Grazia Favret

New risk factors for atherosclerosis in hypertension: focus on the prothrombotic state and lipoprotein(a).

Although adequate control of blood pressure is of basic importance in cardiovascular prevention in hypertensive patients, correction of additional risk factors is an integral part of their management. In addition to classical risk factors, epidemiological research has identified a number of other conditions that might significantly contribute to cardiovascular risk in the general population and might achieve specific relevance in patients with high blood pressure. In fact, more than 20% of patients with premature cardiovascular events do not have any of the traditional risk factors and, although effective intervention on blood pressure and additional risk factors has significantly reduced cardiovascular morbidity and mortality, the contribution to stroke, coronary artery disease and renal failure is still unacceptably high. Evaluation of new risk factors may further expand our capacity to predict atherothrombotic events when these factors are included along with the traditional ones in the assessment of global cardiovascular risk in hypertensive patients. Because it could be anticipated that the role of these novel factors will become increasingly evident in the future, researchers with an interest in hypertension and physicians dealing with problems related to cardiovascular prevention should give them appropriate consideration. This review summarizes the basic biology and clinical evidence of two emerging risk factors that are reciprocally related and contribute to the development and progression of organ damage in hypertension: the prothrombotic state and lipoprotein(a).

Effect of Endothelin 1 on Proximal Reabsorption and Tubuloglomerular Feedback

Endothelin 1 (ET–1) is a powerful constrictor of the afferent glomerular artery, implicated in the occurrence of both functional and acute renal failure. Besides being produced by the endothelium, ET–1 is also secreted by proximal tubular cells, suggesting that it may act as an endoluminal messenger. The present study is intended to verify whether ET–1 may play a role in the tubuloglomerular feedback system. The experiments were performed in rat superficial glomeruli. In 25 nephrons we measured by the total collection technique the single–nephron glomerular filtration rate (SNGFR; nl/min) and reabsorption rates before (control) and during microinjection (MIJ) of ET–1 10–7 M into the first proximal convolution or Bowman’s space. The SNGFR rose from 27±3 to 61±11 nl/min (p<0.01), the percent proximal reabsorption rose from 43 to 74%, and the absolute reabsorption rose from 13±2 to 46±11 nl/min (p<0.01). In additional 23 nephrons the collections were performed at the earliest distal convolution accessible on the renal surface, while MIJ was performed in the last proximal convolution of the same nephrons. The SNGFR rose during MIJ from 22±3 to 40±6 nl/min (p<0.01), the percent reabsorption rose from 61 to 66% (p>0.77), and the absolute reabsorption rose from 12±2 to 26±4 nl/min, (p<0.003). Exposure of the macula densa to intraluminally injected ET–1 causes an abrupt increase in SNGFR of the experimental nephron, in the absence of changes in systemic and renal hemodynamics. During proximal MIJ, ET–1 may have reached the macula densa during the time preceding the beginning of collection and interruption of the delivery of fluid to the distal nephron. ET–1 directly stimulates fractional and absolute volume reabsorption along the proximal tubule. Proximal secretion and/or filtration of ET–1 could represent a physiological mechanism to activate the tubuloglomerular feedback, eliciting a response opposite to that triggered by systemic and intrarenal infusion.

The effect of intraluminal flow rate on glomerulotubular balance in the proximal tubule of the rat kidney

To differentiate intraluminal from peritubular factors in mediating the glomerulotubular balance, single nephron filtration rate (SNGFR) and reabsorption were measured by total collection from the last proximal segment before, and during, externally applied mechanical compression of an early proximal convolution of ninety‐three rat nephrons. SNGFR fell from 55.6 +/− 2.3 nl min‐1 during control conditions to 34.5 +/− 2.0 nl min‐1 during compression (P < 0.0001) in sixty tubules (‘responders’). Absolute reabsorption fell from 41.8 +/− 2.0 to 28.4 +/− 1.8 nl min‐1 (P < 0.0001), while percentage reabsorption rose from 75 +/− 2 to 82 +/𠈒 2% (P < 0.0001). These effects were reversible and independent of whether the compression was applied before or after the control collection. In thirty‐three proximal tubules the compression procedure was not successful (‘non‐responders’), SNGFR remaining unchanged (36.1 +/− 2.9 vs. 36.9 +/− 2.9 nl min‐1, P > 0.3). Absolute and percentage reabsorptions rose slightly, albeit significantly, from 26.1 +/− 2.1 to 30.7 +/− 2.4 nl min‐1 (P < 0.0001) and from 75 +/− 3 to 85 +/− 2% (P < 0.0001). Thus, the response of reabsorption to changes in intraluminal flow is composed of two different adaptive mechanisms. The slowing of flow is present in both sets of tubules and causes a slight increase in resorption; the change in filtration per se is present only in the first set of responders, and causes an SNGFR‐dependent consensual change in resorption.

Proximal reabsorption with changing tubular fluid inflow in rat nephrons

The relative contribution of intraluminal versus peritubular factors in mediating glomerulo‐tubular balance (GTB) is still controversial. We modulated the load of tubular fluid to the proximal tubule of single nephrons of rats by injecting oil into the efferent arteries (EAO). In fifty nephrons the changes in reabsorption induced by obstruction occurred in the same direction as, and were significantly correlated with, the simultaneous changes in single nephron glomerular filtration rate (SNGFR) (y = ‐0.54 + 0.92x, R = 0.91, P < 0.0001). In an additional set of thirty‐nine nephrons the load of tubular fluid was changed, during EAO, by partial collection from Bowmans space or from the early proximal convolution. Thus, the rate of tubular fluid delivery along the proximal tubule was changed in an experimental situation that prevented any modification in the oncotic pressure of peritubular capillaries. The changes in proximal deliveries during this experimental condition were significantly correlated with those during reabsorption (y = ‐2.87 + 0.71x, R = 0.82, P < 0.0001). These data demonstrate that GTB is fully expressed even when the native peritubular environment is kept constant while the rate of perfusion of proximal tubular segments with native tubular fluid is changed.

MICROINJECTION STUDIES ON THE EFFECT OF FUROSEMIDE ON THE RAT NEPHRON

The tubular site of furosemide (F) action was studied by the technique of diuretic microinjection (MIJ) into proximal tubules of the rat nephron. F was injected into the last proximal superficial loop of 51 proximal tubules, at the concentration of 3 x 10(-4) mol/l in an infusate that contained 14C-inulin. Collections were performed at the early distal tubule before and after MIJ. Single nephron filtration rate (SNGFR) remained unchanged, while the percent of filtered volume reabsorbed up to the site of collection was 85+/-2 before, 79+/-2% after MIJ, p < 0.005. The calculated concentration of F in the collected distal tubular fluid during the post-MIJ measurements averaged 3 x 10(-5) mol/l. This experiment was repeated by injecting F into the early proximal convolutions of 43 nephrons, while the collections were performed at the last proximal segments. In these studies of proximal volume absorption, SNGFR was 34+/-3 before, 35+/-4 nl/min after F (p > 0.6). The respective percent reabsorptions were 70+/-3 and 73+/-3% (p +/- 0.3). In order to determine whether the technique per se was suitable to detect changes in reabsorption, the proximal MIJ study was repeated by using the carbonic anhydrase inhibitor dichlorphenamide 3 x 10(-5) mol/l in the microinjectate: while SNGFR remained unchanged, percent reabsorption fell from 63+/-5 to 45+/-7% during injection of the diuretic, p < 0.03. We conclude that the technique is adequate to examine the effects of drugs, and that F does not reduce proximal volume absorption at concentrations of 3 x 10(-5) mol/l. The loop diuretic decreases distal volume absorption by abolishing the osmotic gradient between blood and tubular fluid along the early distal convoluted tubule.

Micropuncture Study of the Effect of Furosemide on Proximal and Distal Tubules of the Rat Nephron

The tubular effects of furosemide were studied by micropuncture and clearance techniques on 20 rats. Collections of tubular fluid (TF) from early distal (ED) and late proximal (LP) segments of the same nephrons and of different nephrons were performed during baseline conditions. Re-collections were taken from the same sites and new collections from different nephrons after 10 mg/kg furosemide. The glomerular filtration rate (GFR) was 1,309 +/- 212 microliters/min during baseline, and 1,348 +/- 199 microliters/min after furosemide (p > 0.89); while the urine flow rate rose from 36 +/- 8 to 167 +/- 30 microliters/min (p < 0.001). The nephron filtration rate (NFR) was not different in 46 paired distal (33.3 +/- 2.6 nl/min) versus proximal samples (34.2 +/- 2.9 nl/min, p > 0.72), neither was it different during baseline (37.2 +/- 1.4, n = 120) as compared to furosemide (37.2 +/- 2.7, n = 91, p > 0.99). The percent reabsorption (PR) at the ED sampling site was 87 +/- 4% during baseline, and 89 +/- 3% in 13 paired samples during furosemide (p > 0.47). PR at the LP sampling sites was 83 +/- 2% during baseline, and 80 +/- 2% in 26 paired samples during furosemide (p > 0.63). In 31 paired ED-LP collections, PR was 82 +/- 4 (ED) versus 72 +/- 4% (LP) during baseline, and 87 +/- 3 versus 74 +/- 6%, respectively, during furosemide. The respective collection rates were 4.6 +/- 1.0 versus 9.5 +/- 1.3 nl/min during baseline (p < 0.0001), 5.8 +/- 2.3 versus 8.7 +/- 3.0 nl/min during furosemide. The LP-ED differences obtained during baseline were not different from those measured during furosemide for the collection rate, PR and NFRs. The absolute LP resorption rate was not significantly different during baseline as compared to furosemide. Thus, furosemide did not affect the difference between ED and LP collection sites in collection rate, absolute and fractional reabsorption, in the absence of changes in GFR and NFR. These data indicate that furosemide acts solely along Henles loop, where it blocks Na+ transport. The urine flow rate rises during furosemide because water abstraction along the distal tubule is reduced by the isotonicity of ED TF, and along the collecting ducts by the isotonicity of the medullary and papillary interstitium caused by the diuretic. We conclude that under the conditions of the present study, furosemide has no proximal effect.

Dual effect of secretin on nephron filtration and proximal reabsorption depending on the route of administration

Secretin is a vasoactive peptide capable of acting on transmembrane volume fluxes. We measured nephron filtration (SNGFR) and resorption during secretin microinjection (MIJ) into the tubular lumen or microperfusion (MP) into peritubular capillaries. In 24 rat nephrons, SNGFR, measured by collections from the distal tubule, rose from 25+/- 4 to 61+/-8 nl/min during MIJ of saline containing secretin 10(-9) M into the last convolution of the proximal tubule (LP). Percent and absolute resorptions rose from 70 to 90% and from 20+/-4 to 56+/-8 nl/min, respectively. During MIJ of secretin, 3 x 10(-)(9) M into the first convolution of the proximal tubule, SNGFR, measured at LP, rose from 32+/-4 to 61+/-8 nl/min, percent and absolute reabsorptions from 52+/-4 to 78+/-3% and from 16+/-2 to 50+/-7 nl/min, respectively (n = 30). During MP of secretin, 1.5x10(-9) M, SNGFR fell from 39+/-6 to 15+/-4, resorption from 19+/-4 to 9+/-2 nl/min, while percent resorption rose from 43+/-6 to 59+/-5% (n = 15). While all MIJ and MP changes were significant (P<0.001), paired pre- versus post-MIJ and MP values were not. Secretin is a powerful vasoconstrictor when perfused into peritubular capillary blood, unlike systemic and intra-arterial injections. When injected into the tubular lumen, it up-regulates SNGFR and increases reabsorption directly.

Systemic and intratubular effects of cyclosporin-A and tacrolimus on the rat kidney

Cyclosporin-A and tacrolimus can cause hypertension and renal failure through endothelin receptors. The importance of tubular function was never investigated. The aim of this study was to compare the effects of intratubular injection of cyclosporin-A and tacrolimus with effects observed during systemic infusion. In 20 rats, either cyclosporin-A or tacrolimus was infused, 30 and 1 mg/kg i.v., respectively, in 30 min. Before and after administration, glomerular filtration rate, single nephron filtration rate, proximal and distal absolute reabsorption and percent reabsorption were measured by clearance and micropuncture techniques. In 22 other rats, single nephron filtration rate, absolute reabsorption, percent reabsorption, were measured at the last proximal and early distal tubules before and during intraluminal microinjection of either cyclosporin-A or tacrolimus. During cyclosporin-A and tacrolimus i.v. infusion, glomerular filtration rate fell from 536+/-43 to 448+/-37 microl/min (P<0.026) and from 408+/-33 to 284+/-81 microl/min (P<0. 02), single nephron filtration rate from 26.4+/-2.0 to 20.6+/-1.9 (P<0.002) and from 21.6+/-2.2 to 17.4+/-2.0 nl/min, respectively (P<0.02). The last proximal absolute reabsorption remained unchanged with cyclosporin-A (16.8+/-2.2 vs. 15.1+/-1.7 nl/min, P>0.444), but was slightly reduced by tacrolimus (14.4+/-1.7 vs. 11.3+/-1.7 nl/min, P<0.05). During microinjection, single nephron filtration rate was increased by cyclosporin-A (20+/-1 vs. 63+/-8 nl/min, P<0.0001), and tacrolimus (from 17+/-2 to 49+/-9 nl/min, P<0.0001), and so was reabsorption, independent of the sampling site. Cyclosporin-A and tacrolimus, indeed, raise single nephron filtration rate directly when injected intraluminally. Since this effect occurs in the direction opposite to that recorded during systemic infusion, it must be mediated through different pathways. The i.v. infusion of cyclosporin-A, but not tacrolimus, impairs glomerulo-tubular balance.

The validity of the recollection technique in micropuncture experiments on the rat kidney

We tested the accuracy of the micropuncture technique by performing total collections of tubular fluid followed by immediate recollections from the same site. We studied thirty‐one Wistar rats under different conditions of hydration and during maintenance infusions. To assure constancy of body fluid volumes the experiments were performed during continuous reinfusion of urine. Overall we performed 190 paired collections and immediate recollections from 147 proximal and 43 distal tubules. The mean values measured during the first collection were not different from the paired means obtained by recollection. Mean values (+/‐ S.E.M.) for first collection and recollection were: single nephron glomerular filtration rate (SNGFR), 34.7 +/− 1.3 vs. 34.2 +/− 1.3 nl min‐1, P > 0.42; percentage reabsorption, 74 +/− 1 vs. 73 +/− 1, P > 0.53; absolute reabsorption, 26.4 +/− 1.2 vs. 25.9 +/− 1.2 nl min‐1, P > 0.47; collection rate, 8.4 +/− 0.6 vs. 8.3 +/− 0.4 nl min‐1, P > 0.92. When subdivided according to different physiological conditions and sampling sites, the results for the paired collection‐recollection means were still not significantly different. The regression between collected and recollected SNGFRs had a slope of 0.96, r = 0.85, P < 0.0001. In order to exclude the possibility that urine reinfusion per se could obscure putative differences in collection‐recollection pairs, the effect of urine reinfusion was separately evaluated. We measured the differences obtained in collection‐recollection pairs before and during urine reinfusion in twenty‐eight tubules from eleven rats, a sample size that allows the detection of 10% difference as significant. While SNGFR did not change, percentage reabsorption fell significantly from 76 +/− 3% before infusion to 60 +/− 3% during reinfusion, P < 0.003. We conclude that the recollection technique yields a reproducible estimate of SNGFR and tubular reabsorption, independent of the sampling site and of the technique used for fluid maintenance. Thus, it can be used to study the effect of different manoeuvres on the rate of proximal tubular transport. Urine infusion per se depresses proximal transport compared with that measured during maintenance with equivalent amounts of isotonic saline.

The effect of reducing proximal tubular fluid delivery on the rate of filtration of single nephrons

The rate of delivery of tubular fluid from the proximal tubule (PT) is thought to reset nephron filtration rate (SNGFR). In micropuncture experiments in rats we tested this hypothesis by reducing the efflux from the PT by simultaneously “double collecting” (DC) tubular fluid from the early distal tubule (DT) and from the last convolution of the PT of the same nephrons. SNGFR measured by total collection of tubular fluid was 34±3 nl/min at the DT and 34±3 nl/min at the PT (p>0.97, n=42). The simultaneous collection from proximal and distal sampling site was performed between these two paired measurements. It yielded an average SNGFR of 40±3 nl/min (p<0.02). This may be due to the collection, at the distal site, of the extra amount of inulin stored between distal and proximal pipette, prior to starting the aspiration of tubular fluid. Since this error would decrease in longer collections, the difference in SNGFR between single and double collections was plotted against the duration of collections. In fact it was negatively correlated with the sampling time (p<0.01), indicating no difference in SNGFRs for collections >4 minutes. Reduction and complete interruption of the delivery of native proximal tubular fluid to the Macula Densa does not seem to influence the measurement of SNGFR. Filtration rate is not significantly different when measured within few minutes at the DT and PT of the same nephrons.