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

2014 ◽  
Vol 307 (10) ◽  
pp. F1136-F1143 ◽  
Author(s):  
Kristinn Sverrisson ◽  
Josefin Axelsson ◽  
Anna Rippe ◽  
Daniel Asgeirsson ◽  
Bengt Rippe
Keyword(s):  
Glomerular Filtration ◽  
High Performance ◽  
Protamine Sulfate ◽  
Size Exclusion ◽  
Endothelial Glycocalyx ◽  
Glomerular Filtration Barrier ◽  
Filtration Barrier ◽  
Charge Selectivity ◽  
The Impact

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± 1.1−5to 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.

Reduced diffusion of charge-modified, conformationally intact anionic Ficoll relative to neutral Ficoll across the rat glomerular filtration barrier in vivo

2011 ◽  
Vol 301 (4) ◽  
pp. F708-F712 ◽  
Author(s):  
Josefin Axelsson ◽  
Kristinn Sverrisson ◽  
Anna Rippe ◽  
William Fissell ◽  
Bengt Rippe
Keyword(s):  
Glomerular Filtration ◽  
High Performance ◽  
Size Exclusion ◽  
Glomerular Filtration Barrier ◽  
Glomerular Permeability ◽  
Charge Effects ◽  
Filtration Barrier ◽  
A Charge

The glomerular filtration barrier (GFB) is commonly conceived as a negatively charged sieve to proteins. Recent studies, however, indicate that glomerular charge effects are small for anionic, carboxymethylated (CM) dextran vs. neutral dextran. Furthermore, two studies assessing the glomerular sieving coefficients (θ) for negative CM-Ficoll vs. native Ficoll have demonstrated an increased glomerular permeability for CM-Ficoll (Asgeirsson D, Venturoli D, Rippe B, Rippe C. Am J Physiol Renal Physiol 291: F1083–F1089, 2006; Guimarães M, Nikolovski J, Pratt L, Greive K, Comper W. Am Physiol Renal Physiol 285: F1118–F1124, 2003.). The CM-Ficoll used, however, showed a larger Stokes-Einstein radius ( ae) than neutral Ficoll, and it was proposed that the introduction of negative charges in the Ficoll molecule had made it more flexible and permeable. Recently, a negative FITC-labeled CM-Ficoll (CMI-Ficoll) was produced with a conformation identical to that of neutral FITC-Ficoll. Using these probes, we determined their θ:s in anesthetized Wistar rats (259 ± 2.5 g). After blood access had been achieved, the left ureter was cannulated for urine sampling. Either polysaccharide was infused (iv) together with a filtration marker, and urine and plasma were collected. Assessment of θ FITC-Ficoll was achieved by high-performance size-exclusion chromatography (HPSEC). CMI-Ficoll and native Ficoll had identical elugrams on the HPSEC. Diffusion of anionic Ficoll was significantly reduced compared with that of neutral Ficoll across the GFB for molecules of ae ∼20–35 Å, while there were no charge effects for Ficoll of ae = 35–80 Å. The data are consistent with a charge effect present in “small pores,” but not in “large pores,” of the GFB and mimicked those obtained for anionic membranes in vitro for the same probes.

Loss of size selectivity of the glomerular filtration barrier in rats following laparotomy and muscle trauma

2009 ◽  
Vol 297 (3) ◽  
pp. F577-F582 ◽  
Author(s):  
Josefin Axelsson ◽  
Irma Mahmutovic ◽  
Anna Rippe ◽  
Bengt Rippe
Keyword(s):  
Glomerular Filtration ◽  
High Performance ◽  
Size Exclusion ◽  
Blunt Injury ◽  
Abdominal Muscles ◽  
Glomerular Filtration Barrier ◽  
Glomerular Permeability ◽  
Filtration Barrier ◽  
Muscle Trauma ◽  
Exclusion Chromatography

Posttraumatic microalbuminuria may be caused by either charge- or size-selective alterations in the glomerular filtration barrier, or both, and/or to a reduction in proximal tubular protein reabsorption. This study was performed to elucidate the pathophysiology of the increases in glomerular permeability occurring in rats exposed to a laparotomy or to a laparotomy and muscle trauma. In anesthetized Wistar rats (250–280 g), the left ureter was cannulated for urine collection, while simultaneously blood access was achieved. Rats were exposed to trauma by a laparotomy (L; n = 8), or by a combination of L and muscle trauma (MT; L+MT) induced by topical blunt injury of the abdominal muscles bilaterally. After L, muscles were crushed using hemostatic forceps at either 2 × 2 sites (“small” MT; n = 9), or at 2 × 5 sites (“large” MT; n = 9). Sham groups ( n = 16), not exposed to a laparotomy, were used as controls. The glomerular sieving coefficients (θ) to polydisperse FITC-Ficoll-70/400 (molecular radius 13–80 Å) were determined at 5 or 60 min after L and L+MT, respectively, from plasma and urine samples, and analyzed by high-performance size-exclusion chromatography. A tissue-uptake technique was used to assess θ for 125I-labeled serum albumin. L, with or without MT, increased θ for Ficoll55–80Å and albumin rapidly and markedly. θ-Ficoll70Å thus increased approximately threefold, and θ for albumin significantly, for all trauma groups. According to the “two-pore model” of glomerular permeability, these changes mainly reflect an increase in the number of large pores in the glomerular filter without any primary changes in the charge-selective properties of the filter.

Size and charge selectivity of the glomerular filter in early experimental diabetes in rats

2007 ◽  
Vol 293 (5) ◽  
pp. F1533-F1538 ◽  
Author(s):  
Catarina Rippe ◽  
Anna Rippe ◽  
Ole Torffvit ◽  
Bengt Rippe
Keyword(s):  
Glomerular Filtration ◽  
Experimental Diabetes ◽  
Renal Plasma Flow ◽  
Size Exclusion ◽  
Glomerular Filtration Barrier ◽  
Functional Changes ◽  
Filtration Barrier ◽  
Charge Selectivity ◽  
Exclusion Chromatography ◽  
Proximal Tubular

Microalbuminuria is an early sign of diabetic nephropathy. The aim of the present study was to investigate whether the changes of the glomerular filtration barrier in early experimental diabetes are due to size- or charge-selective alterations. Wistar rats, made diabetic by streptozotocin (STZ) and having their blood glucose maintained at ∼20 mM for 3 or 9 wk, were compared with age-matched controls. Glomerular clearances of native albumin (Cl-HSA) and neutralized albumin (Cl-nHSA) were assessed using a renal uptake technique. Glomerular filtration rate and renal plasma flow were assessed using 51Cr-EDTA and [125I]iodohippurate, respectively. In a separate set of animals, diabetic for 9 wk, and in controls, glomerular sieving coefficients (θ) for neutral FITC-Ficoll (molecular radius: 15–90 Å) were assessed using size exclusion chromatography. At 3 wk of diabetes, Cl-HSA and Cl-nHSA remained unchanged, indicating no alteration in either size or charge selectivity. By contrast, at 9 wk of diabetes, there was a twofold increase of Cl-HSA, whereas Cl-nHSA remained largely unchanged, at first suggesting a glomerular charge defect. However, according to a two-pore model, the number of large pores, assessed from both Ficoll and Cl-HSA, increased twofold. In addition, a small reduction in proximal tubular reabsorption was observed at 3 wk, which was further reduced at 9 wk. In conclusion, no functional changes were observed in the glomerular filtration barrier at 3 wk of STZ-induced diabetes, whereas at 9 wk there was a decrease in size selectivity due to an increased number of large glomerular pores.

P0053A METHOD FOR THE ISOLATION OF FLUORESCENT GLOMERULI FROM ZEBRAFISH LARVAE

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Anna Iervolino ◽  
Tim Lange ◽  
Florian Siegerist ◽  
Maximilian Schindler ◽  
Giovambattista Capasso ◽  
...  
Keyword(s):  
Glomerular Filtration ◽  
Fluorescent Protein ◽  
Transgenic Zebrafish ◽  
Renal Tubules ◽  
Glomerular Filtration Barrier ◽  
Podocyte Injury ◽  
Zebrafish Larvae ◽  
Filtration Barrier ◽  
Glomerular Damage

Abstract Background and Aims The zebrafish is a powerful animal model to study the glomerular morphology and the function of the permselectivity of the glomerular filtration barrier. Since zebrafish larvae develop quickly and can be bred to transparency, in vivo observation of these animals is possible. At 48 hours post fertilization (dpf), zebrafish develop a single filtering glomerulus which is attached to a pair of renal tubules. Like in mammals, the glomerular filtration barrier consists of a fenestrated endothelium, the glomerular basement membrane (GBM) and interdigitating podocyte foot processes bridged by a molecularly conserved slit diaphragm. By the use of genetically modified zebrafish strains with fluorescently labeled podocytes, it is possible to study alterations of the glomerulus during the development of renal disease directly in vivo and in vitro. As an injury model we used the nitroreductase/metronidazole (NTR/MTZ) zebrafish line to induce podocyte apoptosis and detachment from the GBM. Moreover, treatment of these larvae with MTZ induces glomerular injury that mimics focal segmental glomerulosclerosis (FSGS). The aim of our study was to establish a glomeruli isolation method which allows us to identify deregulation of miRNAs and mRNAs in the injured glomeruli by sequencing. Method The transgenic zebrafish strain Cherry (Tg(nphs2:Eco.nfsB-mCherry); mitfaw2/w2; mpv17a9/a9) which expresses the prokaryotic enzyme nitroreductase (NTR) fused to mCherry, a red fluorescent protein, under the control of the podocyte-specific podocin (nphs2) promoter in a transparent zebrafish strain, was used. The NTR/MTZ is a model of cell ablation to mimic podocyte injury. The prodrug MTZ (80 µM) is converted into a cytotoxin by NTR leading to a dose-dependent apoptosis exclusively in NTR-expressing podocytes. To induce podocyte injury, we treated Cherry larvae at 4 days post fertilization with MTZ (80 µM) freshly dissolved in 0.1% DMSO-E3 medium for 48 hours. Control larvae were treated with 0.1% DMSO-E3 medium. The treatment was stopped by a MTZ washout at 6 dpf. In order to perform the miRNA and mRNA sequencing on glomeruli isolated from MTZ-treated and control larvae we tried to establish a method to obtain total RNA samples of good quality. For this purpose, three different approaches were tested and validated: 1) Sieving method, 2) Fluorescence-Activated Cell Sorting method (FACS), and 3) manual isolation of glomeruli by using a micropipette. Results Zebrafish larvae developed a glomerular damage similar to FSGS after MTZ-treatment. MTZ-treated larvae showed severe pericardial edema, a reduction of the nephrin and podocin expression, proteinuria and an increased mortality rate at 8 dpf. After many tests we showed that glomeruli isolation using the sieving method and FACS were not efficient due to contaminations with other organs (sieving) and a loss of a large amount of cells per sample (FACS), respectively. Samples of the required quality for sequencing resulted only from the manual glomeruli isolation. Conclusion Here we describe methods to isolate fluorescent glomeruli from transgenic zebrafish larvae. For our studies, we used the NTZ/MTR kidney disease model in order to identify mRNAs and miRNAs regulated in response to glomerular damage. This technique will further allow to screen for healing drugs in high-throughput experiments.

The Rat Glomerular Filtration Barrier Does Not Show Negative Charge Selectivity

2002 ◽  
Vol 9 (5) ◽  
pp. 329-342 ◽  
Author(s):  
RICHARD C. SCHAEFFER ◽  
MAX L. GRATRIX ◽  
DAVID R. MUCHA ◽  
JOSÉ M. CARBAJAL
Keyword(s):  
Glomerular Filtration ◽  
Negative Charge ◽  
Glomerular Filtration Barrier ◽  
Filtration Barrier ◽  
Charge Selectivity

Unaltered size selectivity of the glomerular filtration barrier in caveolin-1 knockout mice

2009 ◽  
Vol 297 (2) ◽  
pp. F257-F262 ◽  
Author(s):  
Gustaf Grände ◽  
Catarina Rippe ◽  
Anna Rippe ◽  
Awahan Rahman ◽  
Karl Swärd ◽  
...  
Keyword(s):  
Glomerular Filtration ◽  
High Performance ◽  
Microvascular Permeability ◽  
Size Exclusion ◽  
No Production ◽  
Glomerular Permeability ◽  
Caveolin 1 ◽  
Filtration Barrier ◽  
Transcapillary Escape Rate ◽  
Exclusion Chromatography

The transfer of albumin from blood to tissue has been found to be increased in caveolin-1 knockout (KO) mice. This has been considered to reflect increased microvascular permeability, conceivably caused by an increased endothelial production of nitric oxide (NO) in these mice. To investigate whether such an increase in NO production would also affect glomerular barrier characteristics, the glomerular sieving coefficients (θ) to neutral FITC-Ficoll 70/400 (molecular radius 13–90 Å) were determined in caveolin-1 KO mice vs. their wild-type counterparts. The θ for Ficoll were assessed using high-performance size-exclusion chromatography on blood and urine samples. Furthermore, the transcapillary escape rate (TER) of 125I-labeled albumin and plasma volume (PV) were determined in both types of mice. The kidney expressed low levels of caveolin-1 compared with the lung and bladder, but immunofluorescence associated with vascular structures was evident. Staining was lost in the caveolin-1 KO kidney, as was caveolin-1 expression in the lung and bladder. Despite an increase in the glomerular filtration rate in caveolin-1 KO mice (0.23 ± 0.04 vs. 0.10 ± 0.02 ml/min; both n = 7; P < 0.05), the glomerular Ficoll sieving curves were nearly identical. Furthermore, caveolin-1 KO mice showed an increased PV (6.59 ± 0.42 vs. 5.18 ± 0.13 ml/100 g; P < 0.01) but only a tendency toward an increased TER (14.69 ± 1.59 vs. 11.62 ± 1.62%/h; not significant). It is concluded that in caveolin-1 KO mice the glomerular permeability was not increased, despite the presence of glomerular hyperfiltration. The present data are in line with the concept that the increased transvascular albumin leakage previously found in mice lacking caveolin-1 may be due to an elevation in systemic microvascular pressure due to precapillary vasodilatation, rather than being a consequence of increased microvascular permeability per se.

scholarly journals “Zebrafishing” for Novel Genes Relevant to the Glomerular Filtration Barrier

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Nils Hanke ◽  
Lynne Staggs ◽  
Patricia Schroder ◽  
Jennifer Litteral ◽  
Susanne Fleig ◽  
...  
Keyword(s):  
Glomerular Filtration ◽  
Barrier Function ◽  
Association Studies ◽  
Genome Wide Association Studies ◽  
Glomerular Filtration Barrier ◽  
Novel Genes ◽  
Zebrafish Model ◽  
Filtration Barrier

Data for genes relevant to glomerular filtration barrier function or proteinuria is continually increasing in an era of microarrays, genome-wide association studies, and quantitative trait locus analysis. Researchers are limited by published literature searches to select the most relevant genes to investigate. High-throughput cell cultures and otherin vitrosystems ultimately need to demonstrate proof in anin vivomodel. Generating mammalian models for the genes of interest is costly and time intensive, and yields only a small number of test subjects. These models also have many pitfalls such as possible embryonic mortality and failure to generate phenotypes or generate nonkidney specific phenotypes. Here we describe anin vivozebrafish model as a simple vertebrate screening system to identify genes relevant to glomerular filtration barrier function. Using our technology, we are able to screen entirely novel genes in 4–6 weeks in hundreds of live test subjects at a fraction of the cost of a mammalian model. Our system produces consistent and reliable evidence for gene relevance in glomerular kidney disease; the results then provide merit for further analysis in mammalian models.

scholarly journals MO030IDENTIFICATION OF REGULATED MRNAS AND MIRNAS IN GLOMERULI ISOLATED FROM AN FSGS-LIKE ZEBRAFISH MODEL

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Anna Iervolino ◽  
Tim Lange ◽  
Sabrina Siccardi ◽  
Florian Siegerist ◽  
Francesca Pia Caruso ◽  
...  
Keyword(s):  
Glomerular Filtration ◽  
Slit Diaphragm ◽  
Whole Body ◽  
Transgenic Zebrafish ◽  
Glomerular Filtration Barrier ◽  
Zebrafish Larvae ◽  
Filtration Barrier ◽  
Podocyte Detachment ◽  
Go Terms

Abstract Background and Aims The zebrafish (Danio rerio) is a powerful animal model to study glomerular morphology and the function of the permselectivity of the glomerular filtration barrier. Since zebrafish larvae develop quickly and can be bred to become transparent, in vivo observation of these animals is possible. At 48 hours post fertilization, zebrafish larvae develop a single glomerulus which is attached to a pair of tubules. Like in mammals, the glomerular filtration barrier consists of a fenestrated endothelium, the glomerular basement membrane and interdigitating podocyte foot processes bridged by a slit diaphragm. By using genetically modified zebrafish strains with fluorescently labeled podocytes, it is possible to study alterations of the glomerulus during the development of renal disease like focal segmental glomerulosclerosis (FSGS) directly in vivo. FSGS is characterized by podocyte loss, the effacement of their foot processes as well as scarring of the glomerulus. To study FSGS in zebrafish larvae, we induced podocyte detachment by the use of a zebrafish strain expressing the enzyme nitroreductase converting metronidazole into a toxic substance specifically in podocytes. The aim of our study was to collect glomeruli for the identification of mRNAs as well as miRNAs by RNA_Seq that are up- and down-regulated in the glomeruli of this FSGS-like disease model. Method The transgenic zebrafish strain Cherry (Tg(nphs2:GAL4); Tg(UAS:Eco.nfsB-mCherry); mitfaw2/w2; mpv17a9/a9) which expresses the prokaryotic enzyme nitroreductase (NTR) fused to mCherry, a red fluorescent protein, under the control of the podocyte-specific podocin (nphs2) promoter in a transparent zebrafish strain, was utilized. After addition of metronidazole (MTZ) into the tank water, MTZ is converted into a cytotoxin by NTR leading to dose-dependent apoptosis exclusively in podocytes. Cherry larvae were treated at 4 days post fertilization (dpf) for 48 h with 80 µM MTZ. MTZ-treated and control larvae were homogenized at 6 dpf. The cell suspension was diluted, and red-fluorescent glomeruli were collected using a micropipette and a microscope. Total RNA was isolated, and integrity was checked by a Bioanalyzer. Libraries were generated with a MACE kit and True Quant small RNA seq kit by GenXPro. Constructs were amplified by PCR and sequenced on an Illumina Hiseq 2000. Normalization and statistical analysis for differential gene expression were done using DESeq2. Results Zebrafish larvae showed severe whole-body edema, proteinuria, loss of podocytes and an increased mortality rate after MTZ-treatment. The glomerular histology resembled mammalian FSGS. We found that only the RNA of manually collected glomeruli had an excellent quality. Using RNA_Seq, we identified a total of 16941 genes. DESeq2 analysis showed 494 up-regulated and 473 down-regulated genes. Gene ontology (GO) enrichment analysis of up-regulated genes revealed a total of 167 that are significantly enriched in GO terms (e.g. metabolic processes, immune response and ion transport). Down-regulated genes were enriched in 14 GO terms and most of them are linked to normal glomerular function and the slit diaphragm. DESeq2 analysis identified 200 miRNAs of 777 small RNAs. Some of these miRNA are already described to be regulated in different glomerular diseases like FSGS, lupus nephritis, IgA nephropathy and diabetic nephropathy. Conclusion We analyzed isolated glomeruli from transgenic zebrafish larvae that developed a FSGS-like disease. By sequencing, we have found mRNAs and miRNAs that were significantly regulated after the onset of disease. Detailed knowledge of these mRNAs and miRNA-based gene regulation will help to uncover the pathomechanism as well as to develop therapeutics for the treatment of FSGS.

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

2010 ◽  
Vol 298 (6) ◽  
pp. F1306-F1312 ◽  
Author(s):  
Josefin Axelsson ◽  
Anna Rippe ◽  
Bengt Rippe
Keyword(s):  
Kinase Inhibitor ◽  
Size Exclusion ◽  
Rock Inhibitor ◽  
Glomerular Permeability ◽  
Acute Hyperglycemia ◽  
Hypertonic Glucose ◽  
Filtration Barrier ◽  
Rock Inhibition ◽  
The Impact

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 (πcry) at ∼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 (θ) for polydisperse Ficoll (molecular radius 15–80 Å) by high-performance size-exclusion chromatography. In G there was a marked increase in θ for Ficoll55-80Å 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 θ 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.

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