Regulation of proximal tubular osteopontin in experimental hydronephrosis in the rat.

BACKGROUND: Osteopontin is a tubular-derived glycoprotein with macrophage chemoattractant properties. Our previous observations demonstrate that osteopontin is involved in the accumulation of macrophages within the renal cortex of rats following unilateral ureteral obstruction (UUO). METHODS: The present study performed Northern and Western blot analyses of isolated proximal tubular cells exposed to exogenous angiotensin II, and cultured rat proximal tubular cells subjected to one hour of cyclic mechanical stretch, which provided insight into mechanisms involving the proximal tubular renin-angiotensin system in the increased expression of cortical osteopontin following hydronephrosis. RESULTS: In situ hybridization, using a 35S-labeled antisense riboprobe, showed osteopontin mRNA transcription localized to the cortical tubules of the obstructed kidney. Freshly isolated proximal tubules incubated with angiotensin II (10-5 M) for one hour had increased osteopontin mRNA and protein expression by Northern and Western blot analyses, respectively. Pre-treatment of proximal tubules with losartan (10-5 M) for one hour prior to the addition of exogenous angiotensin II (10-5 M) decreased osteopontin mRNA and protein expression. Rat proximal tubule cells subjected to cyclic mechanical stretch for one hour exhibited a 2.1-fold increment in osteopontin mRNA levels, which was normalized following pre-treatment with losartan. CONCLUSIONS: This study provides evidence that angiotensin II, produced by the proximal tubule in the obstructed kidney as a result of mechanical injury, possibly mechanical stretch, may stimulate angiotensin II type I receptor activation, leading to up-regulated osteopontin expression and secretion by the proximal tubule, thereby facilitating macrophage recruitment into the renal interstitium.

Effectiveness of once-nightly dosing of extended-release niacin alone and in combination for hypercholesterolemia.

We performed a multicenter, open-label study to determine the long-term safety and efficacy of a new extended-release once-a-night niacin preparation, Niaspan, in the treatment of hypercholesterolemia. Niaspan, 0.5 to 3.0 g once a night at bedtime, was used alone or in combination with a statin (inhibitor of hydroxymethylglutaryl coenzyme A reductase), a bile acid sequestrant, or both. Patients included 269 hypercholesterolemic male and female adults enrolled in a 96-week study, and 230 additional adults for whom short-term safety data were available. The dosages of Niaspan attained by 269 patients were 1,000 mg (95% of patients), 1,500 mg (86%), and 2,000 mg (65%). After 48 weeks of treatment, Niaspan alone (median dose 2,000 mg) reduced low-density lipaprotein (LDL) cholesterol (18%), apolipoprotein B (15%), total cholesterol (11%), triglycerides (24%), and lipoprotein(a) (36%), and increased high-density lipoprotein (HDL) cholesterol (29%). Niaspan plus a statin lowered LDL cholesterol (32%), apolipoprotein B (26%), total cholesterol (23%), triglycerides (30%), and lipoprotein(a) (19%), and increased HDL cholesterol (26%). Reversible elevations of aspartate aminotransferase or alanine aminotransferase more than twice the normal range occurred in 2.6% of patients. One patient discontinued Niaspan because of transaminase elevations. Intolerance to flushing, leading to discontinuation of Niaspan, occurred in 4.8% of patients. The overall rate of discontinuance due to flushing in this study combined with 2 previous randomized trials was 7.3%. In the long-term treatment of hypercholesterolemia, Niaspan produced favorable changes in LDL and HDL cholesterol, triglycerides, and lipoprotein(a). Adverse hepatic effects were minor and occurred at rates similar to those reported for statin therapy.

Soluble ICAM-1 (sICAM-1) in biliary atresia and its relationship to disease activity.

BACKGROUND: Intercellular adhesion molecule-1 (ICAM-1) is strongly expressed on the bile ducts and hepatic parenchyma of livers with biliary atresia. A soluble, circulating form of this membrane protein has been found to be elevated in a number of inflammatory hepatic disorders. However, its expression in biliary atresia is unknown. The purpose of this study was to assess the presence of soluble ICAM-1 in infants with biliary atresia in relation to disease activity, degree of cholestasis, and standard liver function tests. MATERIALS AND METHODS: A total of nine patients (n = 9) with biliary atresia (seven) and neonatal hyperbilirubinemia (two) were studied (age range 6 weeks-9 years). Control samples were obtained from three healthy infants (2-10 months). Serum was collected from each patient and stored at -80 degrees C until assayed. Levels of sICAM-1 were measured in duplicate utilizing an ELISA method (Bioscource International). Standard liver function tests (conjugated bilirubin, gamma-glutamyl transpeptidase, alkaline phosphatase, alanine aminotransferase) were determined at the same time. Results are expressed as the means +/- SEM with statistical analysis by Mann-Whitney test. RESULTS: sICAM-1 levels were significantly elevated in all patients with biliary atresia (997 +/- 56 ng/ml) when compared to controls (P < 0.001). No correlation was found between sICAM-1 levels and conjugated bilirubin, gamma-glutamyl transpeptidase, alkaline phosphatase, and alanine aminotransferase levels or with clinical assessment of disease severity. CONCLUSIONS: sICAM-1 is markedly elevated in biliary atresia reflecting the immunopathology of the disease process but does not appear to correlate with markers of liver function. sICAM-1 may be useful in assessing the effects of immunomodulatory therapy.

Diabetic dyslipidemia.

Usual risk factors for coronary artery disease account for only 25-50% of increased atherosclerotic risk in diabetes mellitus. Other obvious risk factors are hyperglycemia and dyslipidemia. However, hyperglycemia is a very late stage in the sequence of events from insulin resistance to frank diabetes, whereas lipoprotein abnormalities are manifested during the largely asymptomatic diabetic prodrome and contribute substantially to the increased risk of macrovascular disease. The insulin-resistant diabetes course affects virtually all lipids and lipoproteins. Chylomicron and very-low-density lipoprotein (VLDL) remnants accumulate, and triglycerides enrich high-density lipoprotein (HDL) and low-density lipoprotein (LDL), leading to high levels of potentially atherogenic particles and low levels of HDL cholesterol. Hyperglycemia eventually impairs removal of triglyceride-rich lipoproteins, the accumulation of which accentuates hypertriglyceridemia. As triglycerides increase-still within the so-called normal range-abnormalities in HDL and LDL became more apparent. Thus, when triglycerides are >200 mg/dL, LDL particles are small and dense (when they are <90 mg/dL, the particles are of the large, buoyant variety). The atherogenicity of small, dense LDL particles is attributed to their increased susceptibility to oxidation, but in many patients they may be a marker for insulin resistance or the presence of atherogenic VLDL. Hypertriglyceridemia is associated with atherosclerosis because (1) it is a marker for insulin resistance and atherogenic metabolic abnormalities; and (2) the small size of triglyceride-enriched lipoproteins enables them to infiltrate the blood vessel wall where they are oxidized, bind to receptors on macrophages, and ingested, leading to the development of the atherosclerotic lesion. Various studies (primary prevention with gemfibrozil: Helsinki Heart Study; secondary prevention with simvastatin and pravastatin: Scandinavian Simvastatin Survival Study [4S] and Cholesterol and Recurrent Events [CARE], respectively) have demonstrated that lipid-lowering therapy in type 2 diabetes is effective in decreasing the number of cardiac events. Risk reduction was 22% to 50% (statins) and approximately 65% (fibrate) relative to placebo. It was also noted (in 4S and CARE) that the risk of major coronary events in untreated diabetic patients was 1.5-1.7-fold greater than in untreated nondiabetic patients. Although gemfibrozil (fibric acid derivative) is more effective in decreasing triglycerides and increasing HDL cholesterol in diabetic patients than the statins, it does not change and may even increase LDL-cholesterol levels (fenofibrate may be an exception, decreasing LDL cholesterol by 20-25% in some studies). However, gemfibrozil does increase LDL particle size. Nevertheless, the statins are the current lipid-lowering drugs of choice because the change in LDL-cholesterol-to-HDL-cholesterol ratio is better than with gemfibrozil. Moreover, the diabetic patient may be more likely to benefit from statin therapy than the nondiabetic patient. It should be noted that, in theory, nicotinic acid can correct or improve all lipid or lipoprotein abnormalities in patients with type 2 diabetes. Unfortunately, it is relatively contraindicated because it causes insulin resistance and may precipitate or aggravate hyperglycemia (in addition to its other well-known side effects such as flushing, gastric irritation, development of hepatotoxicity, and hyperuricemia). It is unknown at present whether newer formulations such as once-daily Niaspan may be better tolerated in diabetes. In any case, most patients with type 2 diabetes have risk factors for coronary artery disease and qualify for aggressive LDL cholesterol-lowering therapy. At the same time, it is presently unknown whether improved glycemic control decreases coronary artery disease risk in such patients.

Efficacy and safety of an extended-release niacin (Niaspan): a long-term study.

Crystalline nicotinic acid (immediate-release niacin) is effective therapy for lipoprotein regulation and cardiovascular risk reduction. However, inconvenient regimens and unpleasant side effects decrease compliance. Sustained-release formulations designed to circumvent these difficulties increase hepatotoxicity. Niaspan, a new US Food and Drug Administration (FDA)-approved, once-daily, extended-release form, has been found effective and safe in short-term trials. The long-term efficacy and safety of Niaspan lipid monotherapy was studied in 517 patients (aged 21-75 years) for < or =96 weeks in dosages < or =3,000 mg/day. Primary efficacy endpoints were low-density lipoprotein (LDL) cholesterol and apolipoprotein B (apo B) changes from baseline; secondary efficacy endpoints were changes in total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, lipoprotein(a), and total cholesterol/HDL-cholesterol ratio; safety data included adverse events and laboratory values over the 2-year study period. LDL-cholesterol levels decreased significantly: 18% at week 48 and 20% at week 96; apo B reduction was similar (16% decrease at week 48 and 19% at week 96). Large elevations in HDL cholesterol (26%, week 48; 28%, week 96) allowed only modest decreases in total cholesterol (12% and 13%, respectively), whereas total cholesterol/HDL-cholesterol ratio decreased by almost one third. Triglyceride and lipoprotein(a) levels were decreased by 27% and 30%, respectively (week 48), and by 28% and 40%, respectively (week 96). All changes from baseline were significant (p <0.001). Niaspan was generally well tolerated, although flushing was common (75%); however, there was a progressive decrease in flushing with time from 3.3 episodes in the first month to < or = 1 episode by week 48. Aspirin was used by one third of patients before Niaspan dosing to minimize flushing episodes. Although serious adverse events occurred in about 10% of patients, none were considered probably or definitely related to Niaspan. Adverse events in general varied widely, but their true relation to the study drug is difficult to ascertain without a placebo (control) group. No deaths occurred. There were statistically significant changes in hepatic transaminases, alkaline phosphatase, direct bilirubin, phosphorus, glucose, amylase, and uric acid. However, these changes were mostly small and are not likely to be biologically or clinically significant (the decrease in phosphorus is a new finding in niacin therapy). No myopathy was observed. Thus, this long-term study confirms the earlier short-term findings that Niaspan is safe and effective as monotherapy in plasma lipoprotein regulation.

Increased expression of decorin in experimental hydronephrosis.

Transforming growth factor (TGF)-beta1 is a potential mediator of tubulointerstitial (TI) fibrosis in the rat unilateral ureteral obstruction (UUO) model. Decorin is a protein composed of a core protein and a chondroitin sulfate side chain and is capable of inactivating TGF-beta. Since TGF-beta strongly induces the synthesis of decorin in experimental glomerulonephritis, it was our intent to investigate whether altered decorin expression is operant in the rat UUO model. Renal cortical decorin mRNA levels initially became elevated (2.5-fold) in obstructed kidney (OBK) versus contralateral unobstructed kidney (CUK) 24 hours post-UUO and remained greater in the OBK specimens at 48 (2.3-fold), 96 (2.2-fold), and 168 (1.9-fold) hours post-ureteral ligation. Whole-body X-irradiation 11 days prior to UUO significantly reduced decorin mRNA at 24 and 96 hours post-UUO. On immunolabeling, decorin was only evident in the adventitia of blood vessels in CUK specimens at any time point after UUO. In contrast, OBK specimens initially demonstrated periglomerular and peritubular interstitial localization of decorin at 96 hours post-ureteral ligation, which became even more intense and diffuse in the tubulointerstitium at 168 hours post-UUO. On Western analysis, there were highly significant increases in decorin protein expression in the OBK versus the CUK specimens at 96 and 168 hours post-UUO. Levels of active TGF-beta1 in the renal cortex of OBK were 1.9- and 3.6-fold higher than CUK at 48 and 96 hours post-UUO. In summary, we demonstrated that post-UUO, decorin mRNA and protein expression is up-regulated in the renal cortex of OBK, but not CUK, specimens in a temporal parallel with active TGF-beta1 levels and macrophage infiltration. We postulate that the development of TI fibrosis in this model may be related to only a physiologic induction of decorin by TGF-beta, and that pharmacologic levels may be required to retard or prevent scarring via TGF-beta inhibition.

Paired natural cysteine mutation mapping: aid to constraining models of protein tertiary structure.

This paper discusses the benefit of mapping paired cysteine mutation patterns as a guide to identifying the positions of protein disulfide bonds. This information can facilitate the computer modeling of protein tertiary structure. First, a simple, paired natural-cysteine-mutation map is presented that identifies the positions of putative disulfide bonds in protein families. The method is based on the observation that if, during the process of evolution, a disulfide-bonded cysteine residue is not conserved, then it is likely that its counterpart will also be mutated. For each target protein, protein databases were searched for the primary amino acid sequences of all known members of distinct protein families. Primary sequence alignment was carried out using PileUp algorithms in the GCG package. To search for correlated mutations, we listed only the positions where cysteine residues were highly conserved and emphasized the mutated residues. In proteins of known three-dimensional structure, a striking pattern of paired cysteine mutations correlated with the positions of known disulfide bridges. For proteins of unknown architecture, the mutation maps showed several positions where disulfide bridging might occur.

Ferritin stimulation of a monokine inhibitor of lipopolysaccharide-augmented myelopoiesis is ferroxidase dependent.

Ferritin inhibition of myelopoiesis has been associated with intrinsic ferroxidase activity of heavy-chain ferritin and with production of a monokine inhibitor of lipopolysaccharide (LPS)-augmented monocytopoiesis. We report here that intrinsic ferroxidase activity of heavy-chain ferritin is required for stimulated production of the monokine inhibitor of LPS-augmented monocytopoiesis.