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 downregulated 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.

No abstract

No abstract

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.

It has been presupposed that the thermodynamic stability constant ( Ktherm) of gadolinium-based MRI chelates relate to the risk of precipitating nephrogenic systemic fibrosis. The present study compared low- Ktherm gadodiamide with high- Ktherm 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- Ktherm chelates have a greater propensity to elicit nephrogenic systemic fibrosis and demonstrate that certain tissues are resistant to these effects.