Jason Kim

Osteopontin modulates angiotensin II–induced inflammation, oxidative stress, and fibrosis of the kidney

Osteopontin, a secreted glycoprotein has been implicated in several renal pathological conditions such as those due to ureteral obstruction, ischemia, and cyclosporine toxicity. We studied its possible role in angiotensin II-mediated renal injury by infusing wild-type and osteopontin knockout mice with angiotensin II and found that it raised blood pressure and increased urinary albumin/creatinine ratios in both strains of mice. However, while wild-type mice responded to the infusion by macrophage infiltration and increased expression of alpha-smooth muscle actin, fibronectin, and transforming growth factor-beta; the osteopontin knockout mice developed none of these. Further, the knockout mice had increased expression of monocyte chemoattractant protein-1; NADPH oxidase subunits such as NOX2, gp47phox, and NOX4; and plasminogen activator inhibitor-1 compared to the wild type animals. Proximal tubule epithelial cells in culture treated with recombinant osteopontin and angiotensin II had increased alpha-smooth muscle actin and transforming growth factor-beta expression. The effect of angiotensin II was blocked by an antibody to osteopontin. In addition, osteopontin attenuated angiotensin II-induced plasminogen activator inhibitor-1 expression. These studies show that osteopontin is a promoter and an inhibitor of inflammation, oxidative stress, and fibrosis that is capable of modulating angiotensin II-induced renal damage.

Critical role for osteopontin in diabetic nephropathy

The profibrotic adhesion molecule, osteopontin (OPN), is upregulated in kidneys of humans and mice with diabetes. The thiazolidinedione (TZD) insulin sensitizers decrease albuminuria in diabetic nephropathy (DN) and reduce OPN expression in vascular and cardiac tissue. To examine whether OPN is a critical mediator of DN we treated db/db mice with insulin, rosiglitazone, or pioglitazone to achieve similar fasting plasma glucose levels. The urine albumin-to-creatinine ratio and glomerular OPN expression were increased in diabetic mice, but both were reduced by the TZDs more than by insulin. We administered streptozotocin to OPN-null and OPN-wild-type mice, and OPN-null mice were bred into both type 1 (Ins2(akita/+)) and 2 (db/db) diabetic mice. In each case, OPN deletion decreased albuminuria, mesangial area, and glomerular collagen IV, fibronectin and transforming growth factor (TGF)-beta in the diabetic mice compared with their respective controls. In cultured mouse mesangial cells, TZDs but not insulin decreased angiotensin II-induced OPN expression, while recombinant OPN upregulated TGF-beta, ERK/MAPK, and JNK/MAPK signaling. These studies show that OPN expression in DN mouse models enhances glomerular damage, likely through the expression of TGF-beta, while its deletion protects against disease progression, suggesting that OPN might serve as a therapeutic target.

Thermoneutral Housing Accelerates Metabolic Inflammation to Potentiate Atherosclerosis but Not Insulin Resistance

Chronic, low-grade inflammation triggered by excess intake of dietary lipids has been proposed to contribute to the pathogenesis of metabolic disorders, such as obesity, insulin resistance, type 2 diabetes, and atherosclerosis. Although considerable evidence supports a causal association between inflammation and metabolic diseases, most tests of this link have been performed in cold-stressed mice that are housed below their thermoneutral zone. We report here that thermoneutral housing of mice has a profound effect on the development of metabolic inflammation, insulin resistance, and atherosclerosis. Mice housed at thermoneutrality develop metabolic inflammation in adipose tissue and in the vasculature at an accelerated rate. Unexpectedly, this increased inflammatory response contributes to the progression of atherosclerosis but not insulin resistance. These findings not only suggest that metabolic inflammation can be uncoupled from obesity-associated insulin resistance, but also point to how thermal stress might limit our ability to faithfully model human diseases in mice.

Adiponectin expression protects against angiotensin II-mediated inflammation and accelerated atherosclerosis.

Adiponectin (APN), an adipocytokine produced by adipose tissue, exerts pleiotropic actions regulating inflammation, metabolism and vascular homeostasis. APN levels are inversely correlated with obesity, type-2 diabetes, hypertension and cardiovascular disease. Although renin angiotensin system (RAS) activation in these interrelated metabolic syndrome components increases angiotensin II (AngII) levels leading to vascular damage, it is unknown whether APN under these conditions provides atheroprotection. We investigated whether increasing plasma APN provides atheroprotection in a hypertensive and accelerated atherosclerosis model. Using adenoviral gene transfer, sustained APN expression increased plasma levels of total and high-molecular weight APN, leading to a significant elevation of plasma HDL-cholesterol (HDL-C). Elevated APN levels were strongly atheroprotective, yet had no impact on blood pressure. Notably, gene expression analyses revealed that APN significantly inhibited the expression of pro-inflammatory and atherogenic genes while it increased the expression of the anti-inflammatory cytokine, IL-10 and the cholesterol efflux transporters, ABCA1 and ABCG1 in the artery wall. These findings suggest that increasing APN levels may be an effective therapeutic strategy to inhibit vascular inflammation and accelerated atherosclerosis associated with RAS activation in the metabolic syndrome.

Endothelial NOTCH1 is suppressed by circulating lipids and antagonizes inflammation during atherosclerosis

Briot et al. show that inflammatory lipids deriving from a high-fat diet suppress NOTCH1 expression and signaling in adult arterial endothelium and propose that reduction of endothelial NOTCH1 is a predisposing factor in the onset of atherosclerosis.

CHOLINE ACETYLTRANSFERASE EXPRESSION DURING A PUTATIVE DEVELOPMENTAL WAITING PERIOD

The relationship between the cholinergic expression, morphological development, and target cell innervation of olivocochlear (OC) efferent neurons was investigated in the postnatal hamster. Similar to what was found in previous studies, tracer injections into the contralateral cochlea labeled cells bodies retrogradely in periolivary regions and labeled cell bodies only rarely in the lateral superior olive (LSO). Few morphological differences were found among cell bodies labeled between postnatal day 1 (P1) and P30. Tracer injections into the crossed OC bundles within the brainstem anterogradely labeled terminals below the inner hair cells of the cochlea prior to P5 and labeled terminals below outer hair cells after P5, consistent with a period of transient innervation, as hypothesized previously. Within the superior olive, choline acetyltransferase (ChAT) was expressed differentially. In periolivary regions, ChAT was expressed as early as P0. ChAT‐immunoreactive cell bodies in periolivary regions were similar morphologically to retrogradely labeled OC neurons. In contrast, within the LSO, ChAT was not expressed until after P2. Consistent with a medial OC projection to the cochlea at early postnatal ages, ChAT immunoreactivity was detected below inner hair cells as early as P2 but was not detected below outer hair cells until after P6. Our results suggest that medial OC neurons not only provide transient connections to inner hair cells but also may express ChAT when they are below inner hair cells. Furthermore, these results raise the possibility that OC neurons may be capable of acetylcholine synthesis and release prior to or simultaneous with their innervation of the cochlea. J. Comp. Neurol. 397:281–295, 1998.

The macrophage LBP gene is an LXR target that promotes macrophage survival and atherosclerosis

The liver X receptors (LXRs) are members of the nuclear receptor superfamily that regulate sterol metabolism and inflammation. We sought to identify previously unknown genes regulated by LXRs in macrophages and to determine their contribution to atherogenesis. Here we characterize a novel LXR target gene, the lipopolysaccharide binding protein (LBP) gene. Surprisingly, the ability of LXRs to control LBP expression is cell-type specific, occurring in macrophages but not liver. Treatment of macrophages with oxysterols or loading with modified LDL induces LBP in an LXR-dependent manner, suggesting a potential role for LBP in the cellular response to cholesterol overload. To investigate this further, we performed bone marrow transplant studies. After 18 weeks of Western diet feeding, atherosclerotic lesion burden was assessed revealing markedly smaller lesions in the LBP−/− recipients. Furthermore, loss of bone marrow LBP expression increased apoptosis in atherosclerotic lesions as determined by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Supporting in vitro studies with isolated macrophages showed that LBP expression does not affect cholesterol efflux but promotes the survival of macrophages in the setting of cholesterol loading. The LBP gene is a macrophage-specific LXR target that promotes foam cell survival and atherogenesis.

342 Cyclic Arg-Gly-Asp (cRGD) peptide prevents diabetic nephropathy in db/db mice.

Purpose Chronic kidney disease and progression to end-stage renal disease (ESRD) impose a significant burden on the health care system. Diabetic nephropathy (DN) accounts for a staggering 45% of all ESRD cases. As the number of individuals with diagnosed diabetes age ≥ 20 years is expected to reach 39 million in 2050, the number of diabetes-related ESRD cases is expected to dramatically rise. Unfortunately, current management can delay but not cure DN. Integrins and their corresponding matrix proteins are increased in DN. We used an Arg-Gly-Asp (RGD)-containing peptide to inhibit integrin function in vitro and examined its effect in diabetic db/db mice. Method Using adhesion cell assays, we inhibited mesangial cell (MC) integrin attachment to fibronectin (FN) by blocking the α5β1-integrin receptor with an RGD hexapeptide in vitro. We customized the peptide by cyclization (cRGD; to enhance its bioavailability) and tested its effect in vivo in diabetic db/db mice by intraperitoneal administration 3MULT/week, since the db/db mouse is the best-accepted and characterized model of progressive human DN. Plasma glucose and systolic blood pressure were measured. Animals were placed in metabolic cages for 24 h urine collection. The right kidney was removed for periodic acid-Schiff staining, Western blot analysis, and immunostaining, and the left kidney was perfused, processed, and embedded for morphometric analysis of electron microscopy images. Primary rat mesangial cells were treated or pretreated with the RGD peptide or PD98059 and used in Western blot analysis for mitogen-activated protein kinase (MAPK) signal transduction. Results Compared to untreated and cRGE-inactive peptide (which has a single amino acid change in the RGD recognition site)-treated diabetic mice and treated and untreated nondiabetic db/m mice, the cRGD peptide prevented the development of DN as determined by improved increased fractional volume of the mesangium, MC and mesangial matrix, as well as albuminuria. There was no effect on GBM widening. These findings were independent of alterations in plasma glucose or blood pressure and were accompanied by decreased expression of α5,β1 integrin, FN, collagen I and collagen IV matrix proteins. RGD peptide inhibited MC adhesion-induced MAPK signal transduction. Conclusion These studies suggest that specifically targeting integrin function with a customized cRGD peptide is an innovative treatment of DN in db/db mice, which could potentially ameliorate or prevent DN in humans.