Stephen Adler

The Calcimimetic AMG 073 as a Potential Treatment for Secondary Hyperparathyroidism of End-Stage Renal Disease

Current treatment of secondary hyperparathyroidism in chronic kidney failure with calcium and active vitamin D is potentially limited by hypercalcemia and hyperphosphatemia. AMG 073 represents a new class of compounds for the treatment of hyperparathyroidism known as calcimimetics, which reduce parathyroid hormone (PTH) synthesis and secretion by increasing the sensitivity of the parathyroid calcium-sensing receptor (CaR) to extracellular calcium. The current study evaluates the efficacy and safety of AMG 073 when added to conventional treatment of secondary hyperparathyroidism in end-stage renal disease (ESRD). Seventy-one hemodialysis patients with uncontrolled secondary hyperparathyroidism, despite standard therapy with calcium, phosphate binders, and active vitamin D sterols, were treated in this 18-wk, dose-titration study with single daily oral doses of AMG 073/placebo up to 100 mg. Changes in plasma PTH, serum calcium, serum phosphorus, and calcium x phosphorus levels were compared between AMG 073 and placebo groups. Mean PTH decreased by 33% in the AMG 073 patients compared with an increase of 3% in placebo patients (P = 0.001). A significantly greater proportion of AMG 073 patients (44%) had a mean PTH < or = 250 pg/ml compared with placebo patients (20%; P = 0.029). Also, a significantly greater proportion of AMG 073 patients (53%) had a decrease in PTH > or =30% compared with placebo patients (23%; P = 0.009). Calcium x phosphorus levels decreased by 7.9% in AMG 073 patients compared with an increase of 11.3% in placebo patients (P = 0.013). Adverse event rates were low and mostly mild to moderate in severity; however, the incidence of vomiting was higher in AMG 073 patients. In this study, the calcimimetic AMG 073 at doses up to 100 mg for 18 wk provided a safe and effective means to attain significant reductions in PTH and calcium x phosphorus levels in ESRD patients. AMG 073 represents a novel and promising therapy to improve the management of secondary hyperparathyroidism.

Cobalt promotes angiogenesis via hypoxia-inducible factor and protects tubulointerstitium in the remnant kidney model.

Tubulointerstitial hypoxia has been implicated in a number of progressive renal diseases, and several lines of evidence indicate that the administration of angiogenic growth factors ameliorates tubulointerstitial injury. We hypothesized that induction of hypoxia-inducible factors (HIF) mediates renoprotection by their angiogenic properties. At 5–9 weeks after subtotal nephrectomy, cobalt was administered to rats to activate HIF. Histological evaluation demonstrated that the tubulointerstitial injury was significantly ameliorated in animals that received cobalt (score: 2.51±0.12 (cobalt) vs 3.21±0.24 (vehicle), P<0.05). Furthermore, animals receiving cobalt had fewer vimentin- and TdT-mediated dUTP nick-end labeling (TUNEL)-positive tubular cells. The renoprotective effect of cobalt was associated with the preservation of peritubular capillary networks (rarefaction index: 13.7±0.4 (cobalt) vs 18.6±0.9 (vehicle), P<0.01). This improvement in capillary networks was accompanied by an increased number of proliferating (PCNA-positive) glomerular and peritubular endothelial cells. The angiogenesis produced by this method was not accompanied by an increase in vascular permeability. Furthermore, in vitro experiments clarified that HIF-1 in tubular epithelial cells promotes proliferation of endothelial cells and that HIF-2 overexpressed in renal endothelial cells mediates migration and network formation. Collectively, these findings demonstrate a renoprotective role of HIF through angiogenesis and provide a rationale for therapeutic approaches to target HIF for activation.

HMG CoA reductase inhibition modulates VEGF-induced endothelial cell hyperpermeability by preventing RhoA activation and myosin regulatory light chain phosphorylation

The beneficial effects of statins are usually assumed to stem from their ability to reduce cholesterol biosynthesis. However, because statins are potent inhibitors of the mevalonate, which governs diverse cell signaling pathways, inhibition of 3‐hydroxy‐3‐methylglutaryl‐coenzyme‐A reductase may also result in pleiotropic effects. The present study describes a novel pleiotropic effect of statins on vascular endothelial growth factor (VEGF)‐induced glomerular endothelial cell (GEnC) hyperpermeability. Using live cell imaging with green fluorescent protein‐tagged myosin regulatory light chain (MLC) and correlative biochemical analyses, we investigated 1) VEGF signaling pathway leading to GEnC hyperpermeability and 2) the modulatory effects of statins on VEGF signaling. Our findings indicate that VEGF stimulation elicits a robust increase in GEnC permeability. The signaling pathway that mediates VEGF‐induced GEnC hyperpermeability involves RhoA activation leading to actin cytoskeletal remodeling, MLC diphosphorylation, and enhanced paracellular gap formation. Remarkably, cotreatment of endothelial cells with simvastatin, a hydrophobic statin, reversed VEGF‐induced GEnC hyperpermeability by preventing MLC diphosphorylation, and cytoskeletal remodeling. In summary, this study identifies RhoA and MLC phosphorylation as key mediators of VEGF‐induced endothelial cell hyperpermeability and demonstrates the modulatory effects of statins on VEGF signaling pathway.

Cell adhesion molecules and the glomerulopathies.

The kidney possesses a unique architecture that allows it to carry out its function of purifying the blood through filtration and tubular reabsorption and secretion. This structure is established and maintained through the interactions of renal cells with the extracellular matrix (ECM) consisting of the glomerular, Bowman’s capsular, tubular and other vascular basement membranes, and the mesangial and tubulointerstitial matrices. These ECMs and basement membranes form a supporting scaffolding for renal cells that help guide the formation, function, and repair of renal structures. Cells, however, do not passively occupy a site on the ECM but interact with it through specific cell adhesion molecules that mediate attachment to the ECM. Work over the past decade has demonstrated that these receptors function as a two-way conduit between the cell and the ECM. Thus, the ECM influences cellular morphology, proliferative, synthetic, and metabolic states, and responsiveness to several extracellular factors while events in the cell can affect how matrix receptors bind to the ECM. Other cell adhesion molecules mediate interactions between cells, playing a role in renal morphogenesis and maintenance of tubular epithelial polarity, as well as providing a foothold for circulating leukocytes and platelets to gain access to areas of injury and inflammation. These receptors help determine the extent and type of inflammation in the kidney in response to diverse injuries, as well as modulating some of the inflammatory mediators that are produced. In this review we will initially discuss receptors that mediate cell-matrix interactions and their roles in the normal kidney and in the response to injury. The second part will concentrate on cell-cell interactions that play an important role in leukocyte and platelet recruitment in inflammation and thrombosis. OVERVIEW OF MATRIX RECEPTORS

Impaired Regulation of Renal Oxygen Consumption in Spontaneously Hypertensive Rats

Abnormalities of nitric oxide (NO) and oxygen radical synthesis and of oxygen consumption have been described in the spontaneously hypertensive rat (SHR) and may contribute to the pathogenesis of hypertension. NO plays a role in the regulation of renal oxygen consumption in normal kidney, so the response of renal cortical oxygen consumption to stimulators of NO production before and after the addition of the superoxide scavenging agent tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) was studied. Baseline cortical oxygen consumption was similar in SHR and Wistar-Kyoto (WKY) rats (SHR: 600 +/- 55 nmol O(2)/min per g, WKY: 611 +/- 51 nmol O(2)/min per g, P > 0.05). Addition of bradykinin, enalaprilat, and amlodipine decreased oxygen consumption significantly less in SHR than WKY (SHR: bradykinin -13.9 +/- 1.9%, enalaprilat -15.3 +/- 1.6%, amlodipine -11.9 +/- 0.7%; WKY: bradykinin -22.8 +/- 1.0%, enalaprilat -24.1 +/- 2.0%, amlodipine -20.7 +/- 2.3%; P < 0.05), consistent with less NO effect in SHR. Addition of tempol reversed the defects in responsiveness to enalaprilat and amlodipine, suggesting that inactivation of NO by superoxide contributes to decreased NO availability. The response to an NO donor was similar in both groups and was unaffected by the addition of tempol. These results demonstrate that NO availability in the kidney is decreased in SHR, resulting in increased oxygen consumption. This effect is due to enhanced production of superoxide in SHR. By lowering intrarenal oxygen levels, reduced NO may contribute to susceptibility to injury and renal fibrosis. Increasing NO production, decreasing oxidant stress, or both might prevent these changes by improving renal oxygenation.

Oxidant Stress Leads to Impaired Regulation of Renal Cortical Oxygen Consumption by Nitric Oxide in the Aging Kidney

Structural and functional changes occur in the kidney with aging. Previous studies have suggested that loss of nitric oxide production contributes to these changes. The authors therefore explored regulation of renal cortical oxygen consumption, a nitric oxide mediated effect, in tissue from Fischer 344 rats at different ages (4, 13, and 23 mo) to characterize changes in renal nitric oxide production with age. Bradykinin, enalaprilat, and amlodipine significantly suppressed cortical oxygen consumption in 4-mo-old rats (bradykinin: -2.5 +/- 0.9% to -21 +/- 1.5%; enalaprilat: -0.7 +/- 0.5% to -26 +/- 1.2%; amlodipine: -1.3 +/- 0.9% to -18 +/- 1.2%; P < 0.05). Similar results were obtained in 13-mo-old animals. However, in 23-mo-old animals, the responses to bradykinin and enalaprilat were attenuated (bradykinin: 0 +/- 0% to -13 +/- 0.9%; enalaprilat: -0.3 +/- 0.3% to -17 +/- 2.1%; P < 0.05), whereas the response to an NO donor was unaffected, suggesting decreased bioavailability of NO. Addition of the superoxide radical scavenger tempol restored the ability of bradykinin, enalaprilat, and amlodipine to suppress oxygen consumption in tissue from 23-mo-old animals to levels seen in younger animals, suggesting NO destruction by superoxide as the reason for decreased NO availability. Apocynin, an inhibitor of NAD(P)H oxidase, similarly restored the ability of all three drugs to suppress oxygen consumption, suggesting NAD(P)H oxidase as the enzyme responsible for enhanced superoxide production in aging. Levels of eNOS protein, assessed by immunoblotting, did not change significantly with age. These results suggest that NO availability is decreased in the aging kidney and that this is due to scavenging of NO by superoxide produced by NAD(P)H oxidase. Oxidant stress, by depleting NO, may contribute to the structural and hemodynamic changes characteristic of the aging kidney.

Hepatitis C in the health care setting. II. Seroprevalence among hemodialysis staff and patients in suburban New York City

BACKGROUND Hepatitis C virus (HCV) is a newly identified blood-borne virus that may pose an occupational hazard for health care workers. Hemodialysis nurses could be anticipated to be at high risk for HCV infection because this group of health care workers frequently comes into contact with blood of a patient population with a seroprevalence rate of at least 10%. METHODS To assess the risk of HCV infection for hemodialysis nurses, serum samples from all of the nurses (22/22, 100%) and patients (125/125, 100%) in one hemodialysis unit (unit A) and 85% (29/34) of nurses from a second unit (unit B), both units in suburban New York City, were tested for HCV antibodies. Samples with positive results of enzyme-linked immunosorbent assay underwent supplemental testing by a first-generation recombinant immunoblot assay. RESULTS Twenty-four (19%) of the hemodialysis patients in unit A were HCV seropositive. Despite an average of 4.7 years spent working in hemodialysis unit A, none of the nurses tested seropositive for HCV antibody. In unit B, despite an average of 6.4 years working in the unit studied, only one nurse tested seropositive for HCV antibody. This nurse reported a long history of elevated liver function values and a negative HBV core antibody status that predated her hemodialysis nursing career. CONCLUSIONS In contrast to the experience with hepatitis B virus infection, hemodialysis nurses appear to be at low risk for occupationally acquired HCV infection.

Glomerular Endothelial Cell Injury Mediated by Shiga-Like Toxin-1

The hemolytic uremic syndrome is an important cause of acute renal failure and is often associated with prior infection with enterotoxigenic strains of Escherichia coli. Significant pathologic changes in the glomerulus include endothelial cell swelling, detachment, and intravascular coagulation. We investigated the potential pathogenetic mechanisms of this disease by exposing cultured rat glomerular endothelial cells to shiga-like toxin 1 (SLT-1). Glomerular endothelial cell viability and protein synthesis were not affected by 10–9 M SLT-1. Synthesis and release of thromboxane (Tx) A2, measured as the stable metabolite TxB2, and of 12-(S)-HETE were each increased 1.6 ± 0.1 fold (p < 0.05). No change was observed in the production of PGE2 or 6-keto-PGF1α, the active metabolite of PGI2. SLT-1 (10–9 M) significantly increased cell retraction and the formation of gaps in the glomerular endothelial cell monolayer for up to 6 h following exposure. A similar effect was seen with 0.1 μM 12-(S)-HETE. SLT-1 and 0.01 μM 12-(S)-HETE also significantly decreased adhesion of glomerular endothelial cells to fibronectin (75 ± 4 and 65 ± 2% of control, respectively; p < 0.001) and laminin (81 ± 5 and 67 ± 4% of control, respectively; p < 0.01). These results support a role for SLT-1 in the production of the pathologic changes seen in the hemolytic uremic syndrome through stimulation of production of procoagulant, vasoconstrictor arachidonic acid metabolites with potential to produce endothelial injury and through alterations in glomerular endothelial cell adhesion to components of the glomerular basement membrane.

Modulation of renal oxygen consumption by nitric oxide is impaired after development of congestive heart failure in dogs.

We investigated the role of nitric oxide (NO) in the modulation of renal O2 consumption in dogs with pacing-induced congestive heart failure (CHF). O2 consumption in the renal cortex (C) and medulla (M) of normal dogs and dogs with CHF was measured under control conditions and in the presence of increasing concentrations of three stimulators of NO production, bradykinin, ramiprilat, and amlodipine, or the NO donor S-nitroso-N-acetyl-penicillamine (SNAP). Baseline O2 consumption (nmol O2/min per gram) was similar in the CHF group (C: 637 ± 65; M: 618 ± 83) and the control group (C: 601 ± 58, M: 534 ± 55). In normal dogs, bradykinin (10−4 M), ramiprilat (10−4 M), amlodipine (10−5 M) and SNAP (10−4 M) all significantly reduced O2 consumption in the cortex (−31.5 ± 3.5%, −33 ± 2.5%, −28.4 ± 4.9%, −49.3 ± 3.1%) and medulla (−26.9 ± 2.2%, −31.4 ± 2.2%, −23.1 ± 1.3%, −48.3 ± 4%), respectively. The responses to bradykinin, ramiprilat and amlodipine were significantly attenuated in dogs with CHF (C: −22.2 ± 1.8%, −20.1 ± 2.6%, −14.2 ± 2.5%; M: −20.8 ± 1.7%, −17.8 ± 1.9%, −15.6 ± 2.6%, respectively; p < 0.05). The responses in dogs with CHF were not altered by NO synthase blockade with L-NAME (10−4 M). In contrast, in normal kidneys treatment with L-NAME significantly attenuated the response to all three stimuli of NO production. Responses to SNAP were not affected either by CHF or L-NAME. These data indicate that the role of NO production in the modulation of tissue O2 consumption in the kidney is impaired after the development of pacing-induced heart failure in dogs.

Enhancement of permeability in endothelial cells for the development of an antithrombogenic bioartificial hemofilter

For the development of an antithrombogenic bioartificial hemofilter, in which the inner surface of hollow fibers is lined by endothelial cells, it is essential to increase the permeability of the cells in order to achieve a sufficient ultrafiltrate. We tried to increase it by using an actin microfilament polymerization inhibitor, cytochalasin B (CyB). Fifty µg/mL CyB was added for 2 h to the culture medium of confluent rat glomerular endothelial cells (RGEC) and human umbilical vein endothelial cells (HUVEC). Under the 130 mmHg hydrostatic pressure, the CyB‐treated group produced significantly more ultrafiltration than the non‐treated control group and this increase was maintained for at least 7 days. Horseradish peroxidase (HRP) permeability acutely and reversibly increased in the CyB‐treated group compared with the non‐treated control group. Scanning electron microscopy revealed a larger average diameter and increased number of fenestrae on the CyB‐treated endothelial cells, compared with the non‐treated cells. This phenomenon also lasted for at least 7 days. The platelet adherence test showed that CyB did not deteriorate the antithrombogenic property of endothelial cells. These results indicate that CyB is potentially applicable for the enhancement of endothelial cell permeability in an antithrombogenic bioartificial hemofilter. Biotechnol. Bioeng. 2008;101: 634–641.