Increased microvascular permeability in mice lacking Epac1 (Rapgef3).

AIM: Maintenance of the blood and extracellular volume requires tight control of endothelial macromolecule permeability, which is regulated by cAMP signalling. This study probes the role of the cAMP mediators rap guanine nucleotide exchange factor 3 and 4 (Epac1 and Epac2) for in vivo control of microvascular macromolecule permeability under basal conditions. METHODS: Epac1-/- and Epac2-/- C57BL/6J mice were produced and compared with wild-type mice for transvascular flux of radio-labelled albumin in skin, adipose tissue, intestine, heart and skeletal muscle. The transvascular leakage was also studied by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) using the MRI contrast agent Gadomer-17 as probe. RESULTS: Epac1-/- mice had constitutively increased transvascular macromolecule transport, indicating Epac1-dependent restriction of baseline permeability. In addition, Epac1-/- mice showed little or no enhancement of vascular permeability in response to atrial natriuretic peptide (ANP), whether probed with labelled albumin or Gadomer-17. Epac2-/- and wild-type mice had similar basal and ANP-stimulated clearances. Ultrastructure analysis revealed that Epac1-/- microvascular interendothelial junctions had constitutively less junctional complex. CONCLUSION: Epac1 exerts a tonic inhibition of in vivo basal microvascular permeability. The loss of this tonic action increases baseline permeability, presumably by reducing the interendothelial permeability resistance. Part of the action of ANP to increase permeability in wild-type microvessels may involve inhibition of the basal Epac1-dependent activity.

Image-based assessment of microvascular function and structure in collagen XV- and XVIII-deficient mice.

Collagen XV and XVIII are ubiquitous constituents of basement membranes. We aimed to study the physiological roles of these two components of the permeability barrier non-invasively in striated muscle in mice deficient in collagen XV or XVIII by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Structural information was obtained with transmission electron microscopy (TEM). MR data were analysed by two different analysis methods to quantify tissue perfusion and microcirculatory exchange parameters to rule out data analysis method-dependent results. Control mice (C57BL/6J Ola/Hsd strain) or mice lacking either collagen XV (Col15a1(-/-)) or XVIII (Col18a1(-/-)) were included in the study. MR images were acquired using a preclinical system using gadodiamide (Gd-DTPA-BMA, molecular weight 0.58 kDa) as a tracer. Exchange capacity (permeability (P)-surface area (S) product relative to blood flow (FB)) was increased in test mice compared to controls, but the contributions from P, S, and FB were different in these two phenotypes. FB was significantly increased in Col18a1(-/-), but slightly decreased in Col15a1(-/-). PS was significantly increased only in Col18a1(-/-) even though P was increased in both phenotypes suggesting S might also be reduced in Col15a1(-/-) mice. Immunohistochemistry and electron microscopy demonstrated alterations in capillary density and morphology in both knockout mouse strains in comparison to the control mice. Both collagen XV and XVIII are important for maintaining normal capillary permeability in the striated muscle. DCE-MRI and the perfusion analyses successfully determined microvascular haemodynamic parameters of genetically modified mice and gave results consistent with more invasive methods.

Increased interstitial protein because of impaired lymph drainage does not induce fibrosis and inflammation in lymphedema.

OBJECTIVE: The pathophysiology of lymphedema is incompletely understood. We asked how transcapillary fluid balance parameters and lymph flow are affected in a transgenic mouse model of primary lymphedema, which due to an inhibition of vascular endothelial growth factor receptor-3 (VEGFR-3) signaling lacks dermal lymphatics, and whether protein accumulation in the interstitium occurring in lymphedema results in inflammation. METHODS AND RESULTS: As estimated using a new optical-imaging technique, we found that this signaling defect resulted in lymph drainage in hind limb skin of K14-VEGFR-3-Ig mice that was 34% of the corresponding value in wild-type. The interstitial fluid pressure and tissue fluid volumes were significantly increased in the areas of visible swelling only, whereas the colloid osmotic pressure in plasma, and thus the colloid osmotic pressure gradient, was reduced compared to wild-type mice. An acute volume load resulted in an exaggerated interstitial fluid pressure response in transgenic mice. There was no accumulation of collagen or lipid in skin, suggesting that chronic edema presented in the K14-VEGFR-3-Ig mouse was not sufficient to induce changes in tissue composition. Proinflammatory cytokines (interleukin-2, interleukin-6, interleukin-12) in subcutaneous interstitial fluid and macrophage infiltration in skin of the paw were lower, whereas the monocyte/macrophage cell fraction in blood and spleen was higher in transgenic compared with wild-type mice. CONCLUSIONS: Our data suggest that a high interstitial protein concentration and longstanding edema is not sufficient to induce fibrosis and inflammation characteristic for the human condition and may have implications for our understanding of the pathophysiology of this condition.

High-salt diet increases hormonal sensitivity in skin pre-capillary resistance vessels.

AIMS: Recent data indicate that the skin of rats on a high-salt diet is able to accumulate Na(+) without commensurate water. This extrarenal mechanism of Na(+) homoeostasis could affect skin vasoregulation. We hypothesized that the major resistance vessel of rat skin, the pre-capillary arterioles, has increased vasoreactivity within the physiological range of circulating ANG II, a hormone relevant to salt-sensitive hypertension. METHODS AND RESULTS: Skin arterioles from skin and muscle were isolated using the agar-infusion technique. Vessels from rats fed high-salt and low-salt diet had similar lumen diameter and media area/lumen area ratio. Contractile sensitivity to ANG II was increased in skin vessels from high-salt vessels at all doses tested starting at 10(-10) m (P < 0.01). Pre-capillary arterioles from muscle displayed similar contractions to ANG II, independent of the diet. As ANG II and the renin-angiotensin system are strongly involved in salt conservation, we explored whether vasoreactivity for noradrenaline was increased as well, because this is a functionally unrelated hormone. At low doses, contractions were similar, but at 10(-5) and 10(-4) m, noradrenaline produced stronger contractions in skin vessels from high-salt compared with low-salt rats (P < 0.01). CONCLUSIONS: Our data demonstrate significantly increased hormonal vasoreactivity of skin vessels from rats on a high-salt diet, which could increase peripheral resistance in many situations and contribute to higher pressure in salt-sensitive hypertension. As vessels from adjacent muscle were unaffected, we raise the interesting possibility that increased vasoreactivity in the skin could be linked to osmotically inactive Na(+) accumulation.

Isolation of interstitial fluid in skin during volume expansion: evaluation of a method in pigs.

The ability to isolate interstitial fluid (IF) from skin would make it possible to study the microcirculation and proteins in this environment both during normal and pathophysiological conditions. Traditional IF sampling using implanted wicks suffer from low volumes with risk of contamination by local inflammatory, intracellular, and vascular proteins. To sample larger volumes of true IF, a recently described tissue centrifugation method was compared with dry and wet wicks from porcine skin under normal conditions and following volume expansion. With all three methods, volume expansion caused a significant lowering of interstitial colloid osmotic pressure as expected, and the fluid was similar to plasma when compared using size-exclusion HPLC. The centrifugation method was superior with respect to isolating larger amounts of true IF for further studies. Mass spectrometry of IF sampled with centrifugation showed that most of the proteins reflected the major plasma proteins with some tissue-specific proteins like decorin, gelsolin, and orosomucoid-1. Lumican, pigment epithelium-derived factor, and fatty acid-binding protein 4 were only identified in IF after volume expansion, possibly reflecting a local response to increased fluid filtration. Tissue centrifugation to collect IF from skin should be applicable to both clinical and experimental studies on IF balance during different pathophysiological conditions and interventions.

Localization of tubular uptake segment of filtered Cystatin C and Aprotinin in the rat kidney.

AIM: The renal tubular uptake of 125I-Aprotinin (*Ap) is on average located more superficially than its filtration site, causing transfer of some of *Ap filtered in deep to more superficial cortical zones. 125I-Cystatin C (*Cy) showed less uptake in deep cortical zones than Ap, suggesting a longer and/or a more superficial tubular uptake site. To test that hypothesis and to quantify the outward transfer of the filtered polypeptides, we estimated the tubular uptake pattern of the tracers in perfusion fixed rat kidneys after intravenous injection of *Cy and *Ap. METHODS: Autoradiographs were made from 10 mum thick slices of Microfil nephron casts from outer (OC), middle (MC) and inner (IC) cortical zones to quantify cortical border-crossing *Ap transfer. Single nephron glomerular filtration rate (snGFR) was estimated as the zonal uptake of *Ap corrected for *Ap transfer, divided by its time-integrated plasma concentration and the zonal number of glomeruli. RESULTS: *Ap and *Cy uptake fell exponentially along the proximal convoluted tubule (PCT), indicating an uptake proportional to luminal concentration. Uptake in IC exceeded that in MC and OC nephrons. The per cent PCT length with *Cy uptake (67.2 +/- 1.6) exceeded that of *Ap (54.6 +/- 1.8). The zonal border-crossing PCT length (29-34% of total PCT) from deep to more superficial cortical zones transferred 4-6% more *Cy than *Ap. CONCLUSION: Greater tubular uptake length of *Cy than of *Ap causes more cortical border-crossing of *Cy. The zonal snGFR estimated from Aprotinin uptake corrected for border-crossing agreed well with that obtained with the Hanssen ferrocyanide technique.

The effect of AVP-V receptor stimulation on local GFR in the rat kidney.

AIM: Arginine vasopressin (AVP) might influence urinary concentration ability by altering the intrarenal distribution of glomerular filtration rate (GFR). METHODS: To study this possibility we have measured the intracortical distribution of GFR following acute AVP-V1 receptor stimulation in anaesthetized female Sprague-Dawley (SPD) rats during euvolemia and water diuresis by the aprotinin method, allowing two consecutive measurements of zonal GFR in the same kidney. RESULTS: Acute i.v. bolus injection of 50 ng V1 receptor agonist ([Phe2, Ile3, Orn8]-vasopressin) followed by a continuous infusion of 5 ng min(-1) in euvolemic rats reduced GFR by 25% in outer cortex (OC), 20% in middle cortex (MC) and 19% in inner cortex (IC) relative to vehicle infusion (all P < 0.05). In water diuretic rats V1 receptor agonist reduced GFR by 22% in OC, 10% in MC and 11% in IC relative to vehicle infusion (P < 0.05). GFR decreased slightly more in OC than in MC and IC in both euvolemic and water diuretic rats (P < 0.05) indicating a distribution of GFR towards MC and IC. Acute infusion of the selective non-peptide V1 receptor antagonist OPC-21268 in euvolemic rats reduced GFR by 14% in OC, 13% in MC and 11% in IC relative to vehicle infusion (P < 0.05), with no significant difference between the layers. CONCLUSIONS: The change in distribution of GFR not only between OC and IC, but also between OC and MC suggests that the afferent/efferent arterioles and not the medullary vasa recta is the main site of resistance change. We conclude that acute i.v. infusion of V1 receptor agonist in high doses reduces GFR more in superficial than in deep cortex in both euvolemic and water diuretic rats and that this may be of some importance for water conservation, adding to the V2- receptor effect on water permeability of the collecting ducts.

Drainage of plasma proteins from the renal medullary interstitium in rats.

1. Lymph vessels are scarce or lacking in the renal inner medulla, raising the question of whether plasma proteins entering the medullary interstitium are removed by diffusion through the interstitium to lymphatics in the outer medulla or cortex, or by convection into the vasa recta. 2. Using micropipettes, we infused 125I-albumin into the papilla of anaesthetized rats and watched its disappearance from the injection site as well as the uptake in the thoracic duct and plasma. 3. Tracer infused into the renal cortex appeared almost immediately in the thoracic duct lymph, and rose to a sevenfold higher concentration than in plasma, whereas tracer infused into the papilla appeared first and increased more sharply in plasma than in the lymph. No spread from the papillary injection site was observed. Tracer injected in renal hilar lymphatics was quantitatively recovered in the thoracic duct. 4. The plasma concentration pattern following papillary infusion was similar to that obtained by intravenous injection, indicating uptake in blood and subsequent distribution to extracellular fluid and lymph from all organs. 5. We conclude that plasma proteins normally diffusing out from the vasa recta are brought back through water flux (1) from the collecting ducts due to the high sodium chloride concentration in the papillary interstitium and (2) from the interstitium into the vasa recta driven by plasma protein osmotic pressure. Accordingly, there is no need for lymph vessels in the inner medulla.

Distribution spaces for hyaluronan and albumin in rat tail tendons.

A low concentration of hyaluronan (HA) in lymph compared with tissue suggests a large bound fraction. To investigate the distribution and mobility of HA and serum albumin (Alb), we eluted the rat tail tendon with a series of l5 successive centrifugations, each preceded by the addition of 0.15 M NaCl (15% of initial wet wt). The eluate concentration fell exponentially versus the accumulated eluate, allowing estimation of the maximal elutable amount (E(HA) and E(Alb)). Alb elution was practically complete from a space of approximately 28% of wet wt at all centrifugation rates. Twenty percent of HA was elutable at 500 rpm, apparently from the same space as Alb, increasing to 40% at >4,000 rpm. This pattern was not significantly influenced by using 2 M NaCl or by the addition of plasma or metabolic inhibitors. Without prehydration and centrifugation at high revolutions per minute, both Alb and HA concentrations fell rapidly toward zero, presumably in part reflecting mobilization of HA- and Alb-free fluid from the collagen intrafibrillar space (3). We conclude that with prehydration the fibrils swell, increasing the intramolecular spaces to become "penetrable" to HA and allowing removal of HA-containing fluid when the fibrils are compressed by the next centrifugation at high revolutions per minute, increasing E(HA) from 23 to 45%. Chemical binding presumably explains the unelutable 55% of tendon HA. Intrafibrillar HA may act to stabilize the fibrillar volume.

Renal hemodynamics in young and old spontaneously hypertensive rats during intrarenal infusion of arginine vasopressin.

BACKGROUND/AIMS: To gain insight into the effect of arginine vasopressin (AVP) on renal hemodynamics in hypertensive rats, we investigated the vasoconstrictive response to AVP on total renal blood flow (RBF) and total and zonal glomerular filtration rate (GFR) in young and old spontaneously hypertensive rats (SHR). A hypothesis of increased AVP sensitivity in the juxtamedullary cortex of SHR was tested. METHODS: Total RBF and total and zonal GFR were studied in 10- and 40-week-old SHR and normotensive Wistar-Kyoto rats (WKY). RBF was recorded by a flowmeter before infusion of AVP and immediately after injection of a bolus dose of 10 ng AVP. Whole kidney GFR and its intracortical distribution was measured by the tubular uptake of 125I- and 131I-labelled aprotinin before and during a continuous infusion of AVP 5 ng/min. Ligand binding measurements of preglomerular V1a receptors were performed in young and old rats. RESULTS: RBF decreased by 43 +/- 3% in 10-week SHR (9.2 +/- 0.5 vs. 5.2 +/- 0.3 ml x min(-1) x g(-1)), significantly more than 10-week WKY where RBF decreased by 35 +/- 3% (9.6 +/- 0.7 vs. 6.5 +/- 0.5 ml x min(-1) x g(-1)) (p < 0.05). The effect of AVP on RBF was attenuated in 40-week-old rats where the decline in RBF was 29 +/- 5% in SHR and 23 +/- 4% in WKY (p > 0.05). GFR decreased by 6 +/- 3% (1.03 +/- 0.04 vs. 0.96 +/- 0.04 ml x min(-1) x g(-1), p < 0.05) in 10-week SHR and was unchanged in 10-week WKY (1.10 +/- 0.07 vs. 1.08 +/- 0.04 ml x min(-1) x g(-1), p > 0.10). GFR decreased by 11 +/- 10% in 40-week SHR and by 4 +/- 4% in 40-week WKY (p > 0.05). AVP infusion significantly increased filtration fraction in all groups except 40-week SHR, indicating that AVP has the strongest vasoconstrictive effect on postglomerular vessels. The intrarenal distribution of GFR was unchanged in the normotensive and hypertensive groups. V1a receptor density was upregulated in young SHR compared to young WKY (p < 0.05), but downregulated in old compared to young SHR (p = 0.05). CONCLUSION: The results indicate that AVP sensitivity is not increased in the juxtamedullary cortex in SHR and the reduced vasoconstrictive effect in old SHR is due to a reduced density of V1a receptors.

Interstitial exclusion of positively and negatively charged IgG in rat skin and muscle.

Volume exclusion, i.e., the space not available for a specific probe, may be dependent on the probe charge. Therefore, interstitial exclusion was measured for positively and negatively charged immunoglobulin (IgG) in skin and muscle of rats by using a continuous infusion method (30). Steady-state concentration of (125)I-labeled IgG 1 (pI = 8.7) and (131)I- labeled IgG 4 (pI = 6.6) was maintained by infusion of tracer for 120-168 h with an implanted osmotic pump. At the end of the infusion period and before tissue sampling, the rat was anesthetized and nephrectomized, and (51)Cr-labeled EDTA was injected and allowed 4 h for equilibration to measure interstitial fluid volume (V(i)). Interstitial fluid was isolated from skin and muscle by using nylon wicks implanted post mortem. The relative IgG available space was measured as the ratio between labeled IgG and (51)Cr-labeled EDTA wick fluid equivalent spaces, and relative excluded volume fraction (V(e)/V(i)) was calculated as 1--V(a)/V(i). V(e)/V(i) in hindlimb skin averaged 0.37 +/- 0.05 (SE) and 0.65 +/- 0.06 (P < 0.01) for IgG 1 and 4, respectively, with corresponding figures of 0.24 +/- 0.05 and 0.51 +/- 0.04 (P < 0.01) in hindlimb muscle (n = 9 for both tissues). These experiments suggest that fixed negative charges, most likely glycosaminoglycans, influence distribution of macromolecules in the interstitium and therefore affect interstitial fluid balance.

Lactoferrin and anti-lactoferrin antibodies: effects of ironloading of lactoferrin on albumin extravasation in different tissues in rats.

Lactoferrin is a cationic iron-binding protein, which is released from activated neutrophils in concert with reactive oxygen species. In vitro, lactoferrin has both anti- and proinflammatory effects; many of them dependent on iron-binding. In vivo, only iron-free lactoferrin reduced inflammatory hyperpermeability in the lung. We therefore examined whether 1 mg iron-free (Apo-Lf) or iron-saturated lactoferrin (Holo-Lf) alone or followed by anti-lactoferrin antibodies (aLf) affected permeability evaluated by extravasation of radiolabelled bovine serum albumin (CBSA) in different tissues of anaesthetized rats. Fifteen minutes after i.v. injection of Lf, aLf or saline was given and circulatory arrest was induced 20 min thereafter. Measurements were performed in control, after Apo-Lf, Holo-Lf, Apo-Lf + aLf, Holo-Lf + aLf and aLf alone (n=6-8 in each group). No intergroup differences were found for plasma volume and haematocrit at the start and end of the 37 min extravasation period or for total tissue water in any of the six different tissues studied, excluding larger transcapillary fluid shifts. However, increases in CBSA were seen without differences in tissue intravascular volume. Iron-free lactoferrin and aLf alone did not change CBSA significantly. Iron-saturated lactoferrin significantly increased CBSA in skin (neck), trachea and left ventricle of the heart to 249 +/- 9, 284 +/- 16 and 160 +/- 7% of control, respectively. When followed by aLf, both Apo- and Holo-Lf increased CBSA significantly in four and five of the tissues studied, respectively. However, no significant effect was seen for Holo-Lf + aLf compared with Holo-Lf alone. In conclusion, iron-saturated, but not iron-free lactoferrin increased CBSA, whereas antilactoferrin increased CBSA compared with lactoferrin alone only when following iron-free lactoferrin.

Interstitial fluid pressure, composition of interstitium, and interstitial exclusion of albumin in hypothyroid rats.

Lack of thyroid hormones may affect the composition and structure of the interstitium. Hypothyrosis was induced in rats by thyroidectomy 4-12 wk before the experiments. In hypothyroid rats (n = 16), interstitial fluid pressure measured with micropipettes in hindlimb skin and muscle averaged +0.1 +/- 0.2 and +0.5 +/- 0.2 mmHg, respectively, with corresponding pressures in control rats (n = 16) of -1.5 +/- 0.1 (P < 0.001) and -0.8 +/- 0.1 mmHg (P < 0.001). Interstitial fluid volume, measured as the difference between the distribution volumes of (51)Cr-EDTA and (125)I-labeled BSA, was similar or lower in skin and higher in hypothyroid muscle. Total protein and albumin concentration in plasma and interstitial fluid (isolated from implanted wicks) was lower in hypothyroid compared with control rats. Hyaluronan content (n = 9) in rat hindlimb skin was 2.05 +/- 0.15 and 1.92 +/- 0.09 mg/g dry wt (P > 0.05) in hypothyroid and control rats, respectively, with corresponding content in hindlimb skeletal muscle of 0.35 +/- 0.07 and 0.23 +/- 0. 01 mg/g dry wt (P < 0.01). Interstitial exclusion of albumin in skin and muscle was measured after (125)I-labeled rat serum albumin infusion for 120-168 h with an implanted osmotic pump. Relative excluded volume for albumin (V(e)/V(i)) was calculated as 1 - V(a)/V(i), and averaged 28 and 28% in hindlimb muscle (P > 0.05), 44 and 45% in hindlimb skin (P > 0.05), and 19 and 32% in back skin (P < 0.05) in hypothyroid and control rats, respectively. Albumin mass was higher in back skin in spite of a lower interstitial fluid albumin concentration, a finding explained by a reduced V(e)/V(i) in back skin in hypothyroid rats. These experiments suggest that lack of thyroid hormones in rats changes the interstitial matrix again leading to reduced interstitial compliance and changes in the transcapillary fluid balance.

The effect of AVP-V2 receptor stimulation on local GFR in the rat kidney.

The effect of AVP-V2 receptor agonist desmopressin, dDAVP, its non-peptide antagonist OPC-31260 and vehicle infusion on glomerular filtration rate (GFR) in the outer, middle and inner cortex was studied in both hydropenic and water diuretic Inactin anaesthetized female Sprague-Dawley rats using the aprotinin method. Two subsequent GFR measurements were carried out in the same kidney by injection of 125I- and 131I-labelled aprotinin before and after i.v. infusion of dDAVP, OPC-31260 or the vehicle. Acute infusion of dDAVP in hydropenic rats increased total GFR by 14% relative to vehicle infusion, whereas in water diuretic rats it had no effect relative to vehicle. No significant changes in arterial pressure (Pa) or renal blood flow (RBF) were recorded. Infusion of OPC-31260 reduced total GFR by 11% compared with vehicle. These results are consistent with the findings that a presensitization of the vasculature by high plasma levels of AVP is necessary for the renal vascular effects mediated by the V2 or V2-like receptors to occur. The ratio between inner and outer cortex GFR remained unchanged from control to experimental condition as follows: dDAVP infusion in hydropenic rats, 0.504 vs. 0.494 in control; vehicle infusion in hydropenic rats, 0. 393 vs. 0.392; OPC-31260 infusion in hydropenic rats, 0.517 vs. 0. 523; dDAVP in water diuretic rats, 0.547 vs. 0.543; vehicle in water diuretic rats, 0.413 vs. 0.417. Thus no significant difference in the GFR response was observed between superficial and deep cortical layers of the rat kidney.

Interstitial exclusion of macromolecules studied by graded centrifugation of rat tail tendon.

Mechanical compression of cartilage and tendon has been shown to expel fluid both from collagen fibrils and from the extrafibrillar space. As reported previously, albumin (Alb) concentration and colloid osmotic pressure in tendon fluid (TF) expelled by repeated centrifugations fell progressively at increasing centrifugation force (G = 600, 2,400, and 13,100), suggesting either molecular sieving in compressed tendon or mobilization of protein-free (excluded) fluid. The present experiments, including analysis of 51Cr-EDTA, aprotinin (Ap), Alb, immunoglobulin G (IgG), and hyaluronan (hyaluronic acid; HA) with molecular weight (MW) ranging from 341 to 5 x 10(6), strongly favored the exclusion hypothesis; the fraction of Alb, IgG, and HA-free fluid (excluded) was already 0.23-0.36 in the first centrifugate, increasing to 0.73-0.82 in the third. The corresponding numbers were, respectively, 0.11 and 0.43 for Ap (MW 6,500), and 0 and 0.08 for 51Cr-EDTA. These data, combined with calculated exclusion by collagen fibrils, proteoglycans, and HA, indicated that the first centrifugate was mainly derived from the extrafibrillar space, with increasing addition of macromolecular free intrafibrillar fluid in the second and third centrifugates, with each space contributing about equally to the total centrifugate volume. The calculations also indicated that Alb-, IgG-, and Ap-free fluid was mobilized from extrafibrillar space by increasing overlap of excluded territories. An excess of HA in tendon compared with that estimated from centrifugate concentrations suggests a large bound or immobilized HA fraction.

Renal handling of radiolabelled human cystatin C in the rat.

Serum cystatin C concentration correlates negatively with glomerular filtration rate as well as or better than that of serum creatinine, suggesting a constant formation, and elimination from extracellular fluid mainly by glomerular filtration. It is not known, however, how well the renal plasma clearance of this 13-kDa basic polypeptide matches the glomerular filtration rate. This was investigated in rats during control conditions and after reduced renal perfusion pressure. 125I-cystatin C and an indicator for glomerular filtration (51Cr-EDTA or 131I-aprotinin) were injected intravenously. The renal accumulation and urinary excretion of the tracers were recorded in periods of 2.5 to 20.0 min. The renal plasma clearance of 125I-cystatin C (Ccy) based on the renal content of 125I correlated well with the glomerular filtration rate (CCr-EDTA) in periods up to 6 min; i.e. Ccy = 0.94 x CCr-EDTA, r = 0.99. Less than 0.5% of the filtered amount appeared in the urine. During more prolonged periods, Ccy increasingly underestimated glomerular filtration rate, reaching about 0.4 x CCr-EDTA in a 20-min period. Free 125I relative to total plasma 125I activity increased from about 2% at 5 min to about 70% at 20 min. In nephrectomized rats, free 125I accumulated in plasma at a slower rate, accounting for about 15% of the total activity 20 min after injection of 125I-cystatin C. We conclude that cystatin C is (a) mainly removed from the extracellular fluid by the kidneys, (b) practically freely filtered in the glomeruli, and (c) completely absorbed and rapidly broken down by the proximal tubular cells.

Effect of furosemide on local and zonal glomerular filtration rate in the rat kidney.

Furosemide has been reported to produce disproportional changes in blood flow in cortical zones and to inhibit tubuloglomerular feedback (TGF), suggesting that furosemide might alter the intracortical distribution of glomerular filtrate. We have tested this hypothesis by a new method for measuring local and total glomerular filtration rate (GFR) based on proximal tubular accumulation of the basic polypeptide aprotinin (mol wt 6513). Local GFR was calculated in tissue samples dissected from outer cortex (OC), inner cortex (IC) and the corticomedullary border zone (CM) from the plasma clearances of two aprotinin tracers injected i.v. before and after a 3 min i.v. infusion of 25 mg kg-1 furosemide. The mean of five samples from each region was used to determine zonal GFR. Isotonic saline was infused at a rate corresponding to urine flow. Furosemide reduced whole kidney GFR from 1.17 to 1.00 mL min-1 and gave a similar reduction of renal artery blood flow. Urine flow increased from 0.6 to 17% of GFR. Haematocrit (approximately 0.48) and plasma protein concentration (approximately 55 mg mL-1) were maintained while the arterial blood pressure tended to decline (118 +/- 5 mmHg to 108 +/- 6 mmHg, P < 0.05). GFR in OC, IC and CM (1.58, 1.18, 0.42 mL min-1 g-1) fell to 87, 88 and 88% of control after furosemide infusion respectively. The furosemide/control ratio for each sample showed a coefficient of variation of about 3%. We conclude that furosemide produced a modest GFR reduction that was uniform throughout the renal cortex. The homogenous GFR response suggests a similar TGF constriction tone in preglomerular vessels of deep and superficial nephrons.

Glomerular filtration and tubular absorption of the basic polypeptide aprotinin.

The basic polypeptide aprotinin (Ap), mol. wt 6500, pI 10.5, is filtered in the glomeruli, virtually completely taken up by the proximal tubular cells and retained there for many hours. This process was studied in rats by determining the renal plasma clearance (CAp) as the amount of [125I]Ap accumulated in the kidney plus that excreted in the urine per unit of time divided by the integrated plasma concentration. In periods lasting 4-20 min after i.v. bolus injection or infusion to constant plasma concentration, CAp was 65% of glomerular filtration rate (GFR) estimated as kidney plus urinary clearance of [51Cr]EDTA (Ccr-EDTA). Less than 0.8% of the filtered Ap appeared in the urine. CAp varied inversely with plasma protein concentration in mg ml-1: CAp/Ccr-EDTA = 0.98-0.0058 x Ppr, corresponding to a glomerular Gibbs-Donnan distribution for a net molecular charge of +6, in agreement with the amino acid composition of Ap. CAp (kidney + urinary) was not altered by inhibiting tubular uptake of [125I]Ap by maleate or by exceeding the uptake capacity with large doses of unlabelled Ap. Neutralized Ap (malonylated) did not accumulate in the kidney, but showed a urinary clearance indistinguishable from that of [51Cr]EDTA. Both CAp and Ccr-EDTA were reduced to 0.04 ml min-1 when glomerular filtration pressure was lowered by ureteral stasis and increased Ppr (80-90 mg ml-1). These findings indicate: (1) no steric or charge restriction to filtration of Ap in the glomerular membrane, (2) the Gibbs-Donnan equilibrium should be considered when estimating glomerular sieving of charged polypeptides in intact animals (3) charge dependent tubular uptake, (4) little or no transtubular transport of intact Ap, (5) no appreciable tubular uptake of Ap from the peritubular side and (6) local renal accumulation of Ap in a period of up to 20 min may be used to estimate local glomerular filtration and/or local proximal tubular reabsorption rates. Model analysis based on the appearance of 125I in plasma, the time course of renal Ap content, and literature data on subcellular Ap distribution are consistent with two populations of endosomes, transporting Ap at widely different rates from the proximal tubular brush border to the lysosomes where breakdown occurs at a high rate.

Repeatable measurement of local and zonal GFR in the rat kidney with aprotinin.

The basic polypeptide aprotinin (Ap), mol. wt 6513, is freely filtered in glomeruli and completely reabsorbed by the proximal tubules. Cellular processing is slow with return to plasma of breakdown products beginning after 20-30 min. When corrected for Gibbs-Donnan distribution of Ap between glomerular filtrate and plasma (i.e. 0.65 at a plasma protein concentration of 50 mg ml-1), the renal clearance of [125I]Ap, estimated as the ratio of kidney uptake and integrated non-protein bound plasma 125I concentration, equals that of [51Cr]EDTA (urine + kidney content). Zonal GFR per gram tissue was obtained from uptake in three to six samples from outer and inner cortex (OC, IC) and the cortico-medullary border zone, 5-30 min after i.v. injection in rats. Control GFR in OC was 2.05 (SD 0.39) ml g-1 min-1 and the IC/OC ratio 0.66 (SD 0.14). Repeated local clearances (CI and CII) were obtained by injecting a second tracer (i.e. [131I]Ap) 15 min after the first injection (i.e. [125I]Ap), which by then had a low plasma concentration. The kidneys were removed at 30 min, frozen and dissected. During control conditions CII/CI averaged 1.06 in OC and IC, and the coefficient of variation (CV) between CII/CI ratios of individual tissue samples was 2% in both zones. Lowering left renal arterial pressure before the second injection reduced GFR proportionally in both zones (34 and 37%) with a CV of intersample CII/CI ratios of 5%. We conclude that the method allows precise and repeatable measurements of local and zonal GFR.