Josefin Axelsson

Acute hyperglycemia induces rapid, reversible increases in glomerular permeability in nondiabetic rats

This study was performed to investigate the impact of acute hyperglycemia (HG) on the permeability of the normal glomerular filtration barrier in vivo. In anesthetized Wistar rats (250-280 g), the left ureter was catheterized for urine collection, while simultaneously blood access was achieved. Rats received an intravenous (iv) infusion of either 1) hypertonic glucose to maintain blood glucose at 20-25 mM (G; n = 8); 2) hypertonic glucose as in 1) and a RhoA-kinase inhibitor (Y-27632; Rho-G; n = 8); 3) 20% mannitol (MANN; n = 7) or 4) hypertonic (12%) NaCl to maintain plasma crystalloid osmotic pressure (pi(cry)) at approximately 320-325 mosmol/l (NaCl; n = 8) or 5) physiological saline (SHAM; n = 8). FITC-Ficoll 70/400 was infused iv for at least 20 min before termination of the experiments, and plasma and urine were collected to determine the glomerular sieving coefficients (theta) for polydisperse Ficoll (molecular radius 15-80 A) by high-performance size-exclusion chromatography. In G there was a marked increase in for Ficoll(55-80A) at 20 min, which was completely reversible within 60 min and abrogated by a Rho-kinase (ROCK) inhibitor, while glomerular permeability remained unchanged in MANN and NaCl. In conclusion, acute HG caused rapid, reversible increases in for large Ficolls, not related to the concomitant hyperosmolarity, but sensitive to ROCK inhibition. The changes observed were consistent with the formation of an increased number of large pores in the glomerular filter. The sensitivity of the permeability changes to ROCK inhibition strongly indicates that the cytoskeleton of the cells in the glomerular barrier may be involved in these alterations.

A1M/α1-Microglobulin Protects from Heme-Induced Placental and Renal Damage in a Pregnant Sheep Model of Preeclampsia.

Preeclampsia (PE) is a serious pregnancy complication that manifests as hypertension and proteinuria after the 20th gestation week. Previously, fetal hemoglobin (HbF) has been identified as a plausible causative factor. Cell-free Hb and its degradation products are known to cause oxidative stress and tissue damage, typical of the PE placenta. A1M (α1-microglobulin) is an endogenous scavenger of radicals and heme. Here, the usefulness of A1M as a treatment for PE is investigated in the pregnant ewe PE model, in which starvation induces PE symptoms via hemolysis. Eleven ewes, in late pregnancy, were starved for 36 hours and then treated with A1M (n = 5) or placebo (n = 6) injections. After injections, the ewes were re-fed and observed for additional 72 hours. They were monitored for blood pressure, proteinuria, blood cell distribution and clinical and inflammation markers in plasma. Before termination, the utero-placental circulation was analyzed with Doppler velocimetry and the kidney glomerular function was analyzed by Ficoll sieving. At termination, blood, kidney and placenta samples were collected and analyzed for changes in gene expression and tissue structure. The starvation resulted in increased amounts of the hemolysis marker bilirubin in the blood, structural damages to the placenta and kidneys and an increased glomerular sieving coefficient indicating a defect filtration barrier. Treatment with A1M ameliorated these changes without signs of side-effects. In conclusion, A1M displayed positive therapeutic effects in the ewe starvation PE model, and was well tolerated. Therefore, we suggest A1M as a plausible treatment for PE in humans.

Extracellular fetal hemoglobin induces increases in glomerular permeability: inhibition with alfa-1-microglobulin and tempol

Extracellular fetal hemoglobin (HbF) and adult hemoglobin (HbA) are proinflammatory and generate ROS. Increased plasma levels of extracellular HbF have recently been reported to occur in early preeclampsia. α1-Microglobulin (A1M) is a physiological heme-binding protein and radical scavenger that has been shown to counteract vascular permeability increases induced by HbA in the perfused placenta. The present study was performed to investigate whether HbF and HbA will increase glomerular permeability in vivo and to test whether A1M and tempol, a ROS scavenger, can prevent their effects. Anesthetized Wistar rats were continuously infused intravenously with either HbA, HbF, or cyano-inactivated HbF together with FITC-Ficoll-70/400, inulin, and (51)Cr-labeled EDTA for 2 h. Plasma samples and urine samples (left ureter) were taken repeatedly and analyzed by high-performance size exclusion chromatography to assess glomerular sieving coefficients for Ficoll of radius 10-80 Å. In separate experiments, A1M or tempol was given before and during Hb infusions. Extracellular HbF caused rapid, transient increases in glomerular permeability to large Ficoll molecules (50-80Å), contrary to the effects of HbA and cyano-inactivated HbF. For HbF, glomerular sieving coefficients for Ficoll of radius 60Å increased from 3.85 ± 0.85 × 10(-5) to 2.60 ± 0.96 × 10(-4) at 15 min, changes that were abrogated by tempol and reduced by A1M. In conclusion, our data demonstrate that extracellular HbF, infused systemically, can acutely increase glomerular permeability through inducing oxidative stress.

Effects of early endotoxemia and dextran-induced anaphylaxis on the size selectivity of the glomerular filtration barrier in rats

This study was performed to investigate the glomerular permeability alterations responsible for the microalbuminuria occurring in endotoxemia and during anaphylactic shock. In anesthetized Wistar rats, the left ureter was catheterized for urine collection while, simultaneously, blood access was achieved. Endotoxemia was induced by lipopolysaccharide (LPS) from Escherichia coli, and glomerular permeability was assessed at 60 and 90 (n = 7) and 120 (n = 7) min. Anaphylaxis was induced by a bolus dose of Dextran-70, and glomerular permeability assessed at 5 min (n = 8) and 40 min (n = 9). Sham animals were followed for either 5 or 120 min. The glomerular sieving coefficients (theta) to fluorescein isothiocyanate-Ficoll (70/400) were determined from plasma and urine samples and assessed using size-exclusion chromatography (HPLC). After start of the LPS infusion (2 h), but not at 60 or 90 min, theta for Ficoll(70A) had increased markedly [from 2.91 x 10(-5) +/- 6.33 x 10(-6) to 7.78 x 10(-5) +/- 6.21 x 10(-6) (P < 0.001)]. In anaphylaxis, there was a large increase in theta for Ficolls >60 A in molecular radius already at 5 min, but the glomerular permeability was completely restored at 40 min. In conclusion, there was a transient, immediate increment of glomerular permeability in dextran-induced anaphylaxis, which was completely reversible within 40 min. By contrast, endotoxemia caused an increase in glomerular permeability that was manifest first after 2 h. In both cases, theta to large Ficoll molecules were markedly increased, reflecting an increase in the number of large pores in the glomerular filter.

A1M Ameliorates Preeclampsia-Like Symptoms in Placenta and Kidney Induced by Cell-Free Fetal Hemoglobin in Rabbit

Preeclampsia is one of the most serious pregnancy-related diseases and clinically manifests as hypertension and proteinuria after 20 gestational weeks. The worldwide prevalence is 3-8% of pregnancies, making it the most common cause of maternal and fetal morbidity and mortality. Preeclampsia lacks an effective therapy, and the only “cure” is delivery. We have previously shown that increased synthesis and accumulation of cell-free fetal hemoglobin (HbF) in the placenta is important in the pathophysiology of preeclampsia. Extracellular hemoglobin (Hb) and its metabolites induce oxidative stress, which may lead to acute renal failure and vascular dysfunction seen in preeclampsia. The human endogenous protein, α1-microglobulin (A1M), removes cell-free heme-groups and induces natural tissue repair mechanisms. Exogenously administered A1M has been shown to alleviate the effects of Hb-induced oxidative stress in rat kidneys. Here we attempted to establish an animal model mimicking the human symptoms at stage two of preeclampsia by administering species-specific cell-free HbF starting mid-gestation until term, and evaluated the therapeutic effect of A1M on the induced symptoms. Female pregnant rabbits received HbF infusions i.v. with or without A1M every second day from gestational day 20. The HbF-infused animals developed proteinuria and a significantly increased glomerular sieving coefficient in kidney that was ameliorated by co-administration of A1M. Transmission electron microscopy analysis of kidney and placenta showed both intracellular and extracellular tissue damages after HbF-treatment, while A1M co-administration resulted in a significant reduction of the structural and cellular changes. Neither of the HbF-treated animals displayed any changes in blood pressure during pregnancy. In conclusion, infusion of cell-free HbF in the pregnant rabbits induced tissue damage and organ failure similar to those seen in preeclampsia, and was restored by co-administration of A1M. This study provides preclinical evidence supporting further examination of A1M as a potential new therapy for preeclampsia.

Scavengers of reactive oxygen species, paracalcitol, RhoA, and Rac-1 inhibitors and tacrolimus inhibit angiotensin II-induced actions on glomerular permeability

Systemic infusions of ANG II rapidly induce large, dynamic increases in the permeability of the glomerular filtration barrier (GFB) in rats. After binding to its receptor(s), ANG II generates reactive oxygen species (ROS) and produces Ca²⁺ influx into cells, leading to activation of a plethora of signaling cascades, including, e.g., calcineurin and small GTPases, such as Rac-1 and RhoA. In the present study we sought to interact with some of these cascades to test potential novel antiproteinuric agents. In anesthetized Wistar rats, the left urether was cannulated for urine collection, and blood access was achieved. Rats were infused with ANG II (16 ng·kg⁻¹·min⁻¹) alone, or together with the ROS scavengers tempol or dimethylthiourea (DMTU) or the D-vitamin analog paracalcitol, the RhoA-kinase inhibitor Y-27632, the Rac-1 inhibitor NSC-23766, or the calcineurin inhibitor tacrolimus. FITC-Ficoll-70/400 (mol.radius 10-80 Å) and ⁵¹Cr-EDTA were infused throughout the experiment. Plasma and urine samples were taken during baseline and at 5 and 15 min after the start of the infusions and analyzed by high-performance size-exclusion chromatography for determination of glomerular sieving coefficients (θ) for Ficoll10-80Å. ANG II infusion into rats caused marked increases in glomerular permeability to large Ficoll molecules (Ficoll50-80Å), which were abrogated by the ROS scavenger tempol and partly by DMTU. Paracalcitol, RhoA, and Rac-1 inhibition, and, to some extent tacrolimus, but not prostacyclin, could also inhibit the glomerular permeability actions of ANG II. Our data suggest that cellular ROS generation and active Ca²⁺ signaling are involved in ANG II-induced increases in glomerular permeability.

Similarity of permeabilities for Ficoll, pullulan, charge‐modified albumin and native albumin across the rat peritoneal membrane

Aim:  Compared to neutral globular proteins, neutral polysaccharides, such as dextran, pullulan and Ficoll, appear hyperpermeable across the glomerular filtration barrier. This has been attributed to an increased flexibility and/or asymmetry of polysaccharides. The present study investigates whether polysaccharides are hyperpermeable also across the continuous capillaries in the rat peritoneum.

Acute reactive oxygen species (ROS)-dependent effects of IL-1β, TNF-α, and IL-6 on the glomerular filtration barrier (GFB) in vivo

This study was performed to investigate the immediate actions of the proinflammatory cytokines IL-1β, TNF-α, and IL-6 on the permeability of the glomerular filtration barrier (GFB) in rats and to test whether these actions are dependent upon the release of reactive oxygen species (ROS). In anesthetized rats, blood access was achieved and the left ureter was cannulated for urine collection. Rats were continuously infused intravenously with either IL-1β (0.4 and 2 μg·kg(-1)·h(-1)), TNF-α (0.4 and 2 μg·kg(-1)·h(-1)), or IL-6 (4 and 8 μg·kg(-1)·h(-1)), together with polydisperse FITC-Ficoll-70/400 and inulin for 1 h. Plasma and urine samples were analyzed by high performance size exclusion chromatography (HPSEC) for determination of glomerular sieving coefficients (θ). The glomerular filtration rate (GFR) was also assessed (51Cr-EDTA). In separate experiments, the superoxide scavenger tempol (30 mg·kg(-1)·h(-1)) was given before and during cytokine infusions. IL-1β and TNF-α caused rapid, partly reversible increases in glomerular permeability to large molecules (Ficoll50-80Å), peaking at 5-30 min, while IL-6 caused a more gradual increase in permeability, leveling off at 60 min. Tempol almost completely abrogated the glomerular permeability effects of the cytokines infused. In conclusion IL-1β, TNF-α, and IL-6, when infused systemically, caused immediate and partly reversible increases in glomerular permeability, which could be inhibited by the superoxide scavenger tempol, suggesting an important role of ROS in acute cytokine-induced permeability changes in the GFB.

mTOR inhibition with temsirolimus causes acute increases in glomerular permeability, but inhibits the dynamic permeability actions of puromycin aminonucleoside

Inhibitors of the mammalian target of rapamycin (mTORi) can produce de novo proteinuria in kidney transplant patients. On the other hand, mTORi has been shown to suppress disease progression in several animal models of kidney disease. In the present study, we investigated whether glomerular permeability can be acutely altered by the mTORi temsirolimus and whether mTORi can affect acute puromycin aminonucleoside (PAN) or angiotensin II (ANG II)-induced glomerular hyperpermeability. In anesthetized Wistar rats, the left ureter was cannulated for urine collection, while simultaneously blood access was achieved. Temsirolimus was administered as a single intravenous dose 30 min before the start of the experiments in animals infused with PAN or ANG II or in nonexposed animals. Polydispersed FITC-Ficoll-70/400 (molecular radius 10-80 Å) and (51)Cr-EDTA infusion was given during the whole experiment. Measurements of Ficoll in plasma and urine were performed sequentially before the temsirolimus injection (baseline) and at 5, 15, 30, 60, and 120 min after the start of the experiments. Urine and plasma samples were analyzed by high-performance size-exclusion chromatography (HPSEC) to assess glomerular sieving coefficients (θ) for Ficoll10-80Å. Temsirolimus per se increased baseline glomerular permeability to Ficoll50-80Å 45 min after its administration, a reactive oxygen species (ROS)-dependent phenomenon. PAN caused a rapid and reversible increase in glomerular permeability, peaking at 5 min, and again at 60-120 min, which could be blocked by the ROS scavenger tempol. mTORi abrogated the second permeability peak induced by PAN. However, it had no effect on the immediate ANG II- or PAN-induced increases in glomerular permeability.

Dynamic, size-selective effects of protamine sulfate and hyaluronidase on the rat glomerular filtration barrier in vivo.

The proteinuric actions of protamine sulfate (PS) have classically been, at least partly, attributed to alterations of the negatively charged glomerular endothelial glycocalyx. To investigate whether the charge-selective properties of the glomerular filtration barrier (GFB) would be altered by PS, we assessed the glomerular sieving of conventional, uncharged, polydispersed Ficoll (n-Ficoll) compared with charge modified, conformationally intact, anionic (carboxymethylated) Ficoll (a-Ficoll) before and after systemic infusions of PS in rats. For comparison, we also investigated the impact of hyaluronidase (hyase), which partially degrades the glycocalyx, on GFB permeability. In anaesthetized Wistar rats, blood access was achieved, and the left ureter was cannulated for urine collection. Rats were infused with either n-Ficoll or a-Ficoll before and during systemic infusions with either PS or hyase. Plasma and urine samples were taken repeatedly and analyzed by high-performance size exclusion chromatography to assess glomerular sieving coefficients (θ) for Ficoll (radius 10-80 Å). The GFB showed a significant glomerular charge selectivity for Ficoll molecules of radius 20-35 Å. PS and hyase infusions reversibly increased θ for large Ficoll molecules (Ficoll molecules of radius 50-80 Å). Thus, for PS, θ for a-Ficoll molecules of radius 70 Å increased from 2.47 × 10(-5) ± 1.1(-5) to 7.25 × 10(-5) ± 1.1(-5) (P < 0.05) at 15 min. For hyase, changes in a-Ficoll molecules of radius 50-80 Å were, however, not statistically significant. Neither PS nor hyase had any effect on θ for n-Ficoll molecules of radius 20-45 Å or a-Ficoll molecules of radius 20-45 Å. It is concluded that systemically administered PS and hyase in moderate doses dynamically decreased the size selectivity of the rat GFB without affecting its charge selective properties.