney in the plasma. If these proteins are of low molecular weight, they will be filtered and, if the concentration in the glomerular filtrate is sufficiently high, tubular reabsorptive processes will be overwhelmed. Examples of proteins that are filtered readily include immunoglobulin light chains, hemoglobin, and myoglobin. Some of these low molecular weight proteins are not detected by standard laboratory methods, which generally are more sensitive to albumin.

Postrenal proteinuria implies protein is added to the urine in the urinary tract after formation by the kidney (i.e. in the ureter, bladder or urethra). Inflammation of the urinary tract, resulting most commonly from bacterial infection, should be considered as a possible cause of proteinuria. Other causes include presence of uroliths and tumours, both of which may cause inflammation directly or may be associated with secondary bacterial infection. In many, but not all cases, the cat will be showing signs of lower urinary tract disease, such as dysuria and pollakiuria. Microscopically, the urine sediment is likely to be active, with evidence of inflammatory cells and possibly bacteria. Samples of relatively dilute urine (urine specific gravity <1.030) should be submitted routinely for bacterial culture (even in the absence of lower urinary tract signs and microscopic evidence of inflammation) to rule out subclinical urinary tract infections before postrenal cause for proteinuria is excluded categorically. However, in cats with CRI, positive urine cultures from samples with noninflammatory urine sediment are uncommon.

Renal proteinuria implies that defective renal function and/or inflammation of parenchymal kidney tissue is the cause of the proteinuria. Active, acute, renal parenchymal inflammation, associated with diseases such as pyelonephritis and acute tubular necrosis, may be suspected from the clinical history. Localisation of the disease to the kidney may be possible based on physical examination findings (painful swollen kidneys on palpation, fever, renal failure), or resulting from the presence of tubular casts on urine microscopy. However, in many instances, a diagnosis of renal proteinuria is made by the exclusion of prerenal and postrenal forms of proteinuria. Renal proteinuria than may be characterised further as being of either glomerular or tubular origin.

Causes

Glomerular proteinuria can occur because of primary glomerular pathology leading to a defective filtration process. Examples of primary glomerular diseases include the following:

In primary glomerular diseases, the magnitude of proteinuria tends to be high (UPCs >2.0, and often above 5 to 10), a feature that may be helpful in distinguishing these diseases from other causes of renal proteinuria. Very high levels of proteinuria (UPC > 10) may be associated with clinical signs of nephrotic syndrome (peripheral oedema or ascites). Cats with severe proteinuria are not always azotemic when their kidney disease is diagnosed. Many proteinuric cats become azotemic over time, because persistent proteinuria of this severity leads undoubtedly to progressive renal injury. In the author's experience, non-azotemic cats that present with signs of the nephrotic syndrome may respond favourably to treatment, at least in the short to medium term.

Low-level proteinuria (UPCs of 0.4 to 2.0) and microalbuminuria also may be caused by increased protein influx across the glomerulus in the absence of classical primary glomerular diseases such as those described above. Increased transglomerular flux of proteins (particularly albumin) may occur because of the following:

A further potential cause of low-level proteinuria (UPCs 0.4 to 2.0) is defective tubular reabsorption of the filtered protein. This may occur as one feature of a more generalised tubular defect, characterised by normoglycemic glucosuria or abnormal electrolyte secretion, or more often as a consequence of a decrease in the number of functional tubules in patients with chronic progressive renal disease. Probably in many patients, the renal defects resulting in mild proteinuria are a combination of increased transglomerular flux and decreased tubular reabsorption.

Role of proteinuria in progression of renal disease

Historically, only moderate-to-sever proteinuria (typically UPC > 1.0) has been considered of clinical significance in veterinary practise. Certainly only protein loss of this magnitude or greater is likely to result in systemic hypoalbuminemia and other features of the nephrotic syndrome. However, most cats with azotemic chronic progressive renal disease have either normal levels of protein in their urine. are microalbuminuric, or have mild proteinuria (UPCs >0.4 but <1.0). Although moderate-to-severe proteinuria has been shown to predict a more rapidly progressive decline in renal function in a number of species, including human beings and dogs, the significance of mild proteinuria, such as is typically present in cats with CRI, is less well documented. Two primary, interrelated questions must be addressed: Is mild proteinuria of clinical significance in cats (i.e. is proteinuria associated with a more rapid decline in renal function) and, if so, is the proteinuria actually damaging to the kidney or is it simply a marker for a more rapidly progressive type of renal injury?

Glomerular capillary hypertension

In rodent models of chronic kidney disease, after loss of a critical amount of renal mass, local changes within the kidney result in hyperfiltration of the remaining functioning nephrons and low-level proteinuria. The hyperfiltration is driven by glomerular capillary hypertension, which in turn is caused, at least in part, by local activation of the renin-angiotensin system (RAS). This is due to the increased local concentration of angiotensin II, which causes selective constriction of the efferent arteriole (capillaries leaving the glomerulus). In addition, angiotensin II stimulates nephron hypertrophy. Ultimately, over time, these adaptive changes are detrimental (maladaptive) and are thought to lead to interstitial fibrosis and inflammation resulting in further nephron loss, even in the absence of any extrinsic factors that damage the kidney (the so-called 'intact nephron hypothesis' - a concept that each nephron is either a fully functional unit or does not function).

Evidence that glomerular capillary hypertension occurs in the feline kidney also has been documented using a remnant kidney model. Surgical reduction in renal mass (3/4 nephrectomy) caused an approximately 10% rise in glomerular capillary pressure. In these cats, development of glomerular hypertension was associated with an increase in protein excretion. Before renal injury, the cats had a mean UPC of 0.07; after subtotal nephrectomy, the mean UPC increased to 0.31. Chronic maintenance of this model is associated with development of interstitial fibrosis, moderate inflammatory infiltrate of the interstitium, and glomerulosclerosis, as is seen in the rodent model described above.

In a cross-sectional epidemiological study of cats at initial diagnosis of their chronic kidney disease, significant risk factors included initial plasma creatinine concentration and increased systolic blood pressure. Age, urine specific gravity, and gender were not associated significantly with the magnitude of proteinuria. Additional, longitudinal studies have demonstrated that proteinuria, as assessed by the UPC ratio, worsens in cats when their renal function deteriorates. These studies suggest that cats with naturally occurring CRI develop proteinuria as a result of development of glomerular hypertension, because hyperfiltration would be expected to be more severe in cats with fewer functioning nephrons and consequently more marked elevation of plasma creatinine concentration. However, interpretation of a changing UPC in the face of loss of functioning nephrons is complex, because the reduced number of nephrons filtering the blood offsets the tendency for protein loss to increase.

Systolic blood pressure also was linked to the severity of proteinuria in these cats. The increased proteinuria in hypertensive cats could be due to an inability of the failing kidney to autoregulate renal blood flow appropriately, with resultant transmission of the elevated systolic blood pressure to the glomerulus. An alternative explanation is that proteinuric renal diseases are more likely to cause systemic hypertension.

Proteinuria and survival time

Proteinuria is associated with shortened survival times in cats with naturally occurring kidney disease. Interventions designed to reduce urinary protein excretion would slow progressive renal injury and therefore improve survival in cats with CRI. This conclusion assumes that protein in the urine is damaging to the remaining functional nephrons and leads to progressive renal injury. Another interpretation of these findings, however, is that the appearance of protein in the urine is merely a marker that progressive renal injury is occurring. In this case, although renoprotective interventions may be expected to reduce protein excretion because they slow progressive renal injury, reducing urine protein excretion per se does not necessarily prove to be renoprotective.

Progressive renal injury as a consequence of proteinuria

In vitro studies have shown that exposure of proximal tubular cells grown in cell culture to albumin and transferrin in concentrations that overwhelm the ability of these cells to digest the proteins within their lysosomes leads to activation of nuclear factor κB. This nuclear factor in turn triggers the expression of genes in these cells and causes them to secrete mediators from their basolateral cell surfaces (i.e. toward the interstitium). These mediators include endothelin-1, monocyte chemotractant protein-1 (MCP-1), and the immunoregulatory cytokine regulated on activation, normal T expressed and secreted (RANTES). Expression and secretion of these proteins can be demonstrated in vivo in animal models of proteinuric renal disease. Interventions that reduce proteinuria in these models also reduce the expression of these and other cytokines involved in the progressive interstitial fibrosis. These molecular details may explain why proteinuria is an independent risk factor for progression of kidney disease in human medicine, and why drugs such as angiotensin-converting enzyme (ACE) inhibitors slow progression successfully in proteinuric kidney disease, independent of their effects on systemic blood pressure.

Proteinuria in non-azotemic animals

Microalbuminuria and mild proteinuria were found to be predictive of all-cause mortality in a population of non-azotemic, apparently healthy cats. The survivors had significantly lower UPC ratios when compared with the non-survivors. Many systemic disease states have been associated with microalbuminuria including a variety of chronic inflammatory diseases and neoplastic conditions. By comparison, the prevalence of microalbuminuria in cats with a known medical problem was 42.9%. Interestingly, although the prevalence of microalbuminuria increased with age, this increase was much more marked in the apparently healthy cats, perhaps because of an increased prevalence of subclinical renal disease in the elderly cat.

One important disease that may cause microalbuminuria and mild proteinuria in cats in hyperthyroidism. The hyperthyroid state leads to glomerular hyperfiltration and probably glomerular capillary hypertension because of inappropriate activation of the renin-angiotensin system. In one study, approximately one half of untreated, nonazotemic, hyperthyroid cats had UPCs on initial diagnosis of >0.5 (i.e. were mildly proteinuric). Management of the hyperthyroid state resulted in a rapid reduction in UPC ratio in the majority of the cats, which suggests that the renal hemodynamic effects of thyroid hormones caused the proteinuria. This finding lends credence to the proposition that if hyperthyroidism is left untreated, it is damaging to the cats' kidneys. However, the degree of proteinuria at diagnosis was not predictive of the occurrence of azotemia once the cats were stabilised in a euthyroid state.

Treatment of proteinuria

Cats with chronic renal insufficiency (CRI)

If proteinuria proves directly injurious top the feline kidney, therapeutic strategies to lower glomerular capillary pressure and reduce proteinuria in cats with CRI should slow progressive renal injury (including interstitial fibrosis) and subsequently improve survival. Benazepril is an ACE inhibitor that is authorised for this indication in Europe a a dose rate of 0.5 to 1.0 mg/kg PO q 24hr. Benazepril has been shown to reduce glomerular capillary pressure in cats with experimentally reduced renal mass without compromising single nephron glomerular filtration rate.. The antiproteinuric effect of this drug also has been demonstrated in clinical cases of naturally occurring feline CRI. However, the effect of benazepril on survival was not statistically significant, except in a very small subgroup of cats with pre-treatment UPCs greater than 1.0. Reducing proteinuria protected against development of interstitial and glomerular lesions in dogs with surgically reduced renal mass treated with enalapril (0.5mg/kg PO q 12 hr). On balance, this evidence suggests that preventing or reducing proteinuria should be a treatment goal for all feline patients with CRI; however, the benefits of ACE inhibitor therapy are likely to be greatest in cats that are more proteinuric. Benefits also are only likely to be manifest in cats that are expected to survive for a reasonable length of time after initiating the treatment. Renal function in cats that are severely azotemic and likely to live for only a few days or weeks actually may be affected adversely by the drop in glomerular capillary pressure associated with ACE inhibition.

Currently, based on the data available, it is suggested treating all cats with UPCs greater than 0.4 and aiming for a post-treatment UPC persistently lower than 0.4.

Cats with CRI and hypertension

Systemic hypertension is a risk factor for proteinuria; effective control of hypertension is important in limiting glomerular capillary hypertension. ACE inhibitors, although effective at lowering pressure within the glomerular capillaries, are not very effective at lowering systemic arterial blood pressure. Therefore, in a hypertensive cat, use of the calcium-channel blocker amlodipine would be the treatment of choice because this is the only agent demonstrated to considerably reduce blood pressure sufficiently to prevent development of hypertensive ocular lesions. However, treatment with calcium-channel blockers has the disadvantage that it dilates the afferent renal arteriole but not the efferent arteriole, which nullifies any protective autoregulatory response and exposes the glomerulus to systemic blood pressure. This is only likely to be of real concern if control of systemic blood pressure is inadequate (i.e systolic blood pressure remains above 160 mm Hg).

A practical approach therefore in hypertensive cats is to manage systemic hypertension with amlodipine initially. Once blood pressure is well controlled, protein excretion should be re-evaluated. If the UPC at this stage is greater than 0.4, addition of an ACE inhibitor to the treatment regimen should be considered. Although no objective data are available to demonstrate that combined therapy with amlodipine and benazepril in cats alters survival time or delays the progression of renal disease, short-term treatment with this combination of drugs has been described and appears well tolerated. Adequate control of hypertension in cats with CRI should be documented by consistent indirect systolic blood pressures below 160 mm Hg. In human medicine, post-treatment blood pressure targets vary with the clinical condition of the patient and targets are lower in patients with more severe proteinuria.

Feeding a diet with a relatively reduced protein and phosphorus content is a cornerstone in the management of CRI in cats. Although the benefits of ACE inhibitors are largely unproven and likely to be small in terms of increases in survival time unless patients are grossly proteinuric, survival time of cats that eat phosphorus-restricted 'renal care' diets is much longer than in cats that do not. The primary benefits of this dietary strategy are amelioration of renal secondary hyperparathyroidism and attenuation of the severity of the uraemia. Additionally, protein ingestion contributes to glomerular hypertension in dogs and people with renal insufficiency; glomerular hypertension may exacerbate proteinuria. Although dietary restriction of protein appears to delay progression of renal disease in laboratory rats, the benefit of protein restriction alone has not been documented consistently in other species. Protein restriction does appear to limit urinary protein loss in dogs and human beings with glomerulonephritis, and is recommended in the treatment of patients that are severely proteinuric (even if they are nonazotemic). Anecdotally, reduction in dietary protein also may be helpful in the management of cats with nephrotic syndrome.

Other potential therapeutic strategies to lower urinary protein excretion include the following:

Nonazotemic cats

No specific, evidence-based recommendations currently can be made for the treatment of cats with mild proteinuria or microalbuminuria that are not azotemic. If, in the future, mild proteinuria or microalbuminuria can be shown definitively to result from renal damage, and if any of the interventions described above can be proven clearly to be beneficial, then their use could be extended logically to nonazotemic patients. At the present time, the author considers using these treatments (dietary protein restriction and ACE inhibition) in nonazotemic cats with moderate-to-marked proteinuria (UPC > 1.0). In nonazotemic cats that are persistently microalbuminuric, or mildly proteinuric (UPC < 1.0), it would be beneficial to confine our efforts to ruling out a prerenal or postrenal cause for the proteinuria and to searching for any associated underlying disease.