In adult horses and foals, renal dysfunction can occur as a secondary complication to gastrointestinal disorders, dehydration, septicemia, endotoxemia and nephrotoxic drug administration. Measurement of renal function is an important feature not only in the diagnosis, but also in the prognosis and management of renal disease. Commonly used drugs such as phenylbutazone and gentamicin can be highly nephrotoxic under certain conditions. Estimation of the glomerular filtration rate (GFR), accepted as one of the earliest and most sensitive assessments of renal function, can be determined in horses using standard techniques such as endogenous or exogenous renal creatinine clearance. These techniques can be time consuming, dangerous to perform on fractious patients, require trained personnel and are subject to errors most often associated with improper or incomplete urine collection. Recently, tests using iohexol, a radiographic contrast agent, have been developed to estimate the GFR in human beings, pigs, sheep, dogs, cats and horse foals with results that have been validated by traditional standards. Serum clearance of a substance that is freely filtered by the kidneys without tubular secretion or reabsorption, that is not protein bound, and that is not metabolized, is a measurement of glomerular filtration rate. Iohexol meets all of these requirements and thus its clearance from serum should accurately estimate GFR. Utilization of serum clearance studies for estimation of GFR provides a clinically feasible and reproducible method in order to measure GFR in horses. Other commonly used methods to assess renal function in horses are fraught with inherent and operator error. Serum clearance of iohexol does not necessitate collection of urine and has been shown to be a safe, reproducible method using collection of timed blood samples to assess renal function in humans and animals. The objectives of this project were 1) to determine a method of estimation of GFR based on serum clearance of a substance that meets the requirements of a marker for GFR, and 2) to make the method clinically applicable by developing a method using two blood samples to derive clearance and thus GFR in normal adult horses. Results of this study showed good agreement between GFR derived by exogenous creatinine clearance and serum clearance of iohexol. In addition, GFR values for all horses using either method were within published reference ranges for this species. The results of this study indicate that a single intravenous injection of iohexol at a dose of 150 mg/kg, followed by collection of 2 serum samples at 3 and 4 hours post injection can be used to estimate the GFR in healthy horses. / Master of Science

<p>Bikunin, a chondroitin sulfate-containing protein of 25 kDa, has protease inhibitory activity and occurs in the plasma in free and complexed form. In inter-α-inhibitor (IαI) and pre-a-inhibitor (PαI) it is covalently linked through its chondroitin sulfate (CS) chain to two or one other polypeptide of about 80 kDa – heavy chains 1 and 2 (H1, H2) and heavy chain 3 (H3) – respectively. Bikunin and the heavy chains are synthesized as precursors, which are proteolytically cleaved and assembled into IαI and PαI in the secretory pathway. The C-terminal extension (CTX) of the heavy chains seems to mediate its own cleavage and theassembly of the complexes. The heavy chains of the IαI family become transferred to hyaluronan during ovulation and inflammation.</p><p>In this thesis, the biosynthesis of PαI, the plasma clearance of bikunin and the binding of IαI to collagen were studied. We found that in H3, a short segment on the N-terminal side of the CTX cleavage site is required for cleavage. Furthermore, the H3 could become linked to free CS chains primed by a xyloside, showing that the bikunin protein core is not needed for coupling. We also identified His649 as a residue essential for coupling, but not for cleavage. </p><p>Bikunin labelled with a residualizing agent, 125I-tyramine cellobiose, was injected into mice to identify tissues involved in its uptake. Half of the radioactivity was recovered in the kidneys, 10% in the liver, and the rest distributed in other tissues. We determined the half-life of bikunin in rat plasma using two independent methods: injection of 125I-bikunin, or hepatectomy followed by assessing the rate of disappearance of endogenous bikunin. Both methods yielded half-time values of 5-7 minutes. Removal of the CS chain did not affect the clearance rate of bikunin.</p><p>IαI and its heavy chains were found to bind to collagen with dissociation constants greater than 2 μM and 0.4-0.6 μM, respectively and this binding was independent of divalent metal ions. We suggest that the interaction of IαI with collagen may play a modulatory role in cell migration or in remodelling of the extracellular matrix.</p>

Bikunin, a chondroitin sulfate-containing protein of 25 kDa, has protease inhibitory activity and occurs in the plasma in free and complexed form. In inter-α-inhibitor (IαI) and pre-a-inhibitor (PαI) it is covalently linked through its chondroitin sulfate (CS) chain to two or one other polypeptide of about 80 kDa – heavy chains 1 and 2 (H1, H2) and heavy chain 3 (H3) – respectively. Bikunin and the heavy chains are synthesized as precursors, which are proteolytically cleaved and assembled into IαI and PαI in the secretory pathway. The C-terminal extension (CTX) of the heavy chains seems to mediate its own cleavage and theassembly of the complexes. The heavy chains of the IαI family become transferred to hyaluronan during ovulation and inflammation. In this thesis, the biosynthesis of PαI, the plasma clearance of bikunin and the binding of IαI to collagen were studied. We found that in H3, a short segment on the N-terminal side of the CTX cleavage site is required for cleavage. Furthermore, the H3 could become linked to free CS chains primed by a xyloside, showing that the bikunin protein core is not needed for coupling. We also identified His649 as a residue essential for coupling, but not for cleavage. Bikunin labelled with a residualizing agent, 125I-tyramine cellobiose, was injected into mice to identify tissues involved in its uptake. Half of the radioactivity was recovered in the kidneys, 10% in the liver, and the rest distributed in other tissues. We determined the half-life of bikunin in rat plasma using two independent methods: injection of 125I-bikunin, or hepatectomy followed by assessing the rate of disappearance of endogenous bikunin. Both methods yielded half-time values of 5-7 minutes. Removal of the CS chain did not affect the clearance rate of bikunin. IαI and its heavy chains were found to bind to collagen with dissociation constants greater than 2 μM and 0.4-0.6 μM, respectively and this binding was independent of divalent metal ions. We suggest that the interaction of IαI with collagen may play a modulatory role in cell migration or in remodelling of the extracellular matrix.

Background: Glomerular filtration rate (GFR) is a clinically important measurement of kidney function and estimating the GFR is of great importance in healthcare. Methods available today either lack in precision or are overly time consuming. Proenkephalin (PENK) has been shown to correlate well with the GFR and has therefore been proposed as a novel biomarker for kidney function. Aim: To evaluate a new point-of-care test for measuring PENK in blood and to assess its correlation to GFR measured by iohexol plasma clearance (mGFRiohexol). Materials and methods: Blood was collected from 21 patients with varying indications for the iohexol plasma clearance test. PENK was measured with IB10 Sphingotest penKid in whole blood, plasma and serum respectively. The concentration was correlated to the mGFRiohexol and results were compared to those from measurement of the routine markers for kidney function, i.e. creatinine and cystatin C. Results: Fourteen men and seven women were included. The median age was 57 years. PENK in plasma correlated weakly with mGFRiohexol (R2=0.22, p=0.042). No significant correlation was shown for PENK in whole blood or serum. Creatinine also showed a weak correlation with mGFRiohexol (R2=0.35, p=0.0046). In contrast, cystatin C was strongly correlated with mGFRiohexol (R2= 0.87, p&lt;0.0001). Conclusion: Our results indicate that PENK is a biomarker of low clinical value for estimating the GFR. However, further studies are needed before this can be assured. Cystatin C, on the contrary, seems to be an accurate biomarker for estimating the GFR.