Kiran Chandrashekar

NOX2 is the primary source of angiotensin II-induced superoxide in the macula densa

Macula densa (MD)-mediated regulation of renal hemodynamics via tubuloglomerular feedback is regulated by interactions between factors such as superoxide (O(2)(-)) and angiotensin II (ANG II). We have reported that NaCl-induced O(2)(-) in the MD is produced by the NOX2 isoform of NADPH oxidase (NOX); however, the source of ANG II-induced O(2)(-) in MD is unknown. Thus we determined the pathways by which ANG II increased O(2)(-) in the MD by measuring O(2)(-) in ANG II-treated MMDD1 cells, a MD-like cell line. ANG II caused MMDD1 O(2)(-) levels to increase by more than twofold (P < 0.01). This increase was blocked by losartan (AT(1) receptor blocker) but not PD-123319 (AT(2) receptor antagonist). Apocynin (a NOX inhibitor) decreased O(2)(-) by 86% (P < 0.01), whereas oxypurinol (a xanthine oxidase inhibitor) and NS-398 (a cyclooxygenase-2 inhibitor) had no significant effect. The NOX-dependent increase in O(2)(-) was due to the NOX2 isoform; a short interfering (si)RNA against NOX2 blunted ANG II-induced increases in O(2)(-), whereas the NOX4/siRNA did not. Finally, we found that inhibiting the Rac1 subunit of NOX blunted ANG II-induced O(2)(-) production in NOX4/siRNA-treated cells but did not further decrease it in NOX2/siRNA-treated cells. Our results indicate that ANG II stimulates O(2)(-) production in the MD primarily via AT(1)-dependent activation of NOX2. Rac1 is required for the full activation of NOX2. This pathway may be an important component of ANG II enhancement of tubuloglomerular feedback.

Protective role of testosterone in ischemia-reperfusion-induced acute kidney injury

Men are at greater risk for renal injury and dysfunction after acute ischemia-reperfusion (I/R) than are women. Studies in animals suggest that the reason for the sex difference in renal injury and dysfunction after I/R is the protective effect of estrogens in females. However, a reduction in testosterone in men is thought to play an important role in mediating cardiovascular and renal disease, in general. In the present study, we tested the hypothesis that I/R of the kidney reduces serum testosterone, and that contributes to renal dysfunction and injury. Male rats that were subjected to renal ischemia of 40 min followed by reperfusion had a 90% reduction in serum testosterone by 3 h after reperfusion that remained at 24 h. Acute infusion of testosterone 3 h after reperfusion attenuated the increase in plasma creatinine and urinary kidney injury molecule-1 (KIM-1) at 24 h, prevented the reduction in outer medullary blood flow, and attenuated the increase in intrarenal TNF-α and the decrease in intrarenal VEGF at 48 h. Castration of males caused greater increases in plasma creatinine and KIM-1 at 24 h than in intact males with renal I/R, and treatment with anastrozole, an aromatase inhibitor, plus testosterone almost normalized plasma creatinine and KIM-1 in rats with renal I/R. These data show that renal I/R is associated with sustained reductions in testosterone, that testosterone repletion protects the kidney, whereas castration promotes renal dysfunction and injury, and that the testosterone-mediated protection is not conferred by conversion to estradiol.

A novel U-STAT3-dependent mechanism mediates the deleterious effects of chronic nicotine exposure on renal injury.

Previous data from our group have demonstrated (Arany I, Grifoni S, Clark JS, Csongradi, Maric C, Juncos LA. Am J Physiol Renal Physiol 301: F125-F133, 2011) that chronic nicotine (NIC) exposure exacerbates acute renal ischemic injury (AKI) in mice that could increase the risk for development and progression of chronic kidney disease (CKD). It has been shown that proximal tubules of the kidney can acquire characteristics that may compromise structural recovery and favor development of inflammation and fibrosis following injury. Chronic NIC exposure can amplify this epithelial process although the mechanism is not identified. Recently, the unphosphorylated form of signal transducer and activator of transcription-3 (U-STAT3) has emerged as a noncanonical mediator of inflammation and fibrosis that may be responsible for the effects of chronic NIC. We found that levels of transforming growth factor β-1 (TGF-β1), α-smooth muscle actin (α-SMA), fibronectin, monocyte chemotactic protein-1 (MCP-1), and expression of U-STAT3 were increased in the ischemic kidneys of NIC-exposed mice. Chronic NIC exposure also increased TGF-β1-dependent F-actin reorganization, vimentin, fibronectin, and α-SMA expression as well as promoter activity of α-SMA and MCP-1 without significant loss of epithelial characteristics (E-cadherin) in cultured renal proximal tubule cells. Importantly, transduction of cells with a U-STAT3 mimetic (Y705F-STAT3) augmented stress fiber formation and also amplified NIC+TGF-β1-induced expression of α-SMA, vimentin, fibronectin, as well as promoter activity of α-SMA and MCP-1. Our results reveal a novel, chronic NIC-exposure-related and U-STAT3-dependent mechanism as mediator of a sustained transcription of genes that are linked to remodeling and inflammation in the kidney during injury. This process may facilitate progression of AKI to CKD. The obtained data may lead to devising therapeutic methods to specifically enhance the protective and/or inhibit adverse effects of STAT3 in the kidney.

Enhanced expression and activity of Nox2 and Nox4 in the macula densa in ANG II-induced hypertensive mice

NAD(P)H oxidase (Nox)2 and Nox4 are the isoforms of Nox expressed in the macula densa (MD). MD-derived superoxide (O₂⁻), primarily generated by Nox2, is enhanced by acute ANG II stimulation. However, the effects of chronic elevations in ANG II during ANG II-induced hypertension on MD-derived O₂⁻ are unknown. We infused a slow pressor dose of ANG II (600 ng·min⁻¹·kg⁻¹) for 2 wk in C57BL/6 mice and found that mean arterial pressure was elevated by 22.3 ± 3.4 mmHg (P < 0.01). We measured O₂⁻ generation in isolated and perfused MDs and found that O₂⁻ generation by the MD was increased from 9.4 ± 0.9 U/min in control mice to 34.7 ± 1.8 U/min in ANG II-induced hypertensive mice (P < 0.01). We stimulated MMDD1 cells, a MD-like cell line, with ANG II and found that O₂⁻ generation increased from 921 ± 91 to 3,687 ± 183 U·min⁻¹·10⁵ cells⁻¹, which was inhibited with apocynin, oxypurinol, or NS-398 by 46%, 14%, and 12%, respectively. We isolated MD cells using laser capture microdissection and measured mRNA levels of Nox. Nox2 and Nox4 levels increased by 3.7 ± 0.17- and 2.6 ± 0.15-fold in ANG II-infused mice compared with control mice. In MMDD1 cells treated with Nox2 or Nox4 small interfering (si)RNAs, ANG II-stimulated O₂⁻ generation was blunted by 50% and 41%, respectively. In cells treated with p22(phox) siRNA, ANG II-stimulated O₂⁻ generation was completely blocked. In conclusion, we found that a subpressor dose of ANG II enhances O₂⁻ generation in the MD and that the sources of this O₂⁻ are primarily Nox2 and Nox4.

Inhibition of Nitric Oxide Synthase 1 Induces Salt-Sensitive Hypertension in Nitric Oxide Synthase 1α Knockout and Wild-Type Mice

We recently showed that &agr;, &bgr;, and &ggr; splice variants of neuronal nitric oxide synthase (NOS1) expressed in the macula densa and NOS1&bgr; accounts for most of the NO generation. We have also demonstrated that the mice with deletion of NOS1 specifically from the macula densa developed salt-sensitive hypertension. However, the global NOS1 knockout (NOS1KO) strain is neither hypertensive nor salt sensitive. This global NOS1KO strain is actually an NOS1&agr;KO model. Consequently, we hypothesized that inhibition of NOS1&bgr; in NOS1&agr;KO mice induces salt-sensitive hypertension. NOS1&agr;KO and C57BL/6 wild-type (WT) mice were implanted with telemetry transmitters and divided into 7-nitroindazole (10 mg/kg/d)–treated and nontreated groups. All of the mice were fed a normal salt (0.4% NaCl) diet for 5 days, followed by a high-salt diet (4% NaCl). NO generation by the macula densa was inhibited by >90% in WT and NOS1&agr;KO mice treated with 7-nitroindazole. Glomerular filtration rate in conscious mice was increased by ≈40% after a high-salt diet in both NOS1&agr;KO and WT mice. In response to acute volume expansion, glomerular filtration rate, diuretic and natriuretic response were significantly blunted in the WT and knockout mice treated with 7-nitroindazole. Mean arterial pressure had no significant changes in mice fed a high-salt diet, but increased ≈15 mm Hg similarly in NOS1&agr;KO and WT mice treated with 7-nitroindazole. We conclude that NOS1&bgr;, but not NOS1&agr;, plays an important role in control of sodium excretion and hemodynamics in response to either an acute or a chronic salt loading.

Oxidative status in the macula densa modulates tubuloglomerular feedback responsiveness in Angiotensin II‐induced hypertension

Tubuloglomerular feedback (TGF) is an important mechanism in control of signal nephron glomerular filtration rate. The oxidative stress in the macula densa, primarily determined by the interactions between nitric oxide (NO) and superoxide ( O2− ), is essential in maintaining the TGF responsiveness. However, few studies examining the interactions between and amount of NO and O2− generated by the macula densa during normal and hypertensive states.

Epidemiology of Intracranial Aneurysms of Mississippi: a 10-year (1997-2007) Retrospective Study

BACKGROUND Despite massive efforts, progress so far has been modest in isolating the genetic determinants for intracranial aneurysm (IA). More detailed epidemiology data might be essential for successful genome-wide association study. Here, we aimed to investigate epidemiology and identify the key risk factors associated with the pathogenesis of IA in a large specific population. METHODS We investigated the epidemiology and analyzed the risk factors of IA pathogenesis by using an International Classification of Diseases, Ninth Revision database search of the patients treated at the University of Mississippi Medical Center, Jackson, MS, within the past 10-year period (1998-2007). All recruited patients were interviewed to assess multiple risk factors and comorbidities (hypertension, tobacco abuse, females sex, diabetes mellitus, coronary artery disease, coronary obstructive pulmonary disease, alcohol abuse, stroke, hyperlipidemia, illicit drug use, and family history). RESULT In this retrospective study, we identified several significant risk factors among well-defined human subjects. The 3 major risk factors identified for our IA population are hypertension, tobacco abuse, and female sex. However, African American race was not a significant risk factor in our study. Furthermore, top two risk factors (hypertension, tobacco abuse) were found to be highly associated with familial cases. CONCLUSIONS In this study, using a specific and well-defined large population, we reported that some key risk factors were further confirmed to be strongly associated with the pathogenesis of IA whereas further investigation into racial factors is apparently needed. Our finding of the confounding effects of top risks with familial cases further complicated the genetic analysis of IA.

The Modulatory Role of Heme Oxygenase on Subpressor Angiotensin II-Induced Hypertension and Renal Injury

Angiotensin II (AngII) causes hypertension (HTN) and promotes renal injury while simultaneously inducing reno-protective enzymes like heme oxygenase-1 (HO-1). We examined the modulatory role of HO on sub-pressor angiotensin II (SP-AngII) induced renal inflammation and injury. We first tested whether the SP-AngII-induced renal dysfunction, inflammation and injury are exacerbated by either preventing (chronic HO-1 inhibition) or reversing (late HO-1 inhibition) SP-AngII-induced HO (using tin protoporphyrin; SnPP). We next examined whether additional chronic or late induction of SP-AngII-induced HO (using cobalt protoporphyrin; CoPP), prevents or ameliorates renal damage. We found that neither chronic nor late SnPP altered blood pressure. Chronic SnPP worsened SP-AngII-induced renal dysfunction, inflammation, injury and fibrosis, whereas late SnPP worsened renal dysfunction but not inflammation. Chronic CoPP prevented HTN, renal dysfunction, inflammation and fibrosis, but surprisingly, not the NGAL levels (renal injury marker). Late CoPP did not significantly alter SP-AngII-induced HTN, renal inflammation or injury, but improved renal function. Thus, we conclude (a) endogenous HO may be an essential determining factor in SP-AngII induced renal inflammation, injury and fibrosis, (b) part of HOs renoprotection may be independent of blood pressure changes; and (c) further induction of HO-1 protects against renal injury, suggesting a possible therapeutic target.

Endothelin Antagonists in Diabetic Nephropathy: Back to Basics

The pivotal discovery of endothelin (ET) by Yanagisawa and colleagues[1][1] in 1988 generated wide interest in this peptide, as evidenced by the nearly 27,000 articles published to date that have examined its role in biology. ET is now recognized as essential to the function of various organs and

Changing Paradigms in Contrast Nephropathy.

The advent of radiologic imaging revolutionized our diagnostic capabilities by allowing us to noninvasively view internal body structures. Its resolution was further augmented by the development of radiographic contrast media. Consequently, contrast-enhanced radiologic imaging has become