Structural Predictors of Lung Function Decline in Young Smokers with Normal Spirometry.

Rationale: Chronic obstructive pulmonary disease (COPD) due to tobacco smoking commonly presents when extensive lung damage has occurred. Objectives: We hypothesized that structural change would be detected early in the natural history of COPD and would relate to loss of lung function with time. Methods: We recruited 431 current smokers (median age, 39 yr; 16 pack-years smoked) and recorded symptoms using the COPD Assessment Test (CAT), spirometry, and quantitative thoracic computed tomography (QCT) scans at study entry. These scan results were compared with those from 67 never-smoking control subjects. Three hundred sixty-eight participants were followed every six months with measurement of postbronchodilator spirometry for a median of 32 months. The rate of FEV1 decline, adjusted for current smoking status, age, and sex, was related to the initial QCT appearances and symptoms, measured using the CAT. Measurements and Main Results: There were no material differences in demography or subjective CT appearances between the young smokers and control subjects, but 55.7% of the former had CAT scores greater than 10, and 24.2% reported chronic bronchitis. QCT assessments of disease probability-defined functional small airway disease, ground-glass opacification, bronchovascular prominence, and ratio of small blood vessel volume to total pulmonary vessel volume were increased compared with control subjects and were all associated with a faster FEV1 decline, as was a higher CAT score. Conclusions: Radiological abnormalities on CT are already established in young smokers with normal lung function and are associated with FEV1 loss independently of the impact of symptoms. Structural abnormalities are present early in the natural history of COPD and are markers of disease progression. Clinical trial registered with www.clinicaltrials.gov (NCT03480347).

Chaos and non-linear phenomena in renal vascular control.

Renal autoregulation of blood flow depends on the functions of the tubuloglomerular feedback (TGF) system and the myogenic response of the afferent arteriole. Studies of the dynamic aspects of these control mechanisms at the level of both the single nephron and the whole kidney have revealed a variety of non-linear phenomena. In halothane-anesthetized, normotensive rats the TGF system oscillates regularly at 2-3 cycles/min because of the non-linearities and the time delays within the feedback system. Oscillations are present in single nephron blood flow, tubular pressure and flow, and in the tubular solute concentrations. Nephrons deriving their afferent arteriole from the same cortical radial artery are entrained, and consequently oscillate at the same frequency. Experimental studies have shown that the synchronization is due to an interaction of the TGF between nephrons. A necessary condition for the interaction is that the nephrons derive their blood supply from the same cortical radial artery. Development of hypertension is associated with a shift from periodic oscillations of tubular pressure to random-like fluctuations. Numerical analyses indicate that these fluctuations are an example of deterministic chaos. Experimental studies show that the development of hypertension is associated with an increase in strength of the interaction between nephrons. Mathematical models suggest that an increased nephron-nephron interaction could cause a bifurcation in the dynamics of TGF from periodic oscillations to deterministic chaos. In addition to the TGF mediated oscillation, experimental studies have also demonstrated the presence of a faster oscillation, this having a frequency of 120-160 mHz. This is caused by a mechanism intrinsic to the vascular wall, and presumably represents the well-known phenomenon of vasomotion. Using newly developed non-linear analytical methods non-linear interactions between vasomotion and the TGF mediated oscillation were detected both in single nephron and in whole kidney blood flow. The physiological significance of these non-linear phenomena in renal vascular control is discussed.

Glucose tolerance and insulin action in rats with renovascular hypertension.

To test whether hypertension can cause hyperinsulinemia or insulin resistance, we performed intravenous glucose tolerance tests at 1 month and euglycemic clamps at 3 months after induction of two-kidney, one clip renovascular hypertension in rats. At 1 month, systolic pressure was higher in 21 clipped than in 12 control animals (161 +/- 5 mm Hg, range 134-187 mm Hg versus 119 +/- 3 mm Hg, range 108-146 mm Hg; p less than 0.001). Glucose tolerance, assessed as the glucose fractional disappearance rate between 3 and 11 minutes after the glucose injection, was similar in the clipped and sham groups (0.059 +/- 0.002 versus 0.056 +/- 0.002 min-1, respectively; p greater than 0.4). The total area under the insulin curve during glucose tolerance tests was also similar in the clipped and sham groups (926 +/- 95 versus 869 +/- 126 microunits/ml x min; p greater than 0.4). There was no significant relation between systolic blood pressure and insulin area during glucose tolerance tests in the clipped group, but there was a positive rectilinear relation in the control group (r = 0.66; p = 0.01). Fourteen animals had euglycemic clamps 2 months after glucose tolerance tests. At that time, systolic pressure (direct femoral measurement) was higher in the seven clipped animals (189 +/- 13 mm Hg versus 122 +/- 5 mm Hg in controls; p less than 0.001). Insulin infusions of 1 and 4 milliunits/min/kg body wt effected similar plasma insulin levels in the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypotensive and renal effects of an extract of the edible mushroom Pleurotus sajor-caju.

An aqueous extract of Pleurotus sajor-caju was found to have a hypotensive effect in rats. Intravenous infusion of the extract into rats caused a decrease of the mean systemic blood pressure in a dose dependent manner. A typical dose of 25 mg of the extract decreased the mean systemic blood pressure from 110 mm Hg to 70 mm Hg. The systolic and diastolic pressure changed proportionally with minimal alteration in heart rate. The hypotensive effect of the extract was not due to its major electrolyte content because a solution reconstituted with the same electrolyte composition had a transient pressor effect rather than lowering the blood pressure. The same extract was also found to affect renal hemodynamics such that it caused a decrease in the glomerular filtration rate by more than 50% after 120 minutes. The effect did not seem to be mediated through changes in systemic blood pressure.

A laser Doppler instrument for in vivo measurements of blood flow in single renal arterioles.

A laser Doppler instrument has been developed to measure the blood flow in single vessels for the study of the dynamics of local control mechanisms. A commercial blood perfusion monitor, designed to measure blood perfusion in a vascular field containing many randomly oriented blood vessels, was modified to perform measurements of blood flow in a single arteriole. In vitro tests of the instrument revealed that the relationship between blood flow and Doppler shift was not a simple linear function. Causes of nonlinearity are revealed and proper use of the device avoids the problem. The device was applied to efferent arterioles that are visible on the surface of the rat kidney. An angiotensin converting enzyme inhibitor and graded doses of angiotensin II were used to perturb kidney blood flow. The induced changes in whole kidney blood flow, measured with an electromagnetic flow probe, and in single efferent arteriolar blood flow, measured with the new instrument, were correlated. An oscillation at approximately 0.035 Hz, previously described in the tubular pressure and attributed to a local feedback mechanism acting on arteriolar resistance, was found in the arteriolar blood flow. The new instrument is easy to use and provides temporal resolution not available with more conventional methods used for flow measurement in the microcirculation.

Time-varying properties of renal autoregulatory mechanisms.

In order to assess the possible time-varying properties of renal autoregulation, time-frequency and time-scaling methods were applied to renal blood flow under broad-band forced arterial blood pressure fluctuations and single-nephron renal blood flow with spontaneous oscillations obtained from normotensive (Sprague-Dawley, Wistar, and Long-Evans) rats, and spontaneously hypertensive rats. Time-frequency analyses of normotensive and hypertensive blood flow data obtained from either the whole kidney or the single-nephron show that indeed both the myogenic and tubuloglomerular feedback (TGF) mechanisms have time-varying characteristics. Furthermore, we utilized the Renyi entropy to measure the complexity of blood-flow dynamics in the time-frequency plane in an effort to discern differences between normotensive and hypertensive recordings. We found a clear difference in Renyi entropy between normotensive and hypertensive blood flow recordings at the whole kidney level for both forced (p < 0.037) and spontaneous arterial pressure fluctuations (p < 0.033), and at the single-nephron level (p < 0.008). Especially at the single-nephron level, the mean Renyi entropy is significantly larger for hypertensive than normotensive rats, suggesting more complex dynamics in the hypertensive condition. To further evaluate whether or not the separation of dynamics between normotensive and hypertensive rats is found in the prescribed frequency ranges of the myogenic and TGF mechanisms, we employed multiresolution wavelet transform. Our analysis revealed that exclusively over scale ranges corresponding to the frequency intervals of the myogenic and TGF mechanisms, the widths of the blood flow wavelet coefficients fall into disjoint sets for normotensive and hypertensive rats. The separation of the scales at the myogenic and TGF frequency ranges is distinct and obtained with 100% accuracy. However, this observation remains valid only for the whole kidney blood pressure/flow data. The results suggest that understanding of the time-varying properties of the two mechanisms is required for a complete description of renal autoregulation.

[Ca2+]i in rat afferent arteriole during constriction measured with confocal fluorescence microscopy.

Using confocal fluorescence microscopy, we followed the time course of intracellular calcium concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC) and endothelial cells (EC) of microperfused afferent arterioles isolated from rat juxtamedullary nephrons. Measurements were made while arterioles were exposed to pharmacological agonists or myogenic stimulation. Luminal addition of acetylcholine triggered an immediate increase of [Ca2+]i in EC and vasodilation. Addition of norepinephrine to the bath solution constricted the vessel and initiated periodic oscillations of [Ca2+]i in VSMC and synchronized vasomotion. Increase of perfusion pressure from 80 to 120 mmHg induced an immediate 9.6 +/- 2.9% (P < 0.05, n = 9) increase of [Ca2+]i in VSMC that was sustained. The arteriole dilated transiently when the perfusion pressure was increased, and persistent vasoconstriction to reduced diameter was observed after 35 s. When nitric oxide (NO) production in the perfused vessel was blocked with nitro-L-arginine methyl ester prior to the pressure step, similar profiles of change in VMSC [Ca2+]i were observed and persistent vasoconstriction began after 28 s. The same pressure step triggered an increase of [Ca2+]i in EC by 11.3 +/- 1.1% (P < 0.05, n = 5). These observations suggest that myogenic constriction of afferent arterioles was associated with an increase of [Ca2+]i in VSMC, and the constriction was delayed by endogenous NO production.

An Arg-Gly-Asp peptide stimulates constriction in rat afferent arteriole.

The potential role of integrins in the myogenic mechanism was studied in the rat afferent arteriole (AA) by fluorescence immunolocalization and microperfusion of isolated AA. Confocal fluorescence images were acquired from frozen sections of rat kidney after indirect immunostaining for various integrin beta- and alpha-subunits. The beta 1-, beta 3-, alpha 3-, alpha 5-, and alpha V-integrins were found on the plasma membrane in smooth muscle of AA, providing the morphological basis for participation of integrins in mechanotransduction. With 1 mM nitro-L-arginine methyl ester (L-NAME) in the luminal perfusate to inhibit endogenous nitric oxide (NO) production from AA, the hexapeptide GRGDSP (10(-7)-10(-3)M) induced immediate vasoconstriction. The constriction was dose dependent and specific or peptides with arginine-glycine-aspartic acid (RGD) motifs, commonly found on the binding sites of extracellular matrix to integrins. In controls, the hexapeptide GRGESP induced no constriction. GRGDSP, 1 mM, induced a 21.6 +/- 2.6% decrease (P < 0.05, n = 6) in lumen diameter for 30 s and an 18.3 +/- 4.1% increase (P < 0.05, n = 6) in smooth muscle intracellular calcium concentration for 18 s, as measured by the emission ratio of Fluo-3/Fura Red. Binding of exogenous RGD motifs with exposed integrins on AA smooth muscle therefore triggers calcium-dependent vasoconstriction. However, the dose response to RGD was not sensitive to the myogenic tone of the vessel, which suggests that the integrin-mediated vasoconstriction is different from myogenic constriction.

NH3 permeability of principal cells and intercalated cells measured by confocal fluorescence imaging.

The cortical collecting duct (CCD) is an important site for NH3 secretion in mammalian nephron. However, given the cellular heterogeneity of this epithelium, the transcellular sites for NH3 secretion are unknown. In the present study, a dual-excitation confocal microscope was designed and optimized to have sufficient temporal resolution to measure the permeability of ammonia (PNH3) across the basolateral and apical membrane of principal cells (PCs) and intercalated cells (ICs) in perfused rabbit CCDs. The rate of cellular NH3 influx was calculated from the time course of increase in intracellular pH (pHi), measured with 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein after 20 mM NH4Cl was added to the bath or luminal perfusate. The time course of increase in pHi was calculated from 488/442 image pairs stored at a rate of 4 Hz. The apparent basolateral and apical PNH3 values of PCs were 36 +/- 5 and 113 +/- 11 microns/s, respectively. The values were 5.0 +/- 0.7 and 34 +/- 3 microns/s after membrane folding correction. The apparent basolateral and apical PNH3 values of ICs were 38 +/- 6 and 132 +/- 15 microns/s. Corrected for membrane folding, the values were 9.0 +/- 1.0 and 47 +/- 5 microns/s, respectively. The results demonstrate that the apical surface was more permeable than the basolateral surface in both cell types. In addition, ICs were more permeable to NH3 than PCs across both membranes. The transcellular PNH3 of PCs and ICs were 27.3 and 29.5 microns/s, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of apical Na(+)/H(+) exchanger activity inhibition in proximal tubules induced by acute hypertension.

We previously showed that acute arterial hypertension induces an inhibition of fluid and NaCl reabsorption in proximal tubules of Sprague-Dawley rats, which is associated with a rapid reversible internalization of apical Na(+)/H(+) exchanger in brush border. To determine whether there is a corresponding inhibition of apical Na(+)/H(+) exchanger activity in proximal tubules to account for the reduced tubular reabsorption, an instrument capable of measuring intracellular pH (pH(i)) ratiometrically and repeatedly on the surface of kidney with high temporal resolution is required. We report the design and validation of such a fluorimetric system based on two ultraviolet nitrogen-pulsed lasers and a photomultiplier. pH(i) of proximal tubules in situ was measured with pH-sensitive fluorescence dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein at 5 Hz. Using the initial rate of change of pH(i) (dpH(i)/dt) during luminal Na(+) removal as an index of apical Na(+)/H(+) exchanger activity, the exchanger activity was found to be reduced by 52 +/- 11% (n = 14, P < 0.05) compared with the baseline after 20 min of induced acute hypertension. The inhibition of Na(+)/H(+) exchange activity was alleviated when the blood pressure was returned to prehypertensive level. These observations indicate that acute changes in arterial pressure can reversibly inhibit apical Na(+)/H(+) exchanger activity, which might contribute to pressure natriuresis in proximal tubule.

Mechanisms of temporal variation in single-nephron blood flow in rats.

Modified laser-Doppler velocimetry was used to determine the number of different mechanisms regulating single-nephron blood flow. Two oscillations were identified in star vessel blood flow, one at 20-50 mHz and another at 100-200 mHz. Tubuloglomerular feedback (TGF) mediates the slower oscillation, and the faster one is probably myogenic in origin. Acute hypertension increased autospectral power in the 20-50 mHz and 100-200 mHz frequency bands to 282 +/- 50 and 248 +/- 64%, respectively, of control even though mean single-nephron blood flow was autoregulated. Mean blood flow increased 24.6 +/- 6.1% when TGF was inhibited by intratubular perfusion with furosemide, and it decreased 42.8 +/- 3.9% when TGF was saturated by tubular perfusion with artificial tubular fluid at high rates. Autospectral power in the low-frequency band decreased 50.5 +/- 9.6% during furosemide and decreased 74.9 +/- 5.9% during TGF saturation, consistent with a TGF origin of the slow oscillation. In contrast, autospectral power of the high-frequency oscillation increased 75.4 +/- 23.9% during TGF inhibition and decreased 35.8 +/- 11% when TGF was saturated, suggesting interactions between the two spontaneously oscillating components in efferent arteriole blood flow.

Chaos in blood flow control in genetic and renovascular hypertensive rats.

Hydrostatic pressure and flow in renal proximal tubules oscillate at 30-40 mHz in normotensive rats anesthetized with halothane. The oscillations originate in tubuloglomerular feedback, a mechanism that provides local blood flow regulation. Instead of oscillations, spontaneously hypertensive rats (SHR) have aperiodic tubular pressure fluctuations; the pattern is suggestive of deterministic chaos. Normal rats made hypertensive by clipping one renal artery had similar aperiodic tubular pressure fluctuations in the unclipped kidney, and the fraction of rats with irregular fluctuations increased with time after the application of the renal artery clip. Statistical measures of deterministic chaos were applied to tubular pressure data. The correlation dimension, a measure of the dimension of the phase space attractor generating the time series, indicated the presence of a low-dimension strange attractor, and the largest Lyapunov exponent, a measure of the rate of divergence in phase space, was positive, indicating sensitivity to initial conditions. These time series therefore satisfy two criteria of deterministic chaos. The measures were the same in SHR as in rats with renovascular hypertension. Since two different models of hypertension displayed similar dynamics, we suggest that chaotic behavior is a common feature of renal vascular control in the natural history of the disease.

Dynamics of TGF-initiated nephron-nephron interactions in normotensive rats and SHR.

Proximal tubular pressure, glomerular filtration rate, and early distal tubule Cl- oscillate at 35 mHz in normotensive rats because of tubuloglomerular feedback (TGF); the oscillation bifurcates to chaos in spontaneously hypertensive rats (SHR). To examine the importance of TGF-initiated vascular interactions between nephrons in these dynamics, we measured tubular pressure simultaneously in two or more nephrons. The oscillations were synchronized in nephrons supplied by a common cortical radial artery. The correlation coefficient of pressure records from coupled nephrons was 0.86 +/- 0.02. Intratubular furosemide perfusion diminished the oscillation in both the perfused and the coupled nephron; total autospectral power in each of the nephrons and cross-spectral power were reduced to 45% of control. The correlation between noncoupled nephrons was not significant, and intratubular furosemide perfused in one nephron had no effect on adjacent but noncoupled nephrons. In SHR, the correlation coefficient of tubular pressure records was high from coupled nephrons only; furosemide diminished the autospectral power of pressure fluctuations to approximately 60-75% of control in both perfused and coupled nephrons, and cross-spectral power was affected by a similar amount. Nephron-nephron interactions, specific to vascular connectivity, persist in SHR and appear to be stronger than in normotensive rats.

Magnitude of TGF-initiated nephron-nephron interactions is increased in SHR.

We compared the tubuloglomerular feedback (TGF)-initiated nephron-nephron interaction in spontaneously hypertensive rats (SHR) and normotensive Sprague-Dawley (SD) rats. Interaction strength was assessed by measuring stop-flow pressure (delta SFP) responses in pairs of nephrons, where only one nephron of the pair was microperfused. The vascular connection was determined from casts of the nephrons and vessels; length of arteriolar separation between the two glomeruli was measured on the casts. When microperfusion rate was increased from 5 to 50 nl/min, delta SFP in perfused nephrons was 10.6 +/- 0.6 and 10.2 +/- 0.7 mmHg [not significant (NS)] in SD and SHR, respectively. In the matched unperfused nephrons from the same cortical radial artery, delta SFP was 1.3 +/- 0.2 and 2.9 +/- 0.7 mmHg (P < 0.05) in SD and SHR. When the perfused and unperfused nephron originated from different cortical radial arteries, delta SFP in the unperfused nephrons was -0.1 +/- 0.2 and 0.0 +/- 0.3 mmHg (NS) in SD and SHR, respectively. In both strains, interaction strength varied inversely with glomerular separation. When the dependence on distance was taken into account, interaction strength was about threefold greater in SHR than in SD. We conclude that the nephron-nephron interaction is significantly greater in SHR. The dependence of interaction strength on distance separating the glomeruli suggests that the interaction is propagated along the preglomerular vasculature.

Redistribution of Na+/H+ exchanger isoform NHE3 in proximal tubules induced by acute and chronic hypertension.

Redistribution of apical Na+/H+ exchangers (NHE) in the proximal tubules as a plausible mechanism of pressure natriuresis was investigated with confocal immunofluorescence microscopy in Sprague-Dawley rats (SD), spontaneously hypertensive rats (SHR), and two-kidney, one-clip Goldblatt hypertensive rats (GH). NHE isoform NHE3 was localized in the brush border of proximal tubules in SD. Twenty minutes of induced acute hypertension (20-40 mmHg) resulted in a pronounced redistribution of isoform NHE3 from the brush border into the base of microvilli, where clathrin-coated pits were localized. Prehypertensive young SHR (5 wk old, mean blood pressure 105 +/- 3 mmHg, n = 11) produced similar findings. However, NHE3 was found to concentrate in the base of microvilli in adult SHR (12 wk old, mean blood pressure 134 +/- 6 mmHg, n = 12) and nonclipped kidneys of GH (mean blood pressure 131 +/- 6 mmHg, n = 6). In clipped kidneys of GH, which were not exposed to the hypertension because of the arterial clips, NHE3 was localized on the brush border as in normal SD. No further redistribution of NHE3 was detected in adult SHR or GH when acute hypertension was induced. Since both acute and chronic increase of arterial pressure can provoke the redistribution of apical NHE in proximal tubules, the pressure-induced NHE redistribution could be a physiological response and an integral part of pressure natriuresis.

Integrin mobilizes intracellular Ca(2+) in renal vascular smooth muscle cells.

Peptides with the Arg-Gly-Asp (RGD) motif induce vasoconstriction in rat afferent arterioles by increasing the intracellular Ca(2+) concentration ([Ca(2+)](i)) in vascular smooth muscle cells (VSMC). This finding suggests that occupancy of integrins on the plasma membrane of VSMC might affect vascular tone. The purpose of this study was to determine whether occupancy of integrins by exogenous RGD peptides initiates intracellular Ca(2+) signaling in cultured renal VSMC. When smooth muscle cells were exposed to 0.1 mM hexapeptide GRGDSP, [Ca(2+)](i) rapidly increased from 91 +/- 4 to 287 +/- 37 nM and then returned to the baseline within 20 s (P < 0.05, 34 cells/5 coverslips). In controls, the hexapeptide GRGESP did not trigger Ca(2+) mobilization. Local application of the GRGDSP induced a regional increase of cytoplasmic [Ca(2+)](i), which propagated as Ca(2+) waves traveling across the cell and induced a rapid elevation of nuclear [Ca(2+)](i). Spontaneous recurrence of smaller-amplitude Ca(2+) waves were found in 20% of cells examined after the initial response to RGD-containing peptides. Blocking dihydropyridine-sensitive Ca(2+) channels with nifedipine or removal of extracellular Ca(2+) did not inhibit the RGD-induced Ca(2+) mobilization. However, pretreatment of 20 microM ryanodine completely eliminated the RGD-induced Ca(2+) mobilization. Anti-beta(1) and anti-beta(3)-integrin antibodies with functional blocking capability simulate the effects of GRGDSP in [Ca(2+)](i). Incubation with anti-beta(1)- or beta(3)-integrin antibodies inhibited the increase in [Ca(2+)](i) induced by GRGDSP. We conclude that exogenous RGD-containing peptides induce release of Ca(2+) from ryanodine-sensitive Ca(2+) stores in renal VSMC via integrins, which can trigger cytoplasmic Ca(2+) waves propagating throughout the cell.