Rance K. Sellon, DVM, PhD
Chronic immune system stimulation that results in the formation of either immune complexes or amyloid can cause glomerular disease and ultimately renal failure. While the hallmark of glomerular disease is proteinuria, the detection of proteinuria should not be the end of the investigational process in affected patients.
Formation of Glomerular Immune Complexes and Amyloid, and Renal Injury
The formation of immune complexes is the predominant feature of type III hypersensitivity. Immune complexes are the products of antibody combined with antigen and form when antigen, and its specific antibody, are present at the same time. Normally, immune complexes are cleared by their binding to erythrocytes and then removed from the erythrocyte by hepatic sinusoidal Kupffer cells as the red blood cell/ immune complex passes through the liver. Very large immune complexes are removed by macrophage phagocytosis. Amyloid, an insoluble protein, is the product of inflammation, and severe or chronic inflammation can be precipitating factors for amyloid formation.
Deposition of soluble immune complexes in the glomerulus occurs when excessive production overwhelms the clearance mechanisms (most common), or when clearance mechanisms fail. In some instances, it is possible that immune complexes form in situ when antibodies recognize a glomerular protein, or a protein trapped in the glomerulus, and bind to it in the glomerulus. Once occupied by antigen, the antibody is capable of binding complement and provoking inflammation (glomerulonephritis, GN) that give rise to the histological appearance of glomerulonephritis. Immunohistochemical examination of renal biopsies can demonstrate the presence of immune complexes.
Once the anatomy of the glomerulus is altered, glomerular function is altered to cause proteinuria. Proteinuria develops as a consequence of loss of the charge selectivity barrier of the glomerulus. Many proteins are negatively charged, and filtration of these proteins is repulsed by negative charge in the glomerulus. Thus, loss of the negative charge in the glomerulus as occurs with GN removes a major obstacle to filtration of proteins. Albumin and other small proteins such as antithrombin (AT) are subsequently lost into the urine to cause the proteinuria and hypoalbuminemia commonly observed in animals with glomerular injury. Loss of larger proteins such as globulins does not typically occur with glomerular diseases because filtration of these proteins is effectively prevented by intact glomerular size selectivity barriers.
The most common reason for the development of immune complexes is chronic antigenic stimulation of both infectious and non-infectious causes. In a retrospective survey canine protein-losing nephropathy (PLN), the diseases most commonly associated with either GN or amyloidosis were tumors, infections (parasitic, bacterial, fungal), hepatitis, polyarthritis, immune-mediated hemolytic anemia, and hyperadrenocorticism. Miscellaneous diseases associated with PLN included inflammatory bowel disease, systemic histiocytosis, degenerative disk disease, diabetes mellitus, chronic pancreatitis, and dilated cardiomyopathy. Despite the many known diseases that are associated with glomerular injury, many patients have no identifiable predisposing cause.
Clinical Features of Glomerulonephritis
There are recognized breed predispositions to the development of glomerular disease, especially in dogs, although any breed can be affected. In some instances, the predisposition is to the development of GN itself. Breeds with familial glomerulonephropathy include: soft-coated Wheaten terriers (GN), Bernese mountain dog (GN), Shar-pei (amyloid), Samoyed (hereditary nephritis), Bull terrier (hereditary nephritis), English Cocker spaniel (hereditary nephritis), Doberman pinscher (idiopathic glomerulonephropathy), and Newfoundland (glomerulosclerosis).
Glomerular disease is often clinically silent and incidentally found during routine health evaluations. Some patients with glomerular disease will have clinical signs referable to the underlying cause and the presence of PLN is discovered during evaluation of the presenting problem. Clinical signs of renal failure, especially vomiting, may also prompt examination by a veterinarian. A history of polyuria or polydipsia may or may not be a feature of affected patients. Affected animals may have weight loss, ascites (pure transudates) and/or peripheral edema, features of the nephrotic syndrome. Other physical examination abnormalities that may be seen include retinal vessel tortuosity or retinal detachments secondary to systemic hypertension, oral ulcers, and uremic breath odor.
Results of laboratory tests in patients with PLN can vary depending on the underlying cause, duration, and the severity of the disease. Non-regenerative anemia may reflect chronic renal failure or anemia of chronic inflammatory disease; inflammatory leukograms may be present. Serum biochemical abnormalities can include hypoalbuminemia, hypercholesterolemia, increased BUN and creatinine, hyperphosphatemia, and hyperglobulinemia. Normal serum albumin would not exclude a diagnosis of PLN in a patient with proteinuria. Proteinuria is usually detected during routine urinalysis. Urine specific gravity can vary among patients with GN. If renal injury is extensive, the urine may be isosthenuric. If injury is confined to the glomerulus, urine specific gravity may exceed 1.025 even in the face of renal origin azotemia, but is seldom truly normal.
Localization of proteinuria to the glomerulus is accomplished by exclusion of other potential sources of proteinuria, especially urinary tract inflammation. Examination of the urine sediment examination is a must for a patient that has proteinuria. If hematuria or pyuria are observed on the urine sediment, the possibility that proteinuria is from inflammatory urinary tract disease has to be considered. Urinary tract inflammation may occur concurrently with GN, but the contribution of the glomeruli to the urinary protein may be difficult to ascertain until inflammation has resolved.
Once urinary tract inflammation has been excluded as a cause of proteinuria, measurement of the urine protein/creatinine ratio (UP/C) is recommended. In dogs, a normal UP/C is less than 0.5; values between 0.5 and 1.0 are considered marginal, and values greater than 1 are considered consistent with renal origin proteinuria. It is emphasized that a UP/C can not be correctly interpreted unless one has also examined a urine sediment; abnormal UP/C has been documented in patients with urinary tract inflammation. A patient with a low protein reading on a dipstick analysis of dilute urine may have an abnormal UP/C. Thus, a patient with a low urine specific gravity and low degree of dipstick proteinuria is a candidate for evaluation of the UP/C to exclude the presence of a pathologic glomerular process. In general, dogs with amyloidosis have higher UP/C than dogs with GN, but overlap in the ranges seen in individual dogs precludes a diagnosis based on UP/C alone. The UP/C does not take the place of the renal biopsy in establishing the presence of a specific glomerular disease. An abnormal UP/C can provoke an investigation to underlying causes of PLN as noted above.
With documentation of abnormal UP/C and failure to find an underlying cause of proteinuria, renal biopsy becomes a legitimate consideration as a next diagnostic step. Several techniques are available for obtaining renal biopsies and the technique used is a function of available equipment and clinician preference. Ultrasound-guided biopsies are accomplished quickly, relatively safely, and if the operator is experienced, are not technically difficult. The major limitation to ultrasound-guided biopsies is the relatively small sample sizes. Since assessment of the glomeruli is critical for the utility of these biopsies, one can first examine the tissue cores obtained for the presence of glomeruli, which are easily seen, in a saline-filled Petri dish under a dissecting microscope before placing in a formalin jar. Evaluation of coagulation panels (platelet numbers, PT and PTT times, and possibly buccal mucosal bleeding times) is suggested prior to ultrasound-guided kidney biopsies. An acceptable alternative for many patients, and perhaps more widely available to the practitioner, is an activated clot time (ACT), requiring only tubes of diatomaceous earth, an incubator, and an accurate watch. The most significant risk of an ultrasound-guided biopsy is excess hemorrhage.
In the absence of ultrasound, renal biopsies may be obtained laparoscopically, or surgically using keyhole, paralumbar or ventral abdominal approaches. These techniques offer the advantages of better visualization of the biopsy site, larger sample sizes, and better control of local hemorrhage. They carry the attendant risks of a more prolonged anesthetic period and usual risks of surgery. In the author's practice, obtaining biopsies by any of these methods would also incur a greater expense for the client.
Biopsies are submitted for histopathological examination. Once a histological diagnosis of GN has been made, the role of immune-complexes in the glomerular injury can potentially be established by requesting that the samples be immunohistochemically stained for immunoglobulin and complement. Detection of either or both of these molecules could suggest, though is not absolutely diagnostic of, immune-mediated injury. The author is not aware, however, of clinical data that would yet indicate that information derived from immunohistochemical staining has any prognostic significance or influences the course of therapy in patients with histologically-confirmed GN.
Management of Patients with PLN
The principle aspects of therapy for patients with protein-losing glomerular diseases are to identify and treat underlying causes, reduce proteinuria, control peripheral edema and ascites, prevent the consequences of a hypercoagulable state and manage progressive renal dysfunction.
Identification of underlying causes of PLN begins with a history and physical examination. Serologic assays for infectious or immune-mediated disease may become part of the diagnostic data base. Abdominal radiographs or ultrasound can identify diseases in the abdominal cavity. Thoracic radiographs should be strongly considered as part of the diagnostic package in the hunt for predisposing diseases, even in patient with no clinical signs of respiratory disease.
To reduce proteinuria, control of dietary intake of protein and administration of drugs that reduce renal protein loss are key. Restriction of dietary protein intake may also be important in slowing the progressive glomerular injury that occurs in animals with PLN. Suggestions for dietary protein content for dogs are 14-20% total protein on a dry matter basis (2-3 g/kg/day); for cats, protein should be 28-32% on a dry matter basis (4 g/kg/day). Protein should be highly digestible to minimize hepatic urea formation and workload on the kidneys to excrete nitrogenous wastes. Patients should be monitored to assure that protein restriction does not lead to protein-malnourishment as evidenced by decreasing serum albumin concentrations in the face of decreasing UP/C, or sudden exacerbations of weight loss.
The most effective drugs in reducing the amount of proteinuria in patients with PLN are the angiotensin converting enzyme inhibitors (ACEi). In the United States, enalapril is the most commonly used drug for this purpose. In addition to serial evaluation of serum albumin levels, the efficacy of therapy may be monitored by serial measurement of UP/C. It is important to keep in mind that the UPC can vary widely with any given patient, and the effectiveness of ACEi therapy will only be made apparent through the trends in the UP/C established over time. Enalapril is administered at 0.5 mg/kg orally once daily, but can be escalated to as much as 1.0 mg/kg every 12 hours if adverse effects are not observed. Monitoring serum BUN and creatinine concentrations is important as ACEi have the potential to worsen azotemia.
Control of peripheral edema/ascites may come with improvements in serum albumin accomplished by the strategies above. Because of the role of renal sodium retention in the formation of edema and ascites in patients with PLN, dietary sodium restriction to 15-40 mg/kg day (0.1-0.4% dietary sodium) may help. The use of diuretics may also be beneficial in control of fluid accumulation. Loop diuretics such as furosemide and thiazide diuretics make reasonable choices in such patients. Patients refractory to loop diuretics may benefit from addition of thiazide diuretics to the treatment regimen.
A potentially devastating consequence of PLN is thromboembolic disease. Dogs with PLN may hypercoagulable secondary to loss of the endogenous anticoagulant AT. AT can be measured and the degree of deficiency of AT may be predictive of the risk of thromboembolism. In the absence AT activity, patients with serum albumin concentrations of less than 2 g/dl are considered to be at greater risk of thromboembolism. Administration of aspirin (0.5-5 mg/kg/ every 24 hours) is also advocated for patients with protein-losing glomerular diseases to inhibit platelet function and reduce the tendency of patients to develop thrombi. The use of immunosuppressive drugs is usually reserved for those patients whose underlying disease would be treated with such drugs.
Renal failure, if present, is managed with symptomatic treatment for the consequences of renal failure, such as gastritis, hypertension, and anemia. In depth discussion is beyond the scope of this manuscript.
References are available upon request.