Introduction
An inherited renal disease that causes juvenile-onsetrenal failure has been recognized in Cocker Spaniels (known in North Americaas English Cocker Spaniels) worldwide for more than 50 years. During thelast decade, research has demonstrated that the disease is caused by certainmolecular abnormalities of the collagen content of the walls of the glomerularcapillaries through which blood filtration occurs in the kidneys. This renaldisease is analogous to a condition called Alport syndrome that occurs inpeople. 청주출장마사지
Glomerular capillary walls consist of several layers; inner and outerlayers of cells, and a middle layer of extracellular matrix material calledthe glomerular basement membrane (GBM). The GBM contains a crucial networkof type IV collagen molecules that is necessary to maintain normal structure and function of glomerular capillary walls. The type IV collagen network inthe GBM is an assembly of three distinct peptide chains, and each of these peptide chains is encoded by separate gene. A mutation in any one of these three genes can disrupt the molecular integrity of the entire collagen IV network in the GBM and cause progressive renal damage that leads to kidney failure. One of these genes (COL4A5) is located on the X-chromosome in both dogs and humans, and when mutations occur in this gene, the disease has an X-linked mode of inheritance in both species. The other two genes (COL4A3 and COL4A4) are located on autosomes (human chromosome 2, and canine chromosome 25), and when a mutation in one of these two genes is responsible, the disease has an autosomal (usually autosomal recessive) mode of inheritance. In Cocker Spaniels, the disease is inherited as an autosomal recessive trait and the peptide products of COL4A3 and COL4A4 are completely absent from the GBM (seebelow). Therefore, COL4A3 and COL4A4 are the candidate genes in which thecausative mutation for FN in Cocker Spaniels is likely to exist, but themutation that causes the disease has not yet been identified. 광주출장마사지
Clinical signs
Dogs with FN develop chronic renal failure, usuallywhile they are between 6 months and 2 years of age. The clinical signs associatedwith chronic renal failure caused by FN are the same as those associatedwith chronic renal failure due to any other cause. Clinical signs that areoften observed include excessive water consumption (polydipsia), excessiveurine volume (polyuria), reduced growth rate or weight loss, poor qualityhair coat, reduced appetite, and vomiting. Such signs can develop insidiouslyand escape recognition until the degree of renal failure is so severe thatovert uremia supervenes. At this late stage of the disease, physical examinationfindings may include thin body condition, dehydration, pallor of mucous membranes,uremic breath odor, and oral ulcerations. Alternatively, especially at earlierstages of the disease, physical exam findings may be normal. 전주출장마사지
Laboratory features
When dogs with FN develop renal failure, serumchemistry test results will be abnormal in proportion to the severity ofrenal failure. Expected abnormalities include azotemia (increased BUN andcreatinine concentrations), hyperphosphatemia, and metabolic acidosis. Hematologytest results usually show some degree of anemia. Urinalysis findings fordogs in renal failure invariably include evidence of impaired urine concentratingability (inappropriately low urine specific gravity). All of the laboratorytest abnormalities mentioned so far might be associated with chronic renalfailure due to any cause; they do not help to differentiate FN from any otherpotential cause of renal failure.
Because FN is a glomerular disease, affected dogs invariably exhibitproteinuria by the time that any clinical illness that can be attributedto FN first develops. Indeed, as renal disease evolves in dogs that haveFN, proteinuria always is the first abnormality that can be detected by non-invasivetesting. Of course, proteinuria can be associated with a variety of differenturinary tract disorders (e.g., bacterial urinary tract infection, urolithiasis,etc.), so an appropriate clinical investigation must be conducted to excludesuch other conditions. Nonetheless, persistent renal proteinuria that isnot otherwise explained in a young (3- to 12-month-old) Cocker Spaniel mostoften proves to be due to FN. If discovered very early in the course of disease,the magnitude of proteinuria may be mild, but it typically becomes markedwithin a month or two. Urine protein-to-creatinine (UPC) ratios commonlyare in the 5-10 range, and occasionally are greater than 10. If detectedearly, proteinuria may be observed while urine concentrating ability is wellpreserved (i.e., urine specific gravity is still high) and serum creatinineconcentrations are still within the normal reference range. However, withcontinued monitoring, dogs with FN will have persistent proteinuria, willsubsequently loose their urine concentrating ability, and will graduallydevelop increasing serum creatinine concentrations. If FN has progressedto renal failure by the time that laboratory tests are performed, the findingswill include marked proteinuria and isosthenuria, as well as the hematologicand serum chemistry abnormalities expected with the severity of renal failurethat then exists. 강남출장마사지
Treatment
The renal disease caused by FN invariably is progressiveand ultimately fatal; however, the rate of disease progression observed inaffected dogs is more rapid in some individuals than in others. Additionally,certain treatments (feeding a diet formulated for dogs with renal failureand administering an angiotensin converting-enzyme inhibitor such as enalaprilor benazepril) may slow the rate of renal disease progression somewhat. Evenif they are effective, however, such therapeutic efforts generally forestallthe development of terminal renal failure by several weeks or a few monthsat best. Moreover, the rate of renal disease progression usually is fairlyrapid during the late stages of the disorder. Once an affected dog developsmoderate azotemia, the disease typically progresses to the terminal stageof renal failure within 3 to 6 more weeks.
Pathologic features
When examined by light microscopy, kidneys fromdogs with FN have a constellation of abnormalities that are indicative ofprogressive glomerular disease. Severity of the abnormalities depends onthe stage of the disease that was present when the tissue was obtained, butthe lesions are non-specific. That is, at the light microscopic level, thekidney changes that are produced by FN are similar to those of other glomerulardisorders in dogs. The distinctive abnormalities that set FN apart from otherglomerular renal diseases are seen only with immunostaining techniques andusing transmission electron microscopy.
Immunostaining of kidney with individual antibodiesthat specifically recognize and bind to each of the different type IV collagenpeptides can be used to identify the collagen content of all renal basementmembranes. Normal GBM stains strongly positive for the alpha-3 (α3) chain,the α4 chain, and the α5 chain of type IV collagen. This staining patternreflects presence of the normal α3-α4-α5 chain network that is required forlong-term maintenance of GBM structure and function. In Cocker Spaniels withFN, however, staining for α3 and α4 chains is totally negative in all basementmembranes where they are normally present, including the GBM. In addition,GBMstaining for α5 chains is greatly reduced, and there is greatly increasedstaining for 3 other chains (α1, α2, and α6) that normally are not prominentcomponents of the GBM. This abnormal staining pattern reflects absence ofthe α3-α4-α5 collagen network from the GBM, which instead contains networkscomposed of α1-α1-α2 and α5-α5-α6 chains. These substitute type IV collagennetworks do not function properly (i.e., they do not function like the α3-α4-α5network), and as a result of this abnormality, the processes of renal injurythat ultimately produce kidney failure are set into motion. This abnormalpattern of immunostaining of renal basement membranes for type IV collagenα-chains is very distinctive of FN in Cocker Spaniels and can be demonstratedat all stages of the disease (i.e., from birth onward). 출장안마
Despite the abnormal type IV collagen content of certain renal basementmembranes, the kidneys of dogs with FN initially develop normally and functionproperly. Within a few months, however, because the α3-α4-α5 network is missing,the ultrastructure of the GBM, which can be seen only by using electron microscopy, starts changing. This is the first structural change in the kidneys that occurs,and it precedes any change in the kidneys that can be seen with routine lightmicroscopy. The ultrastructural GBM changes are at first focal (i.e., presentin some sites but not others) and mild, but they gradually become more extensiveand severe throughout the course of the disease. The ultrastructural GBMchanges also are very distinctive of this disease, especially once they havebecome widespread and at least moderately severe. The GBM becomes greatly thickened and exhibits multilaminar splitting and fragmentation of its internal structure. These GBM changes are associated with other changes in the glomerular capillary wall that alter its filtration properties, which causes the proteinuria to develop.
As the disease progresses, light microscopic lesions develop both inthe glomeruli and in the tubulointerstitium. The morphologic glomerular lesions are those of a membranoproliferative disorder progressing to glomerular sclerosis and obsolescence with periglomerular fibrosis. The tubulointerstitial changes, which always are prominent when renal failure has developed, include extensive tubular degeneration, dilation, and atrophy together with interstitial inflammation and fibrosis. As emphasized previously, these light microscopic lesions are non-specific; they are not sufficient by themselves for definitive diagnosis of FN. 원주출장마사지
Definitive diagnosis
In conjunction with a well documented clinicalcourse of illness that is compatible with FN (and appropriate clinical investigationsthat failed to identify other causes of renal disease), light microscopicfindings that are consistent with FN are sufficient for a presumptive diagnosisof the condition. Nevertheless, definitive diagnosis of FN always rests ondemonstrating at least one, and preferably both, of the distinctive abnormalitiesthat uniquely distinguish FN from all other causes of renal disease. Therefore, the definitive diagnosis of FN always requires special studies (i.e., electron microscopy, or immunostaining, or both) of kidney from the affected dog. Availabilityof centers that can perform such evaluation is limited, but even more importantly,ability to perform (and interpret) such studies depends on having suitabletissue specimens for examination, even if a center that can perform suchstudies is identified. As a rule, tissues that are routinely fixed in 10%formalin, the usual fixative that is used for light microscopic studies,are not satisfactory for electron microscopy or immunostaining. Consequently,obtaining tissue specimens that will be suitable for such studies typicallyrequires prior preparation and planning to become familiar with the appropriateprocedures and assemble the needed materials.
Appendix – Guidelines for collectingrenal tissue specimens for pathologic studies from dogs that are suspectedto have FN
1. Time matters, especially for the electron microscopyspecimens. Post-mortem collection of renal tissue specimens should be accomplishedimmediately after the dog’s death or euthanasia. Renal biopsy specimens alsoshould be processed immediately.
2. Another crucial issue is that the tissue specimensfor electron microscopy and immunostaining must contain glomeruli; that is,they must be samples of renal cortex.
3. The key factor for satisfactory electron microscopicstudies is complete, rapid fixation of the tissue (ultrastructural featuresthe glomerular cells and GBM begin to deteriorate within a few minutes, andthe purpose of “fixation” is to halt this process). Optimum fixation is providedby special fixatives (e.g., 2.5% or 3.0% glutaraldehyde in a phosphate buffer)that are intended for electron microscopy. Fixative solutions for electronmicroscopy must be kept refrigerated and generally have a short shelf-life;we routinely prepare a fresh supply of fixative every 30 days. Besides usingan appropriate fixative solution, good fixation is promoted by cutting thetissue specimen in to sufficiently small pieces (1 mm cubes), but this mustbe done without crushing the specimen. We usually place a piece of renalcortex (about 5-6 mm cube) on a clean glass slide in a small puddle of thefixative (taken from the “EM specimen vial” with a pipette) and then usea fresh single-edge razor (or scalpel) blade to cut the tissue in half, theneach piece in half again, and so on until all the pieces are about 1 mm cubes.These are then put in the “EM specimen vial” (which is promptly refrigeratedagain).
When a fixative that is intended for electron microscopy is not available, the next best fixative solution to use is 10% formalin (i.e., as for light microscopy). Fixation attained in this way can be satisfactory, but starting immediately (i.e., with tissue that is as fresh as possible) and making the pieces sufficiently small is all the more important because formalin fixation proceeds more slowly than fixation with conventional electron microscopy fixatives.
4. Immunostaining is performed using unfixed tissuespecimens that are snap-frozen (with liquid nitrogen) and kept frozen thereafter.To avoid having to ship frozen specimens, we use Michel’s Transport Medium(a non-fixative solution formulated for this purpose) to preserve the specimenuntil it gets to our laboratory; we then freeze it. Michel’s Transport Mediaalso has a short shelf-live and must be kept refrigerated (during storageand use), but it does keep the tissue suitable for eventual immunostainingfor up to 2-3 days (i.e., long enough for it to be delivered to our lab).
5. We also always want to receive conventional formalin-fixedspecimens of kidney for routine light microscopic examinations as well asthe special studies.
Note: the tissue specimens described above are usedfor diagnostic evaluations. For our genetic studies of FN, we also want ablood sample (in a “purple-top” EDTA tube; for DNA) and some pieces of renalcortex in placed in RNALater™ (for RNA isolation). Within the United States,because many of the component materials have a short shelf-life and mustbe kept refrigerated, we prepare and ship kits containing all the necessarymaterials and instructions to veterinarians “upon request.” Using courierservices (e.g., FedEx) for overnight delivery, we can provide such a kitin a timely manner with as little as 24-36 hours of prior notice (barringweekends).
Selected references:
Lees GE, Wilson PD, Helman RG, Homco LD, Frey MS:Glomerular ultrastructural findings similar to hereditary nephritis in 4English cocker spaniels. J Vet Intern Med 1997;11:80-85.
Lees GE, Helman RG, Homco LD, Millichamp NJ, HunterJF, Frey M: Early diagnosis of familial nephropathy in English cocker spaniels.J Am Anim Hosp Assoc 1998;34:189-195.
Lees GE, Helman RG, Kashtan CE, Michael AF, HomcoLD, Millichamp NJ, Ninomiya Y, Sado Y, Naito I, Kim Y: A model of autosomalrecessive Alport syndrome in English cocker spaniel dogs. Kidney Int 1998;54:706-719.