Interaction between NO synthase and NADPH oxidase in control of sodium transport by the renal thick ascending limb during diabetes.

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 ouabain-sensitive O2 consumption and 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.

A true champion of Hungarian kidney research and nephrology education--tribute to László Rosivall.

This article pays tribute to the tremendous achievements of Dr. László Rosivall in renal (patho)physiology research and nephrology education in Hungary on the occasion of his 60th birthday. For the past several decades Dr. Rosivall has been a charismatic leader of academic institutions, national and international societies, foundations in physiology, nephrology and hypertension, but the most important of his many contributions, is his role as a scientist. He earned his MD with Summa cum Laude at Semmelweis University (1973) and was invited immediately after that to join the laboratory of Hársing. He studied the distribution of intra-renal blood flow employing then state-of-the-art methods as well as developed his own technique at Semmelweis University and at the University of Bergen with Knut Aukland. This led to his PhD thesis and degree in 1980. An important determinant of his early basic scientific training and development was his postdoctoral research fellowship and later many visiting professorships in the Nephrology Research and Training Center (NRTC) at the University of Alabama at Birmingham, Birmingham, AL, USA between 1981 and 1983. Actually, this research fellowship not only impacted his own future career, but it also cleared the path for many other young Hungarian scientists who later trained with Dr. Rosivall and then at UAB. The early 1980s were the years of significant scientific discoveries and the NRTC team at UAB made important contributions by their studies on renal and glomerular hemodynamics, the renin-angiotensin system (12, 19, 22) and by the development of classic experimental techniques like renal micropuncture, microperfusion, and the juxtamedullary nephron preparation (3) that are still being used worldwide. When Dr. Rosivall joined UAB in the 1980s, the team at the NRTC included Drs. Navar, Bell, Inscho, Carmines, Casellas, and Oparil, among many others, who share their fond memories of working with Dr. Rosivall in this article.

Renal interstitial hydrostatic pressure and sodium excretion during acute volume expansion in diabetic rats.

Experiments were performed to test the hypothesis that the renal interstitial hydrostatic pressure (RIHP) response to acute volume expansion is suppressed in diabetes mellitus. Sprague-Dawley rats received streptozotocin (STZ rats; 65 mg/kg ip) or vehicle (Sham rats). Two weeks later, RIHP and Na(+) excretion responses to acute graded volume expansion with isotonic saline were quantified under Inactin anesthesia (0.1 mg/kg ip). In Sham rats, acute graded volume expansion to 10% body wt produced increases in RIHP (Delta = 12.2 +/- 2.4 mmHg), urine flow (Delta = 54 +/- 8 microliter. min(-1). g(-1)), and Na(+) excretion (Delta = 11.5 +/- 1.9 mueq. min(-1). g(-1)). In STZ rats, these volume expansion-induced responses were significantly blunted (RIHP by 50%, urine flow by 81%, and Na(+) excretion by 76%). Renal decapsulation eliminated the differences between STZ and Sham rats with regard to volume expansion-induced increases in RIHP, urine flow, and Na(+) excretion. Renal denervation normalized the RIHP response to volume expansion and improved the diuretic and natriuretic responses in STZ rats. Moreover, diuretic and natriuretic responses to direct changes in RIHP (induced by renal interstitial volume expansion) were blunted in STZ rats. We conclude that diminished alterations in RIHP, as well as a reduced impact of RIHP on Na(+) excretion, contribute to the impaired diuretic and natriuretic responses to acute volume expansion during the early stage of diabetes.

Influence of Ca(2+)-activated K(+) channels on rat renal arteriolar responses to depolarizing agonists.

Experiments were performed to evaluate the hypothesis that opening of Ca(2+)-activated K(+) channels (BK(Ca) channels) promotes juxtamedullary arteriolar dilation and curtails constrictor responses to depolarizing agonists. Under baseline conditions, afferent and efferent arteriolar lumen diameters averaged 23.4 +/- 0.9 (n = 36) and 22.8 +/- 1.1 (n = 13) microm, respectively. The synthetic BK(Ca) channel opener NS-1619 evoked concentration-dependent afferent arteriolar dilation. BK(Ca) channel blockade (1 mM tetraethylammonium; TEA) decreased afferent diameter by 15 +/- 3% and prevented the dilator response to 30 microM NS-1619. ANG II (10 nM) decreased afferent arteriolar diameter by 44 +/- 4%, a response that was reduced by 30% during NS-1619 treatment; however, TEA failed to alter afferent constrictor responses to either ANG II or arginine vasopressin. Neither NS-1619 nor TEA altered agonist-induced constriction of the efferent arteriole. Thus, although the BK(Ca) channel agonist was able to curtail afferent (but not efferent) arteriolar constrictor responses to ANG II, BK(Ca) channel blockade did not allow exaggerated agonist-induced arteriolar constriction. These observations suggest that the BK(Ca) channels evident in afferent arteriolar smooth muscle do not provide a prominent physiological brake on agonist-induced constriction under our experimental conditions.

Tyrosine kinase involvement in renal arteriolar constrictor responses to angiotensin II.

Experiments were performed to test the hypothesis that tyrosine kinase activity contributes to renal arteriolar contractile responses to angiotensin (Ang) II. Rats were subjected to short-term enalaprilat treatment to decrease endogenous Ang II formation before tissue was harvested for experiments with the in vitro blood-perfused juxtamedullary nephron technique. Acute surgical papillectomy was used to avoid the indirect afferent arteriolar effect of Ang II that arises through increased tubuloglomerular feedback sensitivity. Arteriolar lumen diameter responses to 1 and 10 nmol/L Ang II were monitored by videomicroscopic methods before and during treatment with various tyrphostin compounds: 100 micromol/L AG18 (broad-spectrum tyrosine kinase inhibitor), 100 nmol/L AG1478 (selective epidermal growth factor receptor tyrosine kinase inhibitor), or 100 micromol/L AG9 (inactive analog). Baseline afferent arteriolar lumen diameter averaged 23.5+/-1.2 micrometer and was not influenced by any tyrphostin. Ang II (10 nmol/L) decreased afferent diameter by 11.1+/-1.0 micrometer under untreated conditions, a response that was not altered by AG9 but significantly blunted by AG18 (34+/-9% inhibition) or AG1478 (52+/-8% inhibition). AG18 did not suppress afferent arteriolar contractile responses to membrane depolarization (20 to 55 mmol/L K(+ )bath). Efferent arteriolar baseline diameter averaged 24.1+/-0.8 micrometer and was unaltered by AG18 or AG1478; however, efferent diameter responses to 10 nmol/L Ang II were diminished 52+/-10% by AG18 and 51+/-13% by AG1478. These observations indicate that Ang II signaling in renal afferent and efferent arteriolar vascular smooth muscle is either mediated or modulated by tyrosine kinase activity, including that of the epidermal growth factor receptor tyrosine kinase.

Regulation of Ca(2+)-activated K(+) channels by multifunctional Ca(2+)/calmodulin-dependent protein kinase.

Activation of mesangial cells by ANG II provokes release of intracellular Ca(2+) stores and subsequent Ca(2+) influx through voltage-gated channels, events that are reflected by a large transient increase in intracellular concentration [Ca(2+)](i) followed by a modest sustained elevation in [Ca(2+)](i). These ANG II-induced alterations in [Ca(2+)](i) elicit activation of large Ca(2+)-activated K(+) channels (BK(Ca)) in a negative-feedback manner. The mechanism of this BK(Ca) feedback response may involve the direct effect of intracellular Ca(2+) on the channel and/or channel activation by regulatory enzymes. The present study utilized patch-clamp and fura 2 fluorescence techniques to assess the involvement of multifunctional calcium calmodulin kinase II (CAMKII) in the BK(Ca) feedback response. In cell-attached patches, KN62 (specific inhibitor of CAMKII) either abolished or reduced to near zero the ANG II-induced BK(Ca) feedback response. This phenomenon did not reflect direct effects of KN62 on the BK(Ca) channel, because this agent alone did not significantly alter BK(Ca) channel activity in inside-out patches. KN62 also failed to alter either the transient peak or sustained plateau phases of the [Ca(2+)](i) response to ANG II. In inside-out patches (1 microM Ca(2+) in bath), calmodulin plus ATP activated BK(Ca) channels in the presence but not the absence of CAMKII. These observations are consistent with the postulate that CAMKII is involved in the BK(Ca) feedback response of mesangial cells, acting to potentiate the influence of increased [Ca(2+)](i) on the BK(Ca) channel or a closely associated regulator of the channel. An additional effect of CAMKII to activate a voltage-gated Ca(2+) channel cannot be ruled out by these experiments.

Store-operated Ca(2+) channels in human glomerular mesangial cells.

Experiments were performed to identify the biophysical properties of store-operated Ca(2+) channels (SOC) in cultured human glomerular mesangial cells (MC). A fluorometric technique (fura 2) was utilized to monitor the change in intracellular calcium concentration ([Ca(2+)](i)) evoked by elevating external [Ca(2+)] from 10 nM to 1 mM (Delta[Ca(2+)]). Under control conditions, Delta[Ca(2+)] averaged 6 nM and was unaffected by elevating bath [K(+)]. After treatment with 1 microM thapsigargin to deplete the intracellular Ca(2+) store, the change in [Ca(2+)](i) (Delta[Ca(2+)](th)) averaged 147 +/- 16 nM. In thapsigargin-treated MC studied under depolarizing conditions (75 mM bath K(+)), Delta[Ca(2+)](th) was 45 +/- 7 nM. The Delta[Ca(2+)](th) response of thapsigargin-treated cells was inhibited by La(3+) (IC(50) = 335 nM) but was unaffected by 5 microM Cd(2+). In patch clamp studies, inward currents were observed in cell-attached patches with either 90 mM Ba(2+) or Ca(2+) in the pipette and 140 mM KCl in the bathing solution. The single-channel conductance was 2.1 pS with Ba(2+) and 0.7 pS with Ca(2+). The estimated selectivities were Ca(2+) > Ba(2+) >> K(+). These channels were sensitive to 2 microM La(3+), insensitive to 5 microM Cd(2+), and voltage independent, with an average channel activity (NP(o)) of 1.02 at command potential (-V(p)) ranging from 0 to -80 mV. In summary, MC exhibited an electrogenic Ca(2+) influx pathway that is suggestive of Ca(2+) entry through SOC, as well as a small-conductance divalent-selective channel displaying biophysical properties consistent with SOC. Based on estimates of whole cell Ca(2+) influx derived from our data, we conclude that SOC with low single-channel conductance must be highly abundant in MC to allow significant capacitative Ca(2+) entry in response to depletion of the intracellular store.

Norepinephrine turnover in peripheral tissues of rats with heart failure.

Experiments were performed to determine if there is regional heterogeneity in sympathetic neural activation of peripheral tissues in rats with chronic heart failure (HF; 6-8 wk after coronary artery ligation). Norepinephrine (NE) turnover, an index of sympathetic activation, was determined on the basis of the decline in tissue NE levels that occurs during the 8-h after tyrosine hydroxylase inhibition (alpha-methyl-DL-p-tyrosine, 300 mg/kg ip at 4-h intervals). Compared with sham-operated rats, NE turnover was increased in the cardiac left ventricle, skeletal muscle, duodenum, and kidney of rats with HF, but was unaltered in liver and spleen. The increased renal NE turnover in HF was largely a reflection of increased turnover in the cortex, with no change evident in the medulla. Blockade of sympathetic ganglionic traffic (hexamethonium, 2 mg/kg sc at 2-h intervals) eliminated the tissue-specific effects of HF on tissue NE levels measured 8-h after tyrosine hydroxylase inhibition. These data support the contention that chronic HF evokes a central nervous system-mediated increase in basal sympathetic tone that exhibits regional heterogeneity (both between and within organs), a phenomenon that likely contributes to the functional consequences of this pathophysiological state.

Dihydropyridine-sensitive Ca(2+) channels in human glomerular mesangial cells.

In mesangial cells (MC), the response of intracellular Ca(2+) concentration ([Ca(2+)](i)) to a contractile agonist is biphasic with a large, transient increase in [Ca(2+)](i) followed by a smaller but sustained elevation as Ca(2+) flows into the cell from the extracellular fluid. It has been postulated that membrane depolarization precedes opening of Ca(2+) channels in the plasmalemmal membrane. However, a role for voltage-gated Ca(2+) channels (VGCC) in human MC has been controversial, and their existence has not been verified with single-channel analysis. We used fura 2 fluorescence and patch-clamp techniques to determine the properties of the Ca(2+) entry pathway responsible for the sustained response of [Ca(2+)](i) in human MC. We found that ANG II at 10 nM, 100 nM, and 1 microM increased [Ca(2+)](i) to sustained levels of 22%, 35%, and 49%, respectively, above baseline. The sustained response to 1 microM ANG II was attenuated by diltiazem and was reduced to a value less than baseline in the absence of external Ca(2+). None of the peak responses (due to release of intracellular stores of Ca(2+)) were affected by removal of external Ca(2+) or addition of diltiazem. Upon elevating the extracellular [K(+)] from 5 mM to 75 mM, [Ca(2+)](i) reached a sustained level of 48% greater than baseline. This effect of high K(+) was attenuated by either Ca(2+) removal or addition of diltiazem. In the presence of 75 or 140 mM K(+), the dihydropyridine agonist BAY K 8644 (1 microM and 10 microM) initiated sustained [Ca(2+)](i) responses averaging 18% and 25%, respectively, greater than baseline. With <10 nM Ca(2+) in the external solution, BAY K 8644 did not significantly affect [Ca(2+)](i). In separate patch-clamp experiments, barium-selective channels were found in cell-attached patches with 90 mM BaCl(2) and 10 microM BAY K 8644 in the pipette solution. The single-channel conductance was 11.2 pS, and the open probability increased steeply at membrane potentials between -30 mV and 0 mV. It is concluded that human glomerular MC contain dihydropyridine-sensitive Ca(2+) channels responsible for the voltage-regulated entry of Ca(2+) into the cell during an agonist-induced contraction.

Superoxide anion curbs nitric oxide modulation of afferent arteriolar ANG II responsiveness in diabetes mellitus.

Experiments were performed to test the hypothesis that the impact of endogenous nitric oxide (NO) on ANG II-induced renal arteriolar constriction is reduced in rats with insulin-dependent diabetes mellitus (65 mg/kg streptozotocin; STZ). Arteriolar diameter responses to exogenous ANG II were quantified before and during NO synthase inhibition (100 microM N(omega)-nitro-L-arginine; L-NNA) by using the in vitro blood-perfused juxtamedullary nephron technique. Afferent arteriolar lumen diameter averaged 20.7 +/- 2.0 micrometer in Sham kidneys and 25.9 +/- 1.3 micrometer in STZ kidneys (P < 0.05). Efferent arteriolar diameter did not differ between Sham and STZ rats. In kidneys from Sham rats, afferent and efferent arteriolar responses to ANG II (0.1-10.0 nM) were exaggerated significantly by L-NNA. L-NNA also augmented efferent arteriolar ANG II responses in kidneys from STZ rats (high-glucose bath) but did not alter ANG II responses in afferent arterioles from STZ rats. L-NNA also accentuated efferent, but not afferent, arteriolar ANG II responses in STZ kidneys during acute restoration of bath glucose to normal levels. Superoxide dismutase (150 U/ml) restored the ability of L-NNA to allow exaggerated afferent arteriolar responses to ANG II in kidneys from STZ rats. These observations indicate that superoxide anion suppresses the modulatory influence of endogenous NO on ANG II-induced afferent arteriolar constriction in diabetes mellitus.

Renal arteriolar contractile responses to angiotensin II in rats with poorly controlled diabetes mellitus.

1. Experiments were performed to test the hypothesis that renal arteriolar responses to angiotensin (Ang)II are altered in insulin-dependent diabetes mellitus. 2. Male Sprague-Dawley rats were treated with streptozotocin (STZ; 65 mg/kg, i.v.) or 0.9% NaCl vehicle (Sham). Partial insulin replacement maintained blood glucose levels at 422 +/- 6 mg/dL (STZ rats) for the ensuing 2 week period (86 +/- 4 mg/dL in Sham rats). The in vitro blood-perfused juxtamedullary nephron technique was used to study renal arteriolar diameter responses to exogenous AngII or K(+)-induced membrane depolarization. 3. Baseline afferent arteriolar diameter did not differ between kidneys harvested from Sham and STZ rats; however, constrictor responses to AngII were accentuated in kidneys from diabetic rats. During exposure to 10 nmol/L AngII, afferent diameter decreased by 37 +/- 5 and 18 +/- 4% in STZ and Sham kidneys, respectively. Efferent arterioles from Sham and STZ rats did not differ with regard to either baseline diameter or AngII responsiveness. 4. In experiments assessing afferent arteriolar responsiveness to membrane depolarization, 40 mmol/L K+ decreased lumen diameter by 73 +/- 11% in kidneys from Sham rats; however, the same depolarizing stimulus only reduced afferent diameter by 28 +/- 7% in STZ kidneys. 5. We conclude that afferent (but not efferent) arteriolar AngII responsiveness is increased during the early stages of poorly controlled diabetes mellitus in the rat. The exaggerated afferent arteriolar responsiveness to AngII occurs despite reduced sensitivity to membrane depolarization, suggesting that the emergence of alternative signalling processes or alterations in vasoactive modulator influences may underlie this phenomenon.

Suppressed impact of nitric oxide on renal arteriolar function in rats with chronic heart failure.

We performed experiments to test the hypothesis that experimental heart failure (HF) is associated with altered nitric oxide (NO)-dependent influences on the renal microvasculature, including diminished modulation of constrictor responses to ANG II. Eight to ten weeks after inducing HF in rats by coronary artery ligation, we administered enalaprilat to suppress ANG II synthesis and studied renal arteriolar function using the in vitro blood-perfused juxtamedullary nephron technique. In kidneys from sham-operated rats, NO synthase inhibition [100 microM Nomega-nitro-L-arginine (L-NNA)] reduced afferent arteriolar diameter by 4.1 +/- 0.6 microm and enhanced ANG II responsiveness (10 nM ANG II decreased afferent diameter by 10.1 +/- 1.4 micrometer before and 12.8 +/- 1.6 micrometer during L-NNA treatment; P < 0.05). In kidneys from HF rats, L-NNA did not alter afferent arteriolar baseline diameter or ANG II responsiveness (10 nM ANG II decreased diameter by 12.5 +/- 1.5 micrometer before and 12.5 +/- 2.3 micrometer during L-NNA). The effects of L-NNA on efferent arteriolar function were also abated in HF rats. In renal cortex of HF rats, NO synthase activity was decreased by 63% and superoxide dismutase activity was diminished by 39% relative to tissue from sham-operated rats. Urinary nitrate/nitrite excretion was also reduced in HF rats. Thus both diminished synthesis and augmented degradation are likely to contribute to a decreased renal microvascular impact of endogenous NO during chronic HF, the consequences of which include loss of NO-dependent modulation of ANG II-induced vasoconstriction.

Enhanced constrictor responses of skeletal muscle arterioles during chronic myocardial infarction.

The goal of this study was to test the hypothesis that chronic myocardial infarction potentiates agonist-induced constrictor responses of rat skeletal muscle arterioles in vivo. Eight weeks after we performed coronary artery ligation or sham (control) surgery, the spinotrapezius muscle was prepared for direct visualization of the microcirculation. Diameter of third-order arterioles (40.7 +/- 0.5 microns) to topical suffusion of angiotensin II (ANG II; 0.1-10 nM), arginine vasopressin (AVP; 0.1-10 nM), endothelin-1 (ET-1; 1.0-100 pM), and the thromboxane analog U-46619 (1.0-100 nM) was measured in both groups. Myocardial-infarcted rats exhibited enhanced arteriolar constrictor responses to ANG II and AVP compared with the responses in controls. In contrast, ET-1- and U-46619-induced constrictor responses were similar in control and myocardial-infarcted rats. Additional experiments explored the impact of NG-monomethyl-L-arginine (L-NMMA; 0.1 mM) on arteriolar reactivity. In control animals, L-NMMA potentiated ANG II- and AVP-induced vasoconstriction, achieving values similar to those observed in myocardial-infarcted rats. L-NMMA did not alter vasoconstrictor responses in rats with chronic myocardial infarction. These observations suggest that enhanced agonist-induced vasoconstriction during heart failure may reflect a loss of nitric oxide-mediated modulation of arteriolar tone.

Characterization of sodium-calcium exchange in rabbit renal arterioles.

Experiments were performed to test the hypothesis that renal arterioles exhibit Na-Ca exchange capability and that this process is regulated by protein kinase C (PKC). Glomeruli with attached arterioles were dissected from rabbit kidney and loaded with fura-2 for measurement of intracellular calcium concentration ([Ca2+]i) using microscope-based photometry. In tissue bathed in Ringer's solution containing 150 mM Na+ and 1.5 mM Ca2+, afferent and efferent arteriolar [Ca2+]i averaged 136 +/- 6 and 154 +/- 7 nM, respectively. Removal of extracellular Na+ increased afferent arteriolar [Ca2+]i by 70 +/- 7 mM, while efferent arteriolar [Ca2+]i only increased by 39 +/- 5 nM (P < 0.01 vs. afferent arteriole). These responses were inhibited by 6 nM Ni2+ and required extracellular Ca2+, but were unaffected by 10 microM diltiazem. After incubation in 500 microM ouabain, 5 microM monensin, and 5 microM nigericin, [Ca2+]i responses to removal of extracellular Na+ were exaggerated significantly, averaging 174 +/- 50 nM in afferent arterioles and 222 +/- 82 nM in efferent arterioles (NS vs. afferent arterioles). Moreover, responses to removal of extracellular Na+ were enhanced by 100 nM phorbol 12-myristate 13-acetate, an affect which was blocked by PKC inhibition (25 nM K252b). These data indicate that both afferent and efferent arterioles express the Na-Ca exchanger, and that PKC activity impacts on exchange capacity in these vessels.

Functional impairment of renal afferent arteriolar voltage-gated calcium channels in rats with diabetes mellitus.

Experiments were performed to test the hypothesis that diabetes mellitus is associated with impaired afferent arteriolar responsiveness to opening of voltage-gated calcium channels. Diabetes was induced by injection of streptozocin (65 mg/kg, i.v.) and insulin was administered via an osmotic minipump to achieve moderate hyperglycemia. Sham rats received vehicle treatments. 2 wk later, the in vitro blood-perfused juxtamedullary nephron technique was used to allow videomicroscopic measurement of afferent arteriolar contractile responses to increasing bath concentrations of either Bay K 8644 or K+. Baseline afferent arteriolar diameter in kidneys from diabetic rats (26.4+/-1.2 microm) exceeded that of Sham rats (19.7+/-1.0 microm). Bay K 8644 evoked concentration-dependent reductions in afferent diameter in both groups of kidneys; however, arterioles from Sham rats responded to 1 nM Bay K 8644 while 100 nM Bay K 8644 was required to contract arterioles from diabetic rats. The EC50 for K+-induced reductions in afferent arteriolar diameter was greater in diabetic kidneys (40+/-4 mM) than in kidneys from Sham rats (28+/-4 mM; P < 0.05). In afferent arterioles isolated by microdissection from Sham rats and loaded with fura 2, increasing bath [K+] from 5 to 40 mM evoked a 98+/-12 nM increase in intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i responses to 40 mM K+ were suppressed in afferent arterioles from diabetic rats (delta = 63+/-5 nM), but were normalized by decreasing bath glucose concentration from 20 to 5 mM. These observations indicate that the early stage of insulin-dependent diabetes mellitus is associated with a functional defect in afferent arteriolar L-type calcium channels, an effect which may contribute to suppressed afferent arteriolar vasoconstrictor responsiveness and promote glomerular hyperfiltration.

Neural influences on renal responses to acute volume expansion in rats with heart failure.

Experiments were performed to test the postulate that neural influences underlie the suppressed excretory response to acute volume expansion (VE) typically observed 3-4 wk after myocardial infarction to induce chronic heart failure (CHF). Responses to VE were assessed in innervated (intact) and denervated (DNX) kidneys of anesthetized CHF rats and sham-operated controls. CHF rats exhibited blunted natriuretic responses to VE in both intact kidneys (35% of sham response) and DNX kidneys (55% of sham DNX response). CHF rats also displayed suppressed excretory responses to atrial natriuretic factor (0.25 microgram.kg-1.min-1 iv) in both intact kidneys (74% of sham response) and DNX kidneys (63% of sham DNX response). Additional experiments confirmed that the compliance of the venoatrial junction did not differ between sham rats (52 +/- 2 mmHg/microliter) and CHF rats (54-2 mmHg/microliter). The observations support the contention that both tonic renal sympathetic renal nerve activity and suppressed renal atrial natriuretic factor responsiveness likely contribute to the blunted excretory response to VE during CHF.

Basal nitric oxide production curtails arteriolar vasoconstrictor responses to ANG II in rat kidney.

Experiments were performed to test the hypothesis that renal arteriolar vasoconstrictor responses to angiotensin II (ANG II) are curtailed through a mechanism that involves stimulation of endogenous nitric oxide (NO) synthesis. The in vitro blood-perfused juxtamedullary nephron technique was exploited to monitor arteriolar lumen diameter responses to exogenous ANG II before and during treatment with the NO synthase inhibitor N omega-nitro-L-arginine (L-NNA). Under control conditions, 1 nM ANG II reduced afferent and efferent arteriolar diameters by 13 and 11%, respectively. In the presence of L-NNA, 1 nM ANG II decreased afferent diameter by 26% and efferent diameter by 35%. This augmentation could not be attributed to the L-NNA-induced decrease in baseline diameter. L-NNA also augmented vasopressin responses, indicating a lack of agonist specificity in this interaction. ANG II reactivity during L-NNA treatment was not enhanced when tissue NO activity was fixed at basal levels (exposure to 1 microM sodium nitroprusside). These results indicate that endogenous NO modulates the vasoconstrictive impact of ANG II on both afferent and efferent arterioles of juxtamedullary nephrons and that this process does not require stimulation of NO synthesis.

Control of the renal microcirculation: cellular and integrative perspectives.

Recent studies targeting ionic signalling events have revealed that considerable segmental heterogeneity exists between pre- and post-glomerular vascular smooth muscle cells, and that even preglomerular vascular smooth muscle cells are not a homogeneous population. Preglomerular branch points enriched with calcium channels may be critical for initiating contractile responses, with subsequent propagation along the vascular wall, and may also facilitate nephron-nephron coupling events. These processes ultimately promote coordinated network behaviour and can underlie development of synergistic vasoactive responses, such as those necessary to provide efficient autoregulation of blood flow and glomerular filtration rate.

Contribution of tubuloglomerular feedback to renal arteriolar angiotensin II responsiveness.

The purpose of the present study was to test the hypothesis that a component of the afferent arteriolar vasoconstrictor response to angiotensin II (Ang II) requires an intact tubuloglomerular feedback (TGF) mechanism. Enalaprilat-treated male Sprague-Dawley rats served as tissue donors for study of renal microvascular function using the in vitro blood-perfused juxtamedullary nephron technique. Arteriolar lumen diameter responses to exogenous Ang II were determined before and after TGF blockade (papillectomy or 50 microM furosemide). Before TGF blockage, 10 nM Ang II significantly reduced diameters of both mid-afferent (53 +/- 5%) and efferent (43 +/- 9%) arterioles. TGF blockade did not alter baseline diameter of either arteriole, but significantly blunted the mid-afferent vasoconstriction evoked by 10 nM Ang II (44 +/- 7% inhibition by papillectomy; 43 +/- 10% inhibition by furosemide). Similar behavior was observed at afferent arteriolar sites near the glomerulus; however, efferent arteriolar Ang II responsiveness was not altered by papillectomy. The impact of TGF blockade on afferent arteriolar Ang II responsiveness was most prominent at high peptide concentrations (10 nM), while not significantly influencing the response to 1 nM Ang II. In contrast, the afferent vasoconstrictor effect of norepinephrine was unaffected by papillectomy. These data indicate that the vasoconstrictor influence of exogenous Ang II on afferent, but not efferent, arterioles of intact juxtamedullary nephrons includes both TGF-dependent and TGF-independent components.

Impact of cyclo-oxygenase blockade on juxtamedullary microvascular responses to angiotensin II in rat kidney.

1. Experiments were designed to evaluate the hypothesis that cyclo-oxygenase products modulate the influence of angiotensin II (AII) on the renal juxtamedullary microvasculature of enalaprilat-treated rats. 2. The in vitro blood-perfused juxtamedullary nephron technique was utilized to provide access to afferent arterioles, efferent arterioles and descending vasa recta located in the outer stripe of the outer medulla. 3. Baseline afferent arteriolar diameter was 20.8 +/- 1.9 microns in kidneys subjected to cyclo-oxygenase blockade (1 mumol/L piroxicam), a value significantly lower than that observed in untreated kidneys (26.1 +/- 1.0 microns). Baseline diameters of efferent arterioles and outer medullary descending vasa recta did not differ between untreated and piroxicam-treated groups. 4. Topical application of 1 nmol/L AII reduced blood flow through outer medullary descending vasa recta by 22 +/- 6% in untreated kidneys and by 24 +/- 7% in piroxicam-treated kidneys. 5. In untreated kidneys, AII (0.01-100 nmol/L) produced concentration-dependent afferent and efferent arteriolar constrictor responses of similar magnitudes. Neither afferent nor efferent arteriolar AII responsiveness was significantly altered in piroxicam-treated kidneys, although afferent responses exceeded efferent responses at AII concentrations > or = 10 nmol/L. 6. We conclude that endogenous cyclo-oxygenase products exert a vasodilator influence on juxtamedullary afferent arterioles under baseline conditions. Although cyclo-oxygenase inhibition had little effect on juxtamedullary microvascular responses to AII, the response to high AII concentrations may be modulated by cyclo-oxygenase products in a manner which delicately alters the relative influence of the peptide on pre- vs postglomerular resistances.