Calcineurin (PP2B) has recently been shown to be upregulated in the medullary thick ascending limb (mTAL) during diabetes. The mTAL expresses all three isoforms of nitric oxide synthase (NOS), which are subject to phosphoregulation and represent substrates for PP2B. Therefore, we hypothesized that diabetes induces PP2B-dependent upregulation of NOS activity and NO production in the mTAL. Three weeks after injection of streptozotocin (STZ rats) or vehicle (sham rats), mTAL suspensions were prepared for use in functional and biochemical assays. PP2B activity and expression were increased in mTALs from STZ rats compared with sham. Nitrite production was significantly reduced in mTALs from STZ rats compared with sham. However, incubation with the free radical scavenger, tempol, unmasked a significant increase in nitrite production by mTALs from STZ rats. Inhibition of PP2B attenuated the increase in nitrite production and NOS activity evident in mTALs from STZ rats. Analysis of specific NOS isoform activity revealed increased NOS1 and NOS2 activities in mTALs from STZ rats. All three NOS isoform activities were regulated in a PP2B-dependent manner. Western blot analysis detected no differences in NOS isoform expression, although phosphorylation of pThr(495)-NOS3 was significantly reduced in mTALs from STZ rats. Phosphorylation of pSer(852)-NOS1, pSer(633)-NOS3, and pSer(1177)-NOS3 was similar in mTALs from STZ and sham rats. Inhibition of PP2B did not alter the phosphorylation of NOS1 or NOS3 at known sites. In conclusion, while NO bioavailability in mTALs is reduced during diabetes, free radical scavenging with tempol unmasks increased NO production that involves PP2B-dependent activation of NOS1 and NOS2.
Aim During type 1 diabetes (T1D), the medullary thick ascending limb (mTAL) displays an NADPH oxidase-dependent increase in sodium transport, in concert with increased NO production by NO synthase 1 (NOS1) and NOS2. We hypothesized that NOS1- and/or NOS2-derived NO blunts T1D-induced activation of sodium transport in the mTAL. Methods T1D was induced by streptozotocin injection (STZ rats); sham rats received vehicle. Three-to-four weeks later, mTAL were isolated from both groups for assay of nitrite and superoxide production, and O2 consumption in the absence or presence of various inhibitors. Results Apocynin (NADPH oxidase inhibitor) normalized superoxide production and ouabainand furosemide-sensitive O2 consumption by mTALs from STZ rats, without altering O2 consumption by mTALs from sham rats. Apocynin also unmasked a T1D-induced increase in nitrite production. NOS inhibition did not alter superoxide production in either group. In sham mTAL, total NOS inhibition, but not isoform-specific inhibition of NOS1 or NOS2, increased ouabain- and furosemide-sensitive O2 consumption, confirming a tonic inhibitory impact of NOS3 on sodium transport. In contrast, neither total nor isoform-specific NOS inhibition altered O2 consumption by STZ mTAL. Apocynin-treatment of STZ mTAL unveiled the ability of isoform-specific NOS inhibition to significantly increase O2 consumption, without further increase in O2 consumption with total NOS inhibition. Conclusion Under normal conditions, NOS3-derived NO inhibits sodium transport in the mTAL. T1D dismantles the impact of NOS-mediated inhibition of sodium transport as a result of NADPH oxidase-dependent NO scavenging. Inhibition of NADPH oxidase to preserve NO bioavailability reveals an inhibitory impact of NOS1- and NOS2-derived NO on sodium transport in the mTAL.
We recently reported that diabetes increases superoxide anion and NO production by the rat renal medullary thick ascending limb (mTAL). We hypothesized that endogenously‐produced NO and superoxide anion exert opposing effects on Na transport by the mTAL during diabetes. O2 consumption (QO2) by mTAL suspensions was measured using the BD™ Oxygen Biosensor System, which discloses QO2 as a time‐related increase in relative fluorescence units (ΔRFU/min/mg protein). Ouabain‐sensitive QO2 (a measure of active Na+ transport) averaged 0.08±0.02 in mTALs from sham rats and 0.22±0.04 in mTALs from rats with STZ‐induced diabetes (P<0.05; n=8–9). NOS inhibition (100 μM 1400W) did not alter ouabain‐sensitive QO2 by mTALs from sham or STZ rats. NADPH oxidase inhibition (100 μM apocynin) had no effect on QO2 by sham mTALs, but caused a 60% decrease in ouabain‐sensitive QO2 by STZ mTALs (P<0.05). In the presence of apocynin, NOS inhibition significantly increased ouabain‐sensitive QO2 by mTALs from STZ rats, with similar effects evoked by isoform‐specific inhibitors of NOS1 and NOS2 (1 μM VNIO and 100 nM 1400W, respectively). Thus, while neither NO nor superoxide anion tonically influences Na transport by mTALs from the normal rat kidney, diabetes triggers superoxide anion stimulation of Na transport that is attenuated by NOS1‐ and NOS2‐dependent mechanisms likely reflecting an inhibitory influence of NO. (Supported by the AHA)
The renoprotective mechanisms of hemeoxygenase‐1 (HO‐1) induction remain unclear. We hypothesize that induction of HO‐1 will decrease blood pressure and proteinuria in spontaneously hypertensive rats (SHR) via antioxidant and anti‐inflammatory mechanisms. Male WKY and SHR were injected with the HO‐1 inducer cobalt protoporphrin (CoPP, 1.5 mg/kg s.c. bi‐weekly) for two weeks. CoPP reduced mean arterial pressure (MAP) (133±2 vs. 144±4 mmHg, p<0.05) and proteinuria (24±2 vs. 14±1mg/day, p<0.05) in SHR rats compared to baseline values, with no effect in WKY. Renal cortical superoxide (O2−) production and urinary 8‐isoprostane excretion were higher in SHR compared to WKY (O2−: 10.5±1 vs.6±1 CPM/mg protein, p<.05; 8‐iso: 7±1.3 vs. 3.4±0.8 ng/day, p<0.05) and CoPP attenuated oxidative stress levels in SHR only (O2−: 5.1±1 CPM/mg, p<0.05; 8‐iso: 4.4±0.7 ng/day). SHR showed a marked elevation in urinary MCP‐1 excretion compared with WKY (19±2 vs. 7.5±2.5 ng/day, p<0.05) and this was attenuated by induction of HO‐1 in SHR (10.5±2.7 ng/day). Plasma nitrite was reduced in SHR compared to WKY (7.9±1.1 vs. 12.7±1.5 μM, p<0.05) and HO‐1 induction increased plasma nitrite levels in SHR (17.5±2.9 μM). These data demonstrate that induction of HO‐1 slows the progression of hypertension and proteinuria in SHR. These changes were associated with reduced renal oxidative stress and inflammation and increased indices of nitric oxide.
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