Eiji Kubota

Renal tubular Sirt1 attenuates diabetic albuminuria by epigenetically suppressing Claudin-1 overexpression in podocytes

Sirtuin 1 (Sirt1), a NAD+-regulated deacetylase with numerous known positive effects on cellular and whole-body metabolism, is expressed in the renal cortex and medulla. It is known to have protective effects against age-related disease, including diabetes. Here we investigated the protective role of Sirt1 in diabetic renal damage. We found that Sirt1 in proximal tubules (PTs) was downregulated before albuminuria occurred in streptozotocin-induced or obese (db/db) diabetic mice. PT-specific SIRT1 transgenic and Sirt1 knockout mice showed prevention and aggravation of the glomerular changes that occur in diabetes, respectively, and nondiabetic knockout mice exhibited albuminuria, suggesting that Sirt1 in PTs affects glomerular function. Downregulation of Sirt1 and upregulation of the tight junction protein Claudin-1 by SIRT1-mediated epigenetic regulation in podocytes contributed to albuminuria. We did not observe these phenomena in 5/6 nephrectomized mice. We also demonstrated retrograde interplay from PTs to glomeruli using nicotinamide mononucleotide (NMN) from conditioned medium, measurement of the autofluorescence of photoactivatable NMN and injection of fluorescence-labeled NMN. In human subjects with diabetes, the levels of SIRT1 and Claudin-1 were correlated with proteinuria levels. These results suggest that Sirt1 in PTs protects against albuminuria in diabetes by maintaining NMN concentrations around glomeruli, thus influencing podocyte function.

Altered pressure-natriuresis in obese Zucker rats

It has not been examined whether the pressure-natriuresis response is altered in the insulin-resistant condition. Furthermore, despite an important role of nitric oxide (NO) in modulating pressure-natriuresis, no investigations have been conducted assessing the renal interstitial NO production in insulin resistance. The present study examined whether pressure-natriuresis was altered in insulin-resistant obese Zucker rats (OZ) and assessed the cortical and medullary nitrate/nitrite (NOx) levels with the use of the renal microdialysis technique. In OZ, serum insulin/glucose ratio (23.0+/-4.0x10(-8), n=9) and blood pressure (119+/-3 mm Hg) were greater than those in lean Zucker rats (LZ; 7.0+/-1.9x10(-8) and 103+/-4 mm Hg, n=9). The pressure-natriuresis curve in OZ was shifted to higher renal perfusion pressure (RPP), and the slope was blunted compared with that in LZ (0.073+/-0.015 vs 0.217+/-0.047 microEq/min kidney weight/mm Hg, P<0.05). The basal renal NOx level was reduced in OZ (cortex, 4.032+/-0.331 micromol/L; medulla, 4. 329+/-0.515 micromol/L) compared with that in LZ (cortex, 7.315+/-1. 102 micromol/L; medulla: 7.698+/-0.964 micromol/L). Furthermore, elevating RPP increased the medullary NOx in LZ, but this pressure-induced response was lost in OZ. Four-week treatment with troglitazone, an insulin-sensitizing agent, improved hyperinsulinemia, systemic hypertension, and basal renal NOx levels (cortex, 5.639+/-0.286 micromol/L; medulla, 5.978+/-0.284 micromol/L), and partially ameliorated the pressure-natriuresis curves; the slope of pressure-natriuresis curves and elevated RPP-induced NOx, however, were not corrected. In conclusion, our study suggests that insulin resistance is closely associated with abnormal pressure-natriuresis and hypertension. These deranged renal responses to insulin resistance are most likely attributed to impaired medullary NO production within the medulla.

Divergent renal vasodilator action of L- and T-type calcium antagonists in vivo.

Objective To assess the in-vivo action on the renal microvasculature of the calcium antagonists nifedipine (L-type blocker), efonidipine (L/T-type blocker), and mibefradil (predominant T-type blocker). Design An intravital needle-type charge-coupled device (CCD) camera videomicroscope was introduced to visualize the renal microcirculation directly in vivo. Methods In anesthetized mongrel dogs, nifedipine (0.01–1 mg/kg per min), efonidipine (0.033–0.33 mg/kg per min), or mibefradil (0.01–1 mg/kg per min) was infused intravenously after the insertion of a CCD probe into the kidney. Renal microvascular responses to calcium antagonists were directly evaluated, with concomitant observation of renal clearance. Results Each calcium antagonist caused modest vasodepressor action without affecting heart rate. Nifedipine (1 mg/kg per min, n = 9) increased renal plasma flow (RPF) (14 ± 4%, P < 0.05) and glomerular filtration rate (GFR) (19 ± 5%, P < 0.05), and tended to increase the filtration fraction (5 ± 2% increment, P = 0.07). Efonidipine (0.33 mg/kg per min, n = 9), however, had no effect on filtration fraction, with 14 ± 6% increments in RPF (P < 0.05) and 14 ± 7% increments in GFR (P = 0.08). Rather, mibefradil (1 mg/kg per min, n = 9) elicited 6 ± 2% decreases in filtration fraction (P < 0.05), with slight increments in RPF (6 ± 3%) and no changes in GFR. In direct in-vivo microvasculature observations, nifedipine caused predominant (22 ± 2%) dilatation of afferent arterioles (from 15.5 ± 0.4 to 18.9 ± 0.4μm, n = 5), compared with that of efferent arterioles (10 ± 2%; from 11.0 ± 0.4 to 12.1 ± 0.3μm). In contrast, efonidipine caused a similar magnitude of vasodilatation (16 ± 4%) compared with 18 ± 2%;n = 6), and mibefradil caused greater dilatation of efferent arterioles (20 ± 4%, n = 7) than that of afferent arterioles (13 ± 4%). Conclusions There exists marked heterogeneity in action of nifedipine, efonidipine and mibefradil on the renal microvascular in canine kidneys in vivo. Furthermore, our current observations suggest an important contribution of T-type calcium channel activity to efferent arteriolar tone in vivo.

Differential Regulation of Elevated Renal Angiotensin II in Chronic Renal Ischemia

The present study was undertaken to clarify the role of intrarenal angiotensin (Ang) II and its generating pathways in clipped and nonclipped kidneys of 4-week unilateral renal artery stenosis in anesthetized dogs. After 4 weeks, renal plasma flow (RPF) decreased in clipped and nonclipped kidneys (baseline, 59±3; clipped, 16±1; nonclipped, 44±2 mL/min;P <0.01, n=22). Renal Ang I levels increased only in clipped, whereas intrarenal Ang II contents were elevated in both clipped (from 0.7±0.1 to 2.0±0.2 pg/mg tissue) and nonclipped kidneys (from 0.6±0.1 to 2.5±0.3 pg/mg tissue). Intrarenal ACE activity was increased in nonclipped kidneys but was unaltered in clipped kidneys. An angiotensin receptor antagonist (olmesartan medoxomil) given into the renal artery markedly restored RPF, and dilated both afferent and efferent arterioles (using intravital videomicroscopy). Furthermore, in clipped kidneys, the elevated Ang II was suppressed by a chymase inhibitor, chymostatin (from 2.1±0.6 to 0.8±0.1 pg/mg tissue;P <0.05), but not by cilazaprilat. In nonclipped kidneys, in contrast, cilazaprilat, but not chymostatin, potently inhibited the intrarenal Ang II generation (from 2.4±0.3 to 1.5±0.2 pg/mg tissue;P <0.05). Finally, [Pro11-d-Ala12]Ang I (an inactive precursor that yields Ang II by chymase but not by ACE; 1 to 50 nmol/kg) markedly elevated intrarenal Ang II in clipped, but not in nonclipped, kidneys. In conclusion, renal Ang II contents were elevated in both clipped and nonclipped kidneys, which contributed to the altered renal hemodynamics and microvascular tone. Furthermore, the mechanisms for intrarenal Ang II generation differ, and chymase activity is enhanced in clipped kidneys, whereas ACE-mediated Ang II generation is possibly responsible for elevated Ang II contents in nonclipped kidneys.

Role of Asymmetrical Dimethylarginine in Renal Microvascular Endothelial Dysfunction in Chronic Renal Failure with Hypertension

We examined whether endothelial function of the renal microcirculation was impaired in a model of chronic renal failure (CRF), and further assessed the role of asymmetrical dimethylarginine (ADMA) and its degrading enzyme, dimethylarginine dimethylaminohydrolase (DDAH), in mediating the deranged nitric oxide (NO) synthesis in CRF. CRF was established in male mongrel dogs by subtotal nephrectomy, and the animals were used in experiments after a period of 4 weeks. The endothelial function of the renal afferent and efferent arterioles was evaluated according to the response to acetylcholine, using an intravital needle-lens charge-coupled device camera. Intrarenal arterial infusion of acetylcholine (0.01 μg/kg/min) elicited 22±2% and 20±2% dilation of the afferent and efferent arterioles in normal dogs. In dogs with CRF, this vasodilation was attenuated (afferent, 12±2%; efferent, 11±1%), and the attenuation paralleled the diminished increments in urinary nitrite+nitrate excretion. In the animals with CRF, plasma concentrations of homocysteine (12.2 ±0.7 vs. 6.8±0.4 μmol/l) and ADMA were elevated (2.60±0.13 vs. 1.50 ±0.08 μmol/l). The inhibition of S-adenosylmethionine-dependent protein arginine N-methyltransferase by adenosine dialdehyde decreased plasma ADMA levels, and improved the acetylcholine-induced changes in urinary nitrite+nitrate excretion and arteriolar vasodilation. Acute methionine loading impaired the acetylcholine-induced renal arteriolar vasodilation in CRF, but not normal dogs, and the impairment in CRF dogs coincided with the changes in plasma ADMA levels. Real-time polymerase chain reaction revealed downregulation of the mRNA expression of DDAH-II in the dogs with CRF. Collectively, these results provide direct in vivo evidence of endothelial dysfunction in canine CRF kidneys. The endothelial dysfunction was attributed to the inhibition of the NO production by elevated ADMA, which involved the downregulation of DDAH-II. The deranged NO metabolic pathway including ADMA and DDAH is a novel mechanism for the aggravation of renal function.

Glucocorticoid-induced hypertension in the elderly relation to serum calcium and family history of essential hypertension

To explore the syndrome of glucocorticoid-induced hypertension in the elderly, we analyzed the clinical findings from 35 patients aged more than 65 years (12 men, 23 women) who received glucocorticoid therapy. Resting blood pressures (BP) were less than 140/90 mm Hg before glucocorticoid therapy, and patients were apparently disease-free apart from the condition for which glucocorticoid therapy was prescribed. Glucocorticoid-induced hypertension is defined as systolic BP more than 160 mm Hg and/or diastolic BP more than 95 mm Hg after glucocorticoid administration. Glucocorticoid-induced hypertension was seen in 13 patients (37.1%); all patients with hypertension [steroid (glucocorticoid)-induced hypertension (SH(+)) group] received more than 20 mg of prednisolone daily, and BP rose rapidly within a week of commencing glucocorticoid administration. The SH(+) group did not differ significantly in terms of age, heart rate, blood count, plasma biochemistry, plasma renin activity, plasma aldosterone, routine urinalysis, or urinary electrolytes from patients who did not show hypertension [SH(-) group]. However, serum total calcium concentrations were significantly lower in the SH(+) group both before and after 2 weeks of glucocorticoid therapy than in the SH(-) group. Furthermore, the SH(+) group showed a significantly higher percentage of patients with a positive family history of essential hypertension than the SH(-) group. In conclusion, although the detailed mechanisms are as yet uncertain, glucocorticoid-induced hypertension occurs often in elderly patients, and is more common in patients with total serum calcium concentrations lower than the normal range, and/or in those with positive family history of essential hypertension.

Role of Endothelium-Derived Hyperpolarizing Factor in ACE Inhibitor-Induced Renal Vasodilation in Vivo

Abstract—Although the angiotensin-converting enzyme (ACE) inhibitor-induced bradykinin enhances nitric oxide (NO) release, bradykinin may also stimulate the production of an additional vasodilator, endothelium-derived hyperpolarizing factor (EDHF). This study examined the role of EDHF in mediating the NO-independent action of ACE inhibitors in canine renal microcirculation in vivo. We used intravital CCD camera videomicroscopy that allowed direct visualization of renal microcirculation in superficial and juxtamedullary nephrons in an in vivo, in situ, and relatively intact setting. In the presence of E4177 (an angiotensin receptor blocker), cilazaprilat (30 &mgr;g/kg) had no effect on diameter of superficial afferent arterioles (Aff), but it increased renal contents of bradykinin and nitrate plus nitrite, and it elicited dilation of juxtamedullary Aff (from 24.0±0.2 to 28.2±0.8 &mgr;m), juxtamedullary efferent arterioles (Eff) (from 24.2±0.2 to 28.0±0.8 &mgr;m), and superficial Eff (from 18.2±0.2 to 19.7±0.2 &mgr;m). These changes in diameters were prevented by N&agr;-adamantaneacetyl-d-Arg-[Hyp3,Thi5,8,D-Phe7]bradykinin, a bradykinin receptor antagonist. The pretreatment with nitro-l-arginine methylester (l-NAME) plus E4177 eliminated the dilator response of juxtamedullary/superficial Eff and the increase in renal nitrate plus nitrite levels induced by cilazaprilat. In contrast, in the presence of E4177+l-NAME, cilazaprilat still caused 8%±3% dilation of juxtamedullary Aff, which was completely eliminated by proadifen, a cytochrome-P450 and KCa channel blocker. Collectively, the ACE inhibitor exerts multiple vasodilator mechanisms, including the inhibition of angiotensin II formation; blockade of angiotensin II activity appears to be a dominant mechanism in superficial Aff, whereas the bradykinin-induced NO acts on superficial Eff and juxtamedullary Aff/Eff. Furthermore, a putative EDHF is an additional mechanism for the ACE inhibitor-induced vasodilation of juxtamedullary Aff in vivo.

Role of nitric oxide and prostaglandin E2 in acute renal hypoperfusion

SUMMARY: Although acute renal ischaemia alters the production of various paracrines, there has been little investigation examining the role of intrarenal vasoactive substances. In the present study, we investigated the role of intrarenal nitric oxide and prostaglandins in modulating the acute renal hypoperfusion‐induced alterations in renal function. After a 90% clipping of the left renal artery for 60 min, the clip was released, and the renal haemodynamics and sodium excretion were evaluated in both clipped and non‐clipped kidneys of anaesthetized dogs. Furthermore, the changes in renal contents of nitrate/nitrite (NOx) and prostaglandin E2 (PGE2) were assessed by using the renal microdialysis technique. The release of the clipping elicited a gradual recovery of renal plasma flow and glomerular filtration rate, and a sustained increase in fractional sodium excretion (FENa) in the clipped kidney. Renal interstitial NOx was reduced in both the cortex (from 8.2 ± 1.1 to 2.5 ± 0.3 µmol/L, P < 0.01) and medulla (from 10.1 ± 0.9 to 3.1 ± 0.2 µmol/L, P < 0.01), but the levels gradually elevated after declamping. The treatment with nitro‐l‐arginine methylester only modestly impaired the recovery of renal plasma flow (RPF; at hour 4) and glomerular filtration rate (GFR; at hours 3 and 4 after declamping), without affecting FENa. Conversely, the renal PGE2 levels increased prominently upon the onset of ischaemia (medulla, from 149 ± 19 to 378 ± 39 pg/mL, P < 0.01; cortex, from 107 ± 13 to 302 ± 34 pg/mL, P < 0.01). Furthermore, the pretreatment with a non‐specific cyclo‐oxygenase (COX) inhibitor, sulpyrine, and a COX‐2‐specific inhibitor, NS398, prominently inhibited the increases in FENa induced by the acute renal arterial clipping in a similar manner. In conclusion, in acute renal hypoperfusion, nitric oxide (NO) plays a permissive role in the recovery of the renal haemodynamics. In contrast, sustained increases in renal PGE2 in both clipped and non‐clipped kidneys indicate that the COX‐2‐mediated PGE2 contributes importantly to the failure of the sodium reabsorption in response to acute renal hypoperfusion.

Divergent natriuretic action of calcium channel antagonists in mongrel dogs : renal haemodynamics as a determinant of natriuresis

This study examined the effects of different types of calcium channel antagonists on renal haemodynamics and natriuresis. The intravenous infusion of nifedipine (L-type blocker), efonidipine (L/T-type blocker) or mibefradil (predominant T-type blocker) into anaesthetized dogs elicited similar, albeit modest, reductions in blood pressure. Nifedipine (1 microgram.min(-1).kg(-1)) increased renal plasma flow (RPF) (23+/-6%; P<0.05) and glomerular filtration rate (GFR) (25+/-5%; P<0.05) (all values are means+/-S.E.M., n=7). Efonidipine (0.33 microgram .min(-1).kg(-1)) also elevated RPF (18+/-6%; P<0.05), and tended to increase GFR (17+/-8%; P=0.08). These antagonists exerted contrasting actions on the filtration fraction (FF), with an increase being elicited by nifedipine, whereas efonidipine had no effect. Furthermore, mibefradil (0.01-1 microgram.min(-1).kg(-1)) slightly elevated RPF (between 5+/-3% and 8+/-3%), but failed to alter GFR, resulting in a decrease in FF. Nifedipine slightly increased urinary sodium excretion (U(Na)V) (29+/-16% increase at 1 microgram .min(-1).kg(-1)) and fractional sodium excretion (FE(Na)) (18+/-14%), whereas efonidipine (0.33 microgram .min(-1).kg(-1)) elicited marked elevations in U(Na)V (110+/-38%; P<0.05) and FE(Na) (102+/-44%; P<0.05). Mibefradil (1 microgram .min(-1).kg(-1)) exerted a moderate natriuretic action [U(Na)V, +60+/-32% (P=0.1); FE(Na), +67+/-20% (P<0.05)]. Furthermore, although a positive correlation was observed between U(Na)V and urinary nitrate/nitrite excretion, no differences were noted between the various calcium channel antagonists. Collectively, this study demonstrates that the glomerular haemodynamic and natriuretic actions of these calcium channel antagonists, which possess diverse blocking activities on L/T-type channels, vary. Based on the divergent actions on FF (i.e. increase, no change and decrease by nifedipine, efonidipine and mibefradil respectively), the natriuretic action of calcium channel antagonists is possibly attributed to the inhibition of tubular sodium reabsorption associated with increased post-glomerular blood flow, rather than increased GFR.

Role of intrarenal angiotensin II in glucocorticoid-induced renal vasodilation

AbstractBackground. Although glucocorticoids elicit systemic hypertension, they are also demonstrated to cause marked increases in renal blood flow. The mechanism of this alteration, however, remains undetermined. Methods. Dogs were treated with dexamethasone (DEX) for 7 days, and renal, as well as systemic hemodynamic, responses to DEX were assessed. In addition, the role of intrarenal angiotensin (ANG) II in mediating the glucocorticoid-induced renal vasodilation was examined in conscious unrestrained dogs. Results. Seven-day treatment with DEX caused prominent increases in mean arterial pressure (MAP; from 80 ± 2 to 98 ± 5 mmHg) and in renal plasma flow (RPF; from 142 ± 4 to 191 ± 7 ml/min), with decreases in renal vascular resistance [RVR; from 0.26 ± 0.01 to 0.22 ± 0.01 mmHg/(ml/min)] and in the filtration fraction (FF; from 0.24 ± 0.01 to 0.20 ± 0.01). DEX treatment did not alter plasma ANG II levels, but enhanced candesartan-induced reduction in MAP. In contrast, the candesartan-induced increase in RPF (19 ± 2% increase) was completely abolished by DEX. DEX treatment markedly reduced renal tissue ANG II content (from 1.09 ± 0.07 to 0.71 ± 0.04 pg/mg tissue), which paralleled the response of renal tissue angiotensin-converting enzyme (ACE) activity (−20 ± 4%). Finally, intravenous ANG II administration caused a greater reduction in RPF during the DEX treatment period (−17 ± 2% vs −11 ± 1% in the control period). Conclusions. Glucocorticoids cause hypertension, but they also cause a paradoxical decrease in RVR and increase in RPF. The renal responses to candesartan and exogenous ANG II during DEX treatment suggest that the attenuation of intrarenal ANG-mediated vascular tone plays an important role in the altered renal hemodynamics. The decreased ANG tone is likely caused by reduced ANG II formation, resulting in part from suppressed ACE activity, but not from decreased sensitivity to ANG II.