Brent Wagner

Nox4 NAD(P)H oxidase mediates hypertrophy and fibronectin expression in the diabetic kidney

Renal hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy. We investigated the role of the NAD(P)H oxidase Nox4 in generation of reactive oxygen species (ROS), hypertrophy, and fibronectin expression in a rat model of type 1 diabetes induced by streptozotocin. Phosphorothioated antisense (AS) or sense oligonucleotides for Nox4 were administered for 2 weeks with an osmotic minipump 72 h after streptozotocin treatment. Nox4 protein expression was increased in diabetic kidney cortex compared with non-diabetic controls and was down-regulated in AS-treated animals. AS oligonucleotides inhibited NADPH-dependent ROS generation in renal cortical and glomerular homogenates. ROS generation by intact isolated glomeruli from diabetic animals was increased compared with glomeruli isolated from AS-treated animals. AS treatment reduced whole kidney and glomerular hypertrophy. Moreover, the increased expression of fibronectin protein was markedly reduced in renal cortex including glomeruli of AS-treated diabetic rats. Akt/protein kinase B and ERK1/2, two protein kinases critical for cell growth and hypertrophy, were activated in diabetes, and AS treatment almost abolished their activation. In cultured mesangial cells, high glucose increased NADPH oxidase activity and fibronectin expression, effects that were prevented in cells transfected with AS oligonucleotides. These data establish a role for Nox4 as the major source of ROS in the kidneys during early stages of diabetes and establish that Nox4-derived ROS mediate renal hypertrophy and increased fibronectin expression.

Angiotensin II-induced ERK1/ERK2 activation and protein synthesis are redox-dependent in glomerular mesangial cells

Angiotensin II (Ang II) stimulates hypertrophy of glomerular mesangial cells. The signalling mechanism by which Ang II exerts this effect is not precisely known. Downstream potential targets of Ang II are the extracellular-signal-regulated kinases 1 and 2 (ERK1/ERK2). We demonstrate that Ang II activates ERK1/ERK2 via the AT1 receptor. Arachidonic acid (AA) mimics the action of Ang II on ERK1/ERK2 and phospholipase A2 inhibitors blocked Ang II-induced ERK1/ERK2 activation. The antioxidant N-acetylcysteine as well as the NAD(P)H oxidase inhibitors diphenylene iodonium and phenylarsine oxide abolished both Ang II- and AA-induced ERK1/ERK2 activation. Moreover, dominant-negative Rac1 (N17Rac1) blocks activation of ERK1/ERK2 in response to Ang II and AA, whereas constitutively active Rac1 resulted in an increase in ERK1/ERK2 activity. Antisense oligonucleotides for Nox4 NAD(P)H oxidase significantly reduce activation of ERK1/ERK2 by Ang II and AA. We also show that protein synthesis in response to Ang II and AA is inhibited by N17Rac1 or MEK (mitogen-activated protein kinase/ERK kinase) inhibitor. These results demonstrate that Ang II stimulates ERK1/ERK2 by AA and Nox4-derived reactive oxygen species, suggesting that these molecules act as downstream signal transducers of Ang II in the signalling pathway linking the Ang II receptor AT1 to ERK1/ERK2 activation. This pathway involving AA, Rac1, Nox4, reactive oxygen species and ERK1/ERK2 may play an important role in Ang II-induced mesangial cell hypertrophy.

Direct and efficient cellular transformation of primary rat mesenchymal precursor cells by KSHV

Infections by viruses are associated with approximately 12% of human cancer. Kaposis sarcoma-associated herpesvirus (KSHV) is causally linked to several malignancies commonly found in AIDS patients. The mechanism of KSHV-induced oncogenesis remains elusive, due in part to the lack of an adequate experimental system for cellular transformation of primary cells. Here, we report efficient infection and cellular transformation of primary rat embryonic metanephric mesenchymal precursor cells (MM cells) by KSHV. Cellular transformation occurred at as early as day 4 after infection and in nearly all infected cells. Transformed cells expressed hallmark vascular endothelial, lymphatic endothelial, and mesenchymal markers and efficiently induced tumors in nude mice. KSHV established latent infection in MM cells, and lytic induction resulted in low levels of detectable infectious virions despite robust expression of lytic genes. Most KSHV-induced tumor cells were in a latent state, although a few showed heterogeneous expression of lytic genes. This efficient system for KSHV cellular transformation of primary cells might facilitate the study of growth deregulation mechanisms resulting from KSHV infections.

Colony-Stimulating Factor-1 (CSF-1) Directly Inhibits Receptor Activator of Nuclear Factor-κB Ligand (RANKL) Expression by Osteoblasts

Colony-stimulating factor-1 (CSF-1), released by osteoblasts, stimulates the proliferation of osteoclast progenitors via the c-fms receptor (CSF-1R) and, in combination with receptor activator of nuclear factor-kappaB ligand (RANKL), leads to the formation of mature osteoclasts. Whether the CSF-1R is expressed by osteoblasts and mediates specific biological effects in osteoblasts has not been explored. Wild-type primary calvaria osteoblasts (OB) were analyzed for CSF-1R expression (RT-PCR and Western blot) and functionality (immunocomplex kinase assay). OB were serum starved for 24 h, and the effect of CSF-1 (0-100 ng/ml) on OB biological activities was determined at 48 h. In wild-type mouse bone marrow cultures, CSF-1 was tested for its effect on RANKL mRNA and osteoclast formation. Because ROS influence osteoblast RANKL expression, studies analyzed the effect of CSF-1 on reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and Nox1 and Nox4 proteins. Results indicate that OB express CSF-1R mRNA and protein and that CSF-1R could be phosphorylated in the presence of CSF-1. In osteoblasts, CSF-1 decreased RANKL mRNA in a dose- and time-dependent manner. Incubation of bone marrow cultures with CSF-1 resulted in a significant decline in tartrate-resistant acid phosphatase (TRACP) activity and CTR expression. RANKL-decreased expression by CSF-1 was correlated with a decrease of NADPH oxidase activity as well as Nox1 and Nox4 protein levels. These findings provide the first evidence that osteoblasts express CSF-1R and are a target for CSF-1 ligand. CSF-1-mediated inhibition of RANKL expression on osteoblasts may provide an important mechanism for coupling bone formation/resorption and preventing excessive osteoclastogenesis during normal skeletal growth.

Nephrogenic systemic fibrosis: Evidence for oxidative stress and bone marrow-derived fibrocytes in skin, liver, and heart lesions using a 5/6 nephrectomy rodent model

Nephrogenic systemic fibrosis (NSF) is associated with gadolinium-based magnetic resonance imaging (MRI) contrast exposure in the setting of acute or chronic renal compromise. It has been proposed that circulating fibrocytes mediate the disease. A study was conducted to determine whether bone marrow-derived fibroblast precursors are involved in contributing to organ fibrosis in MRI contrast-treated rodents with renal insufficiency. Rats status post 5/6 nephrectomy underwent bone marrow transplant from human placental alkaline phosphatase (hPAP)-expressing donors. After engraftment, animals were treated with gadolinium-based MRI contrast (2.5 mmol/kg IP), during weekdays for 4 weeks, or an equivalent volume of normal saline. Dermal cellularity in the contrast-treated group was fourfold that of control. Skin cells from the contrast-treated group demonstrated greater hPAP expression with co-expression of pro-collagen I and α-smooth muscle actin-positive stress fibers. Donor and host cells expressed CD34. Dihydroethidium staining of skin was greater in the contrast-treated animals, indicating oxidative stress. This was abrogated when the animals were co-administered the superoxide dismutase mimetic tempol. In conclusion, a bone marrow-derived cell population is increased in the dermis of MRI contrast-treated rodents. The cell markers are consistent with fibrocytes mediating the disease. These changes correlate with oxidative stress and expression of Nox4, suggestive of a novel therapeutic target. Elucidation of the mechanisms of MRI contrast-induced fibrosis may aid in discovering therapies to this devastating disease.

Type of MRI contrast, tissue gadolinium, and fibrosis

It has been presupposed that the thermodynamic stability constant (K(therm)) of gadolinium-based MRI chelates relate to the risk of precipitating nephrogenic systemic fibrosis. The present study compared low-K(therm) gadodiamide with high-K(therm) gadoteridol in cultured fibroblasts and rats with uninephrectomies. Gadolinium content was assessed using scanning electron microscopy equipped with energy-dispersive X-ray spectroscopy in paraffin-embedded tissues. In vitro, fibroblasts demonstrated dose-dependent fibronectin generation, transforming growth factor-β production, and expression of activated myofibroblast stress fiber protein α-smooth muscle actin. There were negligible differences with respect to toxicity or proliferation between the two contrast agents. In the rodent model, gadodiamide treatment led to greater skin fibrosis and dermal cellularity than gadoteridol. In the kidney, both contrast agents led to proximal tubule vacuolization and increased fibronectin accumulation. Despite large detectable gadolinium signals in the spleen, skin, muscle, and liver from the gadodiamide-treated group, contrast-induced fibrosis appeared to be limited to the skin and kidney. These findings support the hypothesis that low-K(therm) chelates have a greater propensity to elicit nephrogenic systemic fibrosis and demonstrate that certain tissues are resistant to these effects.

PDGF receptor-β modulates metanephric mesenchyme chemotaxis induced by PDGF AA

PDGF B chain or PDGF receptor (PDGFR)-beta-deficient (-/-) mice lack mesangial cells. To study responses of alpha- and beta-receptor activation to PDGF ligands, metanephric mesenchymal cells (MMCs) were established from embryonic day E11.5 wild-type (+/+) and -/- mouse embryos. PDGF BB stimulated cell migration in +/+ cells, whereas PDGF AA did not. Conversely, PDGF AA was chemotactic for -/- MMCs. The mechanism by which PDGFR-beta inhibited AA-induced migration was investigated. PDGF BB, but not PDGF AA, increased intracellular Ca(2+) and the production of reactive oxygen species (ROS) in +/+ cells. Transfection of -/- MMCs with the wild-type beta-receptor restored cell migration and ROS generation in response to PDGF BB and inhibited AA-induced migration. Inhibition of Ca(2+) signaling facilitated PDGF AA-induced chemotaxis in the wild-type cells. The antioxidant N-acetyl-l-cysteine (NAC) or the NADPH oxidase inhibitor diphenyleneiodonium (DPI) abolished the BB-induced increase in intracellular Ca(2+) concentration, suggesting that ROS act as upstream mediators of Ca(2+) in suppressing PDGF AA-induced migration. These data indicate that ROS and Ca(2+) generated by active PDGFR-beta play an essential role in suppressing PDGF AA-induced migration in +/+ MMCs. During kidney development, PDGFR beta-mediated ROS generation and Ca(2+) influx suppress PDGF AA-induced chemotaxis in metanephric mesenchyme.

Pathophysiology of gadolinium-associated systemic fibrosis

Systemic fibrosis from gadolinium-based magnetic resonance imaging contrast is a scourge for the afflicted. Although gadolinium-associated systemic fibrosis is a rare condition, the threat of litigation has vastly altered clinical practice. Most theories concerning the etiology of the fibrosis are grounded in case reports rather than experiment. This has led to the widely accepted conjecture that the relative affinity of certain contrast agents for the gadolinium ion inversely correlates with the risk of succumbing to the disease. How gadolinium-containing contrast agents trigger widespread and site-specific systemic fibrosis and how chronicity is maintained are largely unknown. This review highlights experimentally-derived information from our laboratory and others that pertain to our understanding of the pathophysiology of gadolinium-associated systemic fibrosis.

Centrality of bone marrow in the severity of gadolinium-based contrast-induced systemic fibrosis

Systemic fibrosis can be induced in humans with gadolinium‐based contrast, and cumulative doses correlate with severity. Bone marrow–derived fibrocytes accumulate in the dermis. Whether target organs liberate chemokines to recruit these fibrocytes or whether fibrocytes are stimulated to home to the affected tissue is unknown. Transgenic (tagged) donor rats were treated with gadolinium‐based contrast. Bone marrow was obtained from diseased animals and age‐matched controls. Rats with subtotal nephrectomies were lethally irradiated and underwent salvage transplantation with either the contrast‐naïve or contrast‐exposed bone marrow. Groups were randomly assigned to control or contrast treatment. Contrast treatment led to dermal fibrosis, and this was exacerbated in recipients of contrast‐exposed marrow. Fibronectin, C‐C chemokine receptors (CCRs)2 and 7, and oxidative stress were all increased in skin from contrast‐treated animals—all parameters more severe in recipients of contrast‐treated animals. The respective ligands, monocyte chemoattractant protein and C‐C motif ligand 19, were both elevated in skin from contrast‐treated animals. Coadministration of gadolinium‐based contrast and a CCR2 inhibitor reduced the severity of skin disease as well as dermal cellularity. The functional role of chemokines in the effects of gadolinium‐based contrast was further confirmed in in situ coculture studies using neutralizing CCR2 antibodies. These data implicate dermal liberation of specific chemokines in the recruitment of circulating bone marrow–derived cells. The disease is augmented by bone marrow exposure to contrast, which explains why multiple exposures correlate with severity.—Drel, V. R., Tan, C., Barnes, J. L., Gorin, Y., Lee, D.‐Y., Wagner, B. Centrality of bone marrow in the severity of gadolinium‐based contrast‐induced systemic fibrosis. FASEB J. 30, 3026–3038 (2016). www.fasebj.org

Concomitant Presentation of Adermatopathic Dermatomyositis, Statin Myopathy, Fibromyalgia Syndrome, Piriformis Muscle Myofascial Pain Syndrome, and Diabetic Neuropathy

Background: Differentiation of dermatomyositis from other myopathic conditions depends on characteristic histolo-pathologic findings on muscle biopsy. A 48-year-old diabetic man had a prior history of generalized myalgia and an elevated serum creatine kinase while on a statin. Within a few months after discontinuation of the statin, the myalgia symptoms improved and the creatine kinase normalized. Findings: An incisional biopsy of the gastrocnemius muscle revealed perifascicular muscle fiber damage, microvascular inflammation, pyknotic cytoplasmic inclusion bodies, and neurogenic atrophy with reinnervation consistent with diabetic neuropathy. Conclusion:. We present the clinical findings from a patient with adermatopathic dermatomyositis in the setting of diabetic neuropathy, fibromyalgia syndrome, piriformis syndrome, and 3-hydroxy-3-methyl-glutaryl-coenzyme A [HMG-CoA] reductase inhibitor myopathy.