Rodger Loutzenhiser

Renal Myogenic Response: Kinetic Attributes and Physiological Role

The kinetic attributes of the afferent arteriole myogenic response were investigated using the in vitro perfused hydronephrotic rat kidney. Equations describing the time course for pressure-dependent vasoconstriction and vasodilation, and steady-state changes in diameter were combined to develop a mathematical model of autoregulation. Transfer functions were constructed by passing sinusoidal pressure waves through the model. These findings were compared with results derived using data from instrumented conscious rats. In each case, a reduction in gain and increase in phase were observed at frequencies of 0.2 to 0.3 Hz. We then examined the impact of oscillating pressure signals. The model predicted that pressure signals oscillating at frequencies above the myogenic operating range would elicit a sustained vasoconstriction the magnitude of which was dependent on peak pressure. These predictions were directly confirmed in the hydronephrotic kidney. Pressure oscillations presented at frequencies of 1 to 6 Hz elicited sustained afferent vasoconstrictions and the magnitude of the response depended exclusively on the peak pressure. Elevated systolic pressure elicited vasoconstriction even if mean pressure was reduced. These findings challenge the view that the renal myogenic response exists to maintain glomerular capillary pressure constant, but rather imply a primary role in protecting against elevated systolic pressures. Thus, the kinetic features of the afferent arteriole allow this vessel to adjust tone in response to changes in systolic pressures presented at the pulse rate. We suggest that the primary function of this mechanism is to protect the glomerulus from the blood pressure power that is normally present at the pulse frequency.

Angiotensin II–Induced Ca2+ Influx in Renal Afferent and Efferent Arterioles

Angiotensin II (Ang II)-induced Ca(2+) signaling was studied in isolated rat renal arterioles using fura-2. Ang II (10 nmol/L) caused a sustained elevation in [Ca(2+)](i), which was dependent on [Ca(2+)](o) in both vessel types. This response was blocked by nifedipine in only the afferent arteriole. Using the Mn(2+) quench technique, we found that Ang II stimulates Ca(2+) influx in both vessels. Nifedipine blocked the Ang II-induced Ca(2+) influx in afferent arterioles but not in efferent arterioles. In contrast to Ang II, KCl-induced depolarization stimulated Ca(2+) influx in only the afferent arteriole. Cyclopiazonic acid (CPA, 30 micromol/L) was used to examine the presence of store-operated Ca(2+) entry in myocytes isolated from each arteriole. In efferent myocytes, CPA induced a sustained Ca(2+) increase that was dependent on [Ca(2+)](o) and insensitive to nifedipine. This mechanism was absent in afferent myocytes. SKF 96365 inhibited Ang II-induced Ca(2+) entry in efferent arterioles and CPA-induced Ca(2+) entry in efferent myocytes over identical concentrations. Our findings thus indicate that Ang II activates differing Ca(2+) influx mechanisms in pre- and postglomerular arterioles. In the afferent arteriole, Ang II activates dihydropyridine-sensitive L-type Ca(2+) channels, presumably by membrane depolarization. In the efferent arteriole, Ang II appears to stimulate Ca(2+) entry via store-operated Ca(2+) influx.

Protective Importance of the Myogenic Response in the Renal Circulation

Primary essential hypertension is second only to diabetic nephropathy as a etiology for end-stage renal disease.1 In addition, coexistent/superimposed hypertension plays a major role in the progression of most forms of chronic kidney disease (CKD), including diabetic nephropathy.2–5 Nevertheless, the individual risk is very low, with <1% of the hypertensive population developing end-stage renal disease. Such data indicate that there must be mechanisms that normally protect the kidneys from hypertensive injury of a severity sufficient to result in end-stage renal disease. The following Brief Review summarizes the evidence that indicates that the renal autoregulatory response, primarily mediated by the myogenic mechanism, is largely responsible for such protection. Moreover, the differing patterns of renal damage that are observed in clinical and experimental hypertension are best explained when considered in the context of alterations in the renal autoregulatory capacity. Recent data also indicate that hypertensive renal damage correlates most strongly with systolic blood pressure (BP).6–8 Accordingly, the review further emphasizes the kinetic characteristics of the renal myogenic response to oscillating BP signals that render it particularly capable of providing protection against systolic pressures. Most individuals with primary hypertension develop the modest vascular pathology of benign nephrosclerosis.5 The glomeruli are largely spared, and, therefore, proteinuria is not a prominent feature. Because it progresses fairly slowly with limited ischemic nephron loss, renal function is not seriously compromised, except in some genetically susceptible individuals or groups, such as blacks, in whom a more accelerated course may be seen.2–5 Thus, the slope of the relationship between renal damage and BP through most of the hypertensive range is fairly flat in individuals with benign nephrosclerosis.2–4 However, if the hypertension becomes very severe and exceeds a critical threshold, severe acute disruptive injury of malignant nephrosclerosis to the renal arteries and arterioles develops …

Biphasic actions of prostaglandin E(2) on the renal afferent arteriole : role of EP(3) and EP(4) receptors.

Prostaglandin (PG) E(2) is an important modulator of the actions of angiotensin (Ang) II. In the present study, we investigated the renal microvascular actions of PGE(2) and the EP receptor subtypes involved. Ibuprofen potentiated Ang II-induced vasoconstriction in in vitro perfused normal rat kidneys and augmented afferent arteriolar, but not efferent arteriolar, responses in the hydronephrotic rat kidney model. This preglomerular effect of endogenous prostanoids was mimicked by exogenous PGE(2), which reversed Ang II-induced afferent arteriolar vasoconstriction at concentrations of 0.1 to 10 nmol/L without affecting the efferent arteriole. The PGE(2)-induced vasodilation was potentiated by the phosphodiesterase inhibitor Ro 20-1724 and was mimicked by 11-deoxy-PGE(1) (0.01 to 1 nmol/L). Butaprost, which acts preferentially at EP(2) receptors, was relatively ineffective. Whereas 0.1 to 10 nmol/L PGE(2) elicited vasodilation, higher concentrations (1 to 10 micromol/L) restored Ang II-induced afferent arteriolar vasoconstriction. This response was blocked by pertussis toxin (200 microg/mL) and was mimicked by the EP(1)/EP(3) agonist sulprostone (1 to 300 nmol/L). Reverse transcription-polymerase chain reaction of individually isolated afferent arterioles revealed the presence of message for EP(4) and all 3 EP(3) splice variants (alpha, beta, and gamma) but not EP(1) or EP(2). Our findings thus indicate that PGE(2) elicits both vasodilatory and vasoconstrictor actions on the afferent arteriole. The vasodilation is mediated by EP(4) receptors coupled to cAMP, presumably via G(alphas). The vasoconstriction is mediated by an EP(3) receptor coupled to G(alphai) and appears to reflect a functional antagonism of the EP(4)-induced vasodilation.

Renal microvascular dysfunction, hypertension and CKD progression.

Purpose of reviewDespite apparent blood pressure (BP) control and renin–angiotensin system (RAS) blockade, the chronic kidney disease (CKD) outcomes have been suboptimal. Accordingly, this review is addressed to renal microvascular and autoregulatory impairments that underlie the enhanced dynamic glomerular BP transmission in CKD progression. Recent findingsClinical data suggest that failure to achieve adequate 24-h BP control is likely contributing to the suboptimal outcomes in CKD. Whereas evidence continues to accumulate regarding the importance of preglomerular autoregulatory impairment to the dynamic glomerular BP transmission, emerging data indicate that nitric oxide-mediated efferent vasodilation may play an important role in mitigating the consequences of glomerular hypertension. By contrast, the vasoconstrictor effects of angiotensin II are expected to potentially reduce glomerular barotrauma and possibly enhance ischemic injury. When adequate BP measurement methods are used, the evidence for BP-independent injury initiating mechanisms is considerably weaker and the renoprotection by RAS blockade largely parallels its antihypertensive effectiveness. SummaryAdequate 24-h BP control presently offers the most feasible intervention for reducing glomerular BP transmission and improving suboptimal outcomes in CKD. Investigations addressed to improving myogenic autoregulation and/or enhancing nitric oxide-mediated efferent dilation in addition to the more downstream mediators may provide additional future therapeutic targets.

Hypoxia inhibits myogenic reactivity of renal afferent arterioles by activating ATP-sensitive K+ channels.

Recent findings implicate K+ channels as important modulators of myogenic tone and possible mediators of the vasodilatory effects of hypoxia. In the present report, we examined the effects of hypoxia on myogenic vasoconstriction of renal afferent arterioles. Using the in vitro perfused hydronephrotic rat kidney model, we observed precisely graded decreases in arteriolar diameter when renal perfusion pressure was increased. Normal myogenic reactivity was observed over PO2 levels of 150 to 80 mm Hg. Reducing PO2 to 60, 40, and 30 mm Hg resulted in a significant progressive inhibition of myogenic reactivity. At approximately 20 mm Hg, myogenic vasoconstriction was essentially abolished, whereas the vasoconstriction induced by 30 mmol/L KCl was unaffected. The addition of 1.0 mumol/L glibenclamide completely restored myogenic vasoconstriction during hypoxia. In contrast, 1.0 mmol/L tetraethylammonium did not alter the effects of hypoxia. To investigate the relation between hypoxia-induced vasodilation and smooth muscle oxidative phosphorylation, we monitored changes in arteriolar levels of reduced NADH during exposure to hypoxia. Arterioles preconstricted by elevated pressure were optically isolated for simultaneous monitoring of vessel diameter and NADH fluorescence (360-nm excitation, 450-nm emission). Reducing perfusate PO2 from 150 to 20 mm Hg resulted in progressive loss of myogenic tone with no change in arteriolar NADH. These findings indicate that lowering PO2 within a physiological range attenuates myogenic reactivity of the renal afferent arteriole by causing the activation of ATP-sensitive K+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)

A highly sensitive technique to measure myosin regulatory light chain phosphorylation: the first quantification in renal arterioles

Phosphorylation of the 20-kDa myosin regulatory light chains (LC(20)) plays a key role in the regulation of smooth muscle contraction. The level of LC(20) phosphorylation is governed by the relative activities of myosin light chain kinase and phosphatase pathways. The regulation of these two pathways differs in different smooth muscle types and in the actions of different vasoactive stimuli. Little is known concerning the regulation of LC(20) phosphorylation in the renal microcirculation. The available pharmacological probes are often nonspecific, and current techniques to directly measure LC(20) phosphorylation are not sensitive enough for quantification in small arterioles. We describe here a novel approach to address this important issue. Using SDS-PAGE with polyacrylamide-bound Mn(2+)-phosphate-binding tag and enhanced Western blot analysis, we were able to detect LC(20) phosphorylation using as little as 5 pg (250 amol) of isolated LC(20). Phosphorylated and unphosphorylated LC(20) were detected in single isolated afferent arterioles, and LC(20) phosphorylation levels could be accurately quantified in pooled samples of three arterioles (<300 cells). The phosphorylation level of LC(20) in the afferent arteriole was 6.8 +/- 1.7% under basal conditions and increased to 34.7 +/- 5.1% and 44.6 +/- 6.6% in response to 30 mM KCl and 10(-8) M angiotensin II, respectively. The application of this technique will enable investigations of the different determinants of LC(20) phosphorylation in afferent and efferent arterioles and provide insights into the signaling pathways that regulate LC(20) phosphorylation in the renal microvasculature under physiological and pathophysiological conditions.

Dual endothelium-dependent vascular activities of proteinase-activated receptor-2-activating peptides : evidence for receptor heterogeneity

The vascular actions of the proteinase‐activated receptor‐2‐activating peptides (PAR2APs), SLIGRL‐NH2 (SL‐NH2) and SLIGKV‐NH2 (KV‐NH2) as well as the reverse‐sequence peptide, LSIGRL‐NH2 (LS‐NH2) and an N‐acylated PAR2AP derivative, trans‐cinnamoyl‐LIGRLO‐NH2 (tcLI‐NH2), were studied in rat intact and endothelium‐denuded artery ring preparations, primarily from the pulmonary artery (RPA). In RPA rings with but not without a functional endothelium, SL‐NH2 (but not LS‐NH2) caused either an endothelium‐dependent relaxation (at concentrations: <10 μm) or (at higher concentrations: >10 μm), an endothelium‐dependent contraction. No contractile response was observed in endothelium‐denuded preparations, that otherwise contracted in response to the PAR1AP, TFLLR‐NH2. The endothelium‐dependent contractile response to SL‐NH2 was not blocked by the α‐adrenoceptor antagonist prazosin, the endothelin antagonist BQ123, the angiotensin II antagonist DuP753, by tetrodotoxin; nor by the enzyme inhibitors, Nω‐nitro‐l‐arginine‐methylester (NO‐synthase), indomethacin (cyclo‐oxygenase), SKF‐525A (epoxygenase) and MK886 (leukotriene synthesis inhibitor). In the relaxation assay, KV‐NH2 was 5 fold less potent than SL‐NH2, whereas in the contractile assay KV‐NH2 was about equipotent with SL‐NH2. However, the maximal contractile response to KV‐NH2 was lower than that of SL‐NH2. The PAR2AP analogue, tcLI‐NH2, was as active as SL‐NH2 in the relaxation assay but was inactive as a contractile agonist in the endothelium‐intact RPA. The relaxant responses caused by SL‐NH2 and trypsin, as well as the contractile response caused by SL‐NH2, did not desensitize in the course of repeated exposures of the tissue to agonist; whereas the contractile response to trypsin, only observed at concentrations greater than 30 u ml−1, was desensitized by previous exposure of the tissue to either thrombin or trypsin. In a contractile assay, where the tissue was desensitized to a concentration of trypsin that would otherwise cause a relaxant response, the preparation still contracted in response to SL‐NH2. However, the trypsin‐desensitized preparations were no longer contracted by thrombin. From the cross‐desensitization by thrombin of the contractile response to trypsin (and vice versa), we concluded that the contractile effect of trypsin was due to activation of the thrombin receptor and not PAR2. We concluded that the endothelium‐dependent contraction caused by high concentrations of SL‐NH2 is due to an as yet unidentified contracting factor; whereas the endothelium‐dependent relaxation response observed at low concentrations of SL‐NH2 (10 μm) is mediated by nitric oxide. The distinct structure activity profiles for the contractile response (potency of KV‐NH2SL‐NH2) compared with the relaxant response (potency of KV‐NH2<5SL‐NH2); the contractile responsiveness to SL‐NH2 of an endothelium‐intact RPA preparation, that did not contract in response to trypsin; and the lack of contractile activity of the PAR2AP analogue tcLI‐NH2, that was as active as SL‐NH2 in the relaxation assay all argue in favour of receptor heterogeneity in the vasculature for the PAR2APs. It remains to be determined if the distinct endothelial receptor responsible for the contractile action of SL‐NH2 can be proteolytically activated, like PAR1 and PAR2.

Functional Evidence for an Inward Rectifier Potassium Current in Rat Renal Afferent Arterioles

Abstract — An inward rectifier potassium current, Kir, has been identified in cerebral and coronary resistance vessels, where it is considered to be an important determinant of resting membrane potential (RMP) and to play a role in blood flow regulation. We investigated the functional role of Kir in the renal afferent arteriole using the in vitro–perfused hydronephrotic rat kidney. Increasing external KCl from 5 to 15 mmol/L induced afferent arteriolar vasodilation. This response was inhibited by 10 to 100 &mgr;mol/L Ba2+, concentrations selective for blockade of Kir, and by chloroethylclonidine (100 &mgr;mol/L) but was not blocked by glibenclamide (10 &mgr;mol/L) or ouabain (3 mmol/L). Reducing external KCl from 5 to 1.5 mmol/L to enhance rectification of Kir caused vasoconstriction at low renal arterial pressure (40 mm Hg) and vasodilation during myogenic vasoconstriction (120 mm Hg), suggesting that this current dominates RMP at low perfusion pressures. When administered to kidneys perfused at 40 mm Hg renal arterial pressure, 30 &mgr;mol/L Ba2+ elicited afferent arteriolar depolarization, reducing RMP from −47±2 to −34±2 mV (n=10, P <0.0001), and vasoconstriction, reducing diameters from 14.5±1 to 10.9±0.8 &mgr;m (n=10, P =0.0016). Although Ba2+ reduced resting diameter, blockade of Kir did not prevent myogenic signaling in this vessel. Our findings thus demonstrate the presence of Kir in rat renal afferent arterioles and suggest that this current is an important determinant of RMP in situ.

Dynamic autoregulation in the in vitro perfused hydronephrotic rat kidney

Renal autoregulation is mediated by tubuloglomerular feedback, operating at 0.03-0.05 Hz, and a faster system, operating at 0.1-0.2 Hz, that has been attributed by exclusion to myogenic vasoconstriction. In this study, we examined dynamic autoregulation in the hydronephrotic rat kidney, which lacks tubuloglomerular feedback but exhibits pressure-induced afferent arteriolar vasoconstriction. Kidneys were harvested under anesthesia from Sprague-Dawley rats and perfused in vitro using defined, colloid-free medium. Renal perfusate flow was assessed during forced pressure fluctuations at mean pressures of 60-140 mmHg. Transfer function analysis revealed passive behavior at 60 mmHg and active, pressure-dependent responses at higher pressures. In all cases, coherence was high (0.89 ± 0.03 between 0.01 and 0.9 Hz). There was a resonance peak in admittance gain at ≈0.3 Hz and an associated broad peak in phase angle. Below this frequency, gain declined progressively. The minimum gain achieved at 0.01-0.05 Hz was pressure sensitive, being 1.08 ± 0.02 at 60 mmHg and 0.71 ± 0.04 at 140 mmHg. These findings are consistent with in vivo results and with model-based predictions of the dynamics of myogenic autoregulation, supporting the postulate that the rapid component of autoregulation reflects operation of a myogenic mechanism.Renal autoregulation is mediated by tubuloglomerular feedback, operating at 0.03-0.05 Hz, and a faster system, operating at 0.1-0.2 Hz, that has been attributed by exclusion to myogenic vasoconstriction. In this study, we examined dynamic autoregulation in the hydronephrotic rat kidney, which lacks tubuloglomerular feedback but exhibits pressure-induced afferent arteriolar vasoconstriction. Kidneys were harvested under anesthesia from Sprague-Dawley rats and perfused in vitro using defined, colloid-free medium. Renal perfusate flow was assessed during forced pressure fluctuations at mean pressures of 60-140 mmHg. Transfer function analysis revealed passive behavior at 60 mmHg and active, pressure-dependent responses at higher pressures. In all cases, coherence was high (0.89 +/- 0.03 between 0.01 and 0.9 Hz). There was a resonance peak in admittance gain at approximately 0.3 Hz and an associated broad peak in phase angle. Below this frequency, gain declined progressively. The minimum gain achieved at 0.01-0.05 Hz was pressure sensitive, being 1.08 +/- 0.02 at 60 mmHg and 0.71 +/- 0.04 at 140 mmHg. These findings are consistent with in vivo results and with model-based predictions of the dynamics of myogenic autoregulation, supporting the postulate that the rapid component of autoregulation reflects operation of a myogenic mechanism.