Background: Serial monitoring of acute phase protein (APP) concentrations in canine autoimmune hemolytic anemia (AIHA) has not been reported.Hypotheses: Acute canine AIHA is accompanied by an acute phase response (APR) characterized by increased C-reactive protein (CRP) and a1-acid glycoprotein (AAG) concentrations and decreased albumin concentrations.Animals: Twenty-seven dogs with AIHA and 11 control dogs. Methods: Prospective, cohort study. CRP, AAG, and albumin concentrations, white blood cell (WBC) count, and packed cell volume (PCV) were determined at admission (day 1), every 48 hours until death or discharge, and on days 30, 90, 180, and 365.Results: Compared with controls, CRP and AAG concentrations were increased and albumin concentration was decreased in dogs with AIHA (days 1-7; P o .002) and normalized with disease stabilization (days 9-365; P 4 .05). APP concentrations on day 1 were not predictive of survival, duration of hospitalization, or number of blood transfusions (P 5 .153-.940). PCV correlated with APP concentrations over time (CRP r 5 À.600, AAG r 5 À.665, albumin r 5 .533; P o .0001) as did WBC count (CRP r 5 .253, AAG r 5 .486, albumin r 5 À.246; P o .006). Day 1 CRP concentration was lower for dogs that received corticosteroids before referral (115.3 mg/mL) compared with dogs that did not (191.2 mg/mL; P 5 .02).Conclusions: An APR occurs in canine AIHA. Initial APP concentrations are not predictive of acute survival, correlate with hematologic markers of remission, and normalize rapidly with disease stabilization.

Abstract. Alport syndrome refers to a hereditary disorder characterized by progressive renal disease and a multilaminar appearance to the glomerular basement membrane (GBM). In a small group of patients with Alport syndrome, cyclosporine A was reported to decrease proteinuria and maintain stable renal function over 7 to 10 yr of follow-up. The present study examined the effect of cyclosporine A on GBM structure and the progression to renal failure in a canine model of X-linked Alport syndrome. Affected male dogs and normal male dogs treated with cyclosporine A underwent serial renal biopsies. Body weight, serum concentrations of creatinine and albumin, and GFR were sequentially determined. Controls consisted of untreated dogs that developed end-stage renal failure by 8 mo of age. Renal biopsies were assessed for glomerulosclerosis and the percent of multilaminar GBM as measured by image analysis. Significant differences were found between treated and untreated affected dogs for weight, serum creatinine, and GFR. There was a significant delay in the progression of multilaminar change to the GBM, although treated affected dogs at termination had attained approximately 100% split GBM as did untreated affected dogs. A significant difference in the number of sclerotic glomeruli was also noted; treated dogs rarely developed obsolete glomeruli during the period studied. Interstitial fibrosis was not significantly affected by cyclosporine A treatment. These findings indicate that cyclosporine A is beneficial in slowing, but not stopping, the clinical and pathologic progression of Alport syndrome. At least part of this beneficial effect comes from a delayed deterioration of GBM structure, which in turn may be related to glomerular hemodynamics altered by cyclosporine A.

Bone marrow-derived stromal stem cells (BMSC) can differentiate along a variety of mesenchymal lines, including mesangial cells. For determining whether BMSC can be induced to differentiate along podocytic lines in vitro, canine BMSC were cultured on plastic, type I collagen, and NC1 hexamers of type IV collagen from normal and Alport canine glomerular basement membrane. Results were compared with a mouse podocyte cell line. In the case of the podocyte line, differentiation occurred on all three matrices as indicated by the expression of synaptopodin and CD2-associated protein (CD2AP) and organization of myosin heavy chain IIA into a linear pattern. BMSC proliferated equally well on all matrices, but cells that were grown on type IV collagen NC1 hexamers became larger and stellate. Evidence for podocytic differentiation occurred on all three collagen matrices as indicated by the redistribution of myosin IIA to a linear pattern and expression of synaptopodin, CD2AP, and ␣-actinin. A punctate distribution of CD2AP was seen only in cells that were grown on normal and Alport glomerular basement membrane NC1 hexamers. Differentiated podocytes expressed the ␣1, ␣2, and ␣5 chains of type IV collagen but at higher levels in cells that were grown on NC1 hexamers. Similar results were obtained in BMSC for the ␣1 and ␣2 chains only. The ␣3, ␣4, and ␣6 chains were never detected in the podocyte line or BMSC. These results indicate that BMSC undergo a degree of podocytic differentiation in vitro and greater when grown on type IV collagen NC1 hexamers than type I collagen. Alport and normal NC1 hexamers seem equally permissive to BMSC growth and differentiation, suggesting that these processes are not influenced specifically by the ␣3/␣4/␣5 network. BMSC may be useful in the development of stem cell-based reconstitution of glomeruli that are damaged by disease and for gene therapy of genetic glomerular diseases such as Alport syndrome.

Alport syndrome is an inherited disorder of type IV collagen with progressive nephropathy, ocular abnormalities, and high-tone sensorineural deafness. In X-linked Alport syndrome, mutations in the COL4A5 gene encoding the ␣5 chain of type IV collagen lead to loss of the ␣3/␣4/␣5 network and increased susceptibility of the glomerular basement membrane to long-term damage. The molecular defects that underlie the otopathology in this disease remain poorly understood. We used a canine model of X-linked Alport syndrome to determine the expression of type IV collagen ␣-chains in the inner ear. By 1 month in normal adult dogs, the ␣3, ␣4, and ␣5 chains were co-expressed in a thin continuous line extending along the basilar membrane and the internal and external sulci, with the strongest expression along the lateral aspect of the spiral ligament in the basal turn of the cochlea. Affected dogs showed complete absence of the ␣3/␣4/␣5 network. The lateral aspect of the spiral ligament is populated by tension fibroblasts that express ␣-smooth muscle actin and nonmuscle myosin and are postulated to generate radial tension on the basilar membrane via the extracellular matrix for reception of high frequency sound. We propose that in Alport syndrome, the loss of the ␣3/␣4/␣5 network eventually weakens the interaction of these cells with their extracellular matrix, resulting in reduced tension on the basilar membrane and the inability to respond to high frequency sounds.

Serum nonesterified fatty acid (NEFA) and beta-hydroxybutyrate (BHBA) concentrations are used to evaluate energy status in peripartum dairy cows. Blood samples from 37 cows in the week before parturition and 47 cows in the first week after parturition from 3 dairy herds were taken 1h before the first feeding (-1h) as well as 4 and 10h after the first feeding. Nonesterified fatty acid concentrations were measured in samples from cows before calving and BHBA was measured in samples from lactating cows. Mean NEFA concentrations in the prepartum cows were significantly higher at -1h (0.20 mmol/L) than at 4h (0.14 mmol/L), but were not different between 4 and 10h (0.17 mmol/L). Using a cutpoint of NEFA > or = 0.4 mmol/L, 32% of cows had high concentrations at -1h compared with 16% of the same cows at 4 and 10h. There were no differences in mean BHBA between -1h (646 micromol/L) and 4h (596 micromol/L), but mean BHBA was higher at 10h (711 micromol/L) than at -1h. Using a cutpoint of BHBA > or = 1,400 micromol/L, there were no differences in the proportions of high BHBA, which were 9, 11, and 13% of cows at -1, 4, and 10h, respectively. Prandial effects on serum NEFA may affect interpretation of this analyte. In order not to misclassify cows when assessing energy status, samples for NEFA must at least be taken at a consistent time relative to feeding within a given herd. When sampling cows to monitor elements of energy metabolism in the prepartum period, there was twice the probability of detecting animals with NEFA values > or = 0.4 mmol/L if they were sampled 1h before the first feed delivery compared with sampling the same cows 4 or 10h after feeding.