Effects of the antioxidant drug tempol on renal oxygenation in mice with reduced renal mass.

We tested the hypothesis that reactive oxygen species (ROS) contributed to renal hypoxia in C57BL/6 mice with ⅚ surgical reduction of renal mass (RRM). ROS can activate the mitochondrial uncoupling protein 2 (UCP-2) and increase O(2) usage. However, UCP-2 can be inactivated by glutathionylation. Mice were fed normal (NS)- or high-salt (HS) diets, and HS mice received the antioxidant drug tempol or vehicle for 3 mo. Since salt intake did not affect the tubular Na(+) transport per O(2) consumed (T(Na/)Q(O2)), further studies were confined to HS mice. RRM mice had increased excretion of 8-isoprostane F(2α) and H(2)O(2), renal expression of UCP-2 and renal O(2) extraction, and reduced T(Na/)Q(O2) (sham: 20 ± 2 vs. RRM: 10 ± 1 µmol/µmol; P < 0.05) and cortical Po(2) (sham: 43 ± 2, RRM: 29 ± 2 mmHg; P < 0.02). Tempol normalized all these parameters while further increasing compensatory renal growth and glomerular volume. RRM mice had preserved blood pressure, glomeruli, and patchy tubulointerstitial fibrosis. The patterns of protein expression in the renal cortex suggested that RRM kidneys had increased ROS from upregulated p22(phox), NOX-2, and -4 and that ROS-dependent increases in UCP-2 led to hypoxia that activated transforming growth factor-ß whereas erythroid-related factor 2 (Nrf-2), glutathione peroxidase-1, and glutathione-S-transferase mu-1 were upregulated independently of ROS. We conclude that RRM activated distinct processes: a ROS-dependent activation of UCP-2 leading to inefficient renal O(2) usage and cortical hypoxia that was offset by Nrf-2-dependent glutathionylation. Thus hypoxia in RRM may be the outcome of NADPH oxidase-initiated ROS generation, leading to mitochondrial uncoupling counteracted by defense pathways coordinated by Nrf-2.

Prevalence of atrial fibrillation and its predictors in nondialysis patients with chronic kidney disease.

BACKGROUND AND OBJECTIVES: Chronic kidney disease (CKD) increases systemic inflammation, which is implicated in development and maintenance of atrial fibrillation (AF); therefore, we hypothesized that the prevalence of AF would be increased among nondialysis patients with CKD. This study also reports independent predictors of the presence of AF in this population. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A retrospective, cross-sectional analysis of 1010 consecutive nondialysis patients with CKD from two community-based hospitals was conducted. Estimated GFRs (eGFRs) were calculated using the Modification of Diet in Renal Disease (MDRD) equation. Multivariate logistic regression was used to determine independent predictors. RESULTS: Of 1010 nondialysis patients with CKD, 214 (21.2%) had AF. Patients with AF were older than patients without AF (76 +/- 11 versus 63 +/- 15 yr). The prevalence of AF among white patients (42.7%) was higher than among black patients (12.7%) or other races (5.7%). In multivariate analyses, age, white race, increasing left atrial diameter, lower systolic BP, and congestive heart failure were identified as independent predictors of the presence of AF. Although serum high-sensitivity C-reactive protein levels were elevated in our population (5.2 +/- 7.4 mg/L), levels did not correlate with the presence of AF or with eGFR. Finally, eGFR did not correlate with the presence of AF in our population. CONCLUSIONS: The prevalence of AF was increased in our population, and independent predictors were age, white race, increasing left atrial diameter, lower systolic BP, and congestive heart failure.

Humoral autoimmunity to basement membrane antigens is regulated in C57BL/6 and MRL/MpJ mice transgenic for anti-laminin Ig receptors.

Basement membrane proteins are targeted in organ-limited and systemic autoimmune nephritis, yet little is known about the origin or regulation of immunity to these complex extracellular matrices. We used mice transgenic for a nephrotropic systemic lupus erythematosus (SLE) Ig H chain to test the hypothesis that humoral immunity to basement membrane is actively regulated. The LamH-Cmu Ig H chain transgene combines with diverse L chains to produce nephrotropic Ig reactive with murine laminin alpha1. To determine the fate of transgene-bearing B cells in vivo, transgenic mice were outcrossed onto nonautoimmune B6 and SLE-prone MRL backgrounds and exposed to potent mitogen or Ag in adjuvant. In this work we demonstrate that transgenic autoantibodies are absent in serum from M6 and M29 lineage transgenic mice and transgenic B cells hypoproliferate and fail to increase Ig production upon exposure to endotoxin or when subjected to B cell receptor cross-linking. Administration of LPS or immunization with autologous or heterologous laminin, maneuvers that induce nonoverlapping endogenous anti-laminin IgG responses, fails to induce a transgenic anti-laminin response. The marked reduction in splenic B cell number suggests that selected LamH-Cmu H chain and endogenous L chain combinations generate autospecificities that lead to B cell deletion. It thus appears that SLE-like anti-laminin B cells have access to and engage a tolerizing self-Ag in vivo. Failure to induce autoimmunity by global perturbations in immune regulation introduced by the MRL autoimmune background and exposure to potent environmental challenge suggests that humoral immunity to nephritogenic basement membrane epitopes targeted in systemic autoimmunity is tightly regulated.

Proteinase 3 sidesteps caspases and cleaves p21(Waf1/Cip1/Sdi1) to induce endothelial cell apoptosis.

BACKGROUND: Emerging data raise possibilities of a complex and specific biologic role for leukocyte-derived proteases in substrate processing and in signaling pathways. Neutrophil proteinase 3 (PR3) is a caspase-like protease that enters endothelial cells, cleaves nuclear factor-kappaB (NF-kappaB), and induces sustained JNK activation, implying that the major cell cycle inhibitor p21 may be inactivated. Cleavage of p21 by caspase-3 is reported to be required for endothelial cell apoptosis. We hypothesized that PR3 may target p21. METHODS: Human umbilical vein endothelial cells (HUVEC) were treated with or without PR3 (5 microg/mL) from 0 hours or up to 8 hours, and analyzed for changes in cell cycle control proteins by immunoblotting, immunofluorescence and flow cytometry. RESULTS: PR3 exposure resulted in cleavage of p21 between Thr80 and Gly81, loss of nuclear p21 by cytoplasmic sequestration and depletion of p21 from cyclin/cyclin-dependent kinase (CDK) complexes. Examination of cyclins D and E, p53, Rb, and p27 revealed a largely nonproliferative expression profile. Cells arrested in G1 were more susceptible to PR3 effects. We examined inflamed human colonic tissue and found a fragment similar in size to that generated by PR3 in HUVEC. Granzyme B, a T-cell homologue of PR3 that cleaves caspase substrates, also cleaves p21 between Asp62 and Phe63. A reported substrate of granzyme B and caspases, Bid, is cleaved by PR3 signifying commonality of substrates among these proteases. CONCLUSION: A theme is developing that the granulocyte protease, PR3, is an exogenous caspase-like molecule that can sidestep intracellular caspase functions at sites of inflammation.

Kappa editing rescues autoreactive B cells destined for deletion in mice transgenic for a dual specific anti-laminin Ig.

We explored mechanisms involved in B cell self-tolerance in a double- and triple-transgenic mouse model bearing the LamH-C mu Ig H chain conventional transgene and a gene-targeted replacement for a functional V kappa 8J kappa 5 L chain gene. Whereas the H chain is known to generate anti-laminin Ig in combination with multiple L chains, the H + L Ig binds ssDNA in addition to laminin. Immune phenotyping indicates that H + L transgenic B cells are regulated by clonal deletion, receptor editing via secondary rearrangements at the nontargeted kappa allele, and anergy. Collectively, the data suggest that multiple receptor-tolerogen interactions regulate autoreactive cells in the H + L double-transgenic mice. Generation of H + LL triple-transgenic mice homozygous for the targeted L chain to exclude secondary kappa rearrangements resulted in profound B cell depletion with absence of mature B cells in the bone marrow. We propose that the primary tolerogen of dual reactive B cells in this model is not ssDNA, but a strongly cross-linking tolerogen, presumably basement membrane laminin, that triggers recombination-activating gene activity, L chain editing, and deletion.

Novel effects of neutrophil-derived proteinase 3 and elastase on the vascular endothelium involve in vivo cleavage of NF-kappaB and proapoptotic changes in JNK, ERK, and p38 MAPK signaling pathways.

Leukocyte-derived proteases have long been considered simply degradative. However, emerging data raise possibilities of a complex and specific biologic role for these proteases in substrate processing and in signaling pathways within cells. This study reports that the release of neutrophilic and monocytic proteases, such as proteinase 3 (PR3) and human neutrophil elastase (HNE), can result in their entry into endothelial cells coincident with the activation of proapoptotic-signaling events through ERK, JNK, and p38 MAPK. Inhibition of JNK blocked PR3-induced apoptosis, and inhibition of p38 MAPK blocked PR3- and HNE-induced apoptosis, indicating that these pathways are required for activation of apoptosis. It is here shown that protease entry results in direct cleavage of p65 NF-kappaB in the N-terminal region by PR3 and in the C-terminal region by HNE. This cleavage results in diminished transcriptional activity by NF-kappaB as demonstrated by diminished levels of TNF-alpha-induced IL-8 message in the presence of PR3 or HNE. Inhibition of caspases did not block the cleavage of p65 NF-kappaB, and sequence analysis showed that the PR3 and HNE cleavage sites are unique with respect to reported caspase sites. The data demonstrate that PR3 and HNE have specific, fundamental roles in endothelial responses during inflammation. Upon entry, they can usurp the cell's control of its own fate by directly intervening into caspase cascades. This provides a unique mechanism of crosstalk between leukocytes and endothelial cells at sites of inflammation that impacts both cytokine networks and cell viability.