Simultaneous assessment of glomerular filtration and barrier function in live zebrafish

Abstract

brafish pronephros is a well-established model to study glomerular development, structure, and function. A few methods have been described to evaluate glomerular barrier function in zebrafish larvae so far. However, there is a need to assess glomerular filtration as well.In the present study, we extended the available methods by simultaneously measuring the intravascular clearances of Alexa fluor 647-conjugated 10-kDa dextran and FITC-conjugated 500-kDa dextran as indicators of glomerular filtration and barrier function, respectively. After intravascular injection of the dextrans, mean fluorescence intensities of both dextrans were measured in the cardinal vein of living zebrafish (4 days postfertilization) by confocal microscopy over time. We demonstrated that injected 10-kDa dextran was rapidly cleared from the circulation, became visible in the lumen of the pronephric tubule, quickly accumulated in tubular cells, and was detectably excreted at the cloaca. In contrast, 500-kDa dextran could not be visualized in the tubule at any time point. To check whether alterations in glomerular function can be quantified by our method, we injected morpholino oligonucleotides (MOs) against zebrafish nonmuscle myosin heavy chain IIA (zMyh9) or apolipoprotein L1 (zApol1). While glomerular filtration was reduced in zebrafish nonmuscle myosin heavy chain IIA MO-injected larvae, glomerular barrier function remained intact. In contrast, in zebrafish apolipoprotein L1 MO-injected larvae, glomerular barrier function was compromised as 500-kDa dextran disappeared from the circulation and became visible in tubular cells. In summary, we present a novel method that allows to simultaneously assess glomerular filtration and barrier function in live zebrafish. glomerular filtration; in vivo observations; kidney; pronephros; zebrafish GENOME-WIDE CLINICAL SCREENINGS have produced numerous gene candidates that are currently being discussed for their relevance as risk factors for chronic kidney disease (5). Validating such candidates in mouse knockout models, however, is an expensive and time-consuming task. It would therefore be of great interest to establish a rapid screening method that is easy to handle, accurate, and dependable. The zebrafish seems to ideally fulfill this request. The zebrafish pronephros contains a single glomerulus that is structurally identical to mouse and human glomeruli and is fully developed after 3-4 days (1, 6). Moreover, its high reproduction rate and the possibility of knocking down specific genes via injection of morpholino oligonucleotides (MOs) make the zebrafish ideally suited for rapid screening experiments. Being highly transparent, the zebrafish larva is a powerful model for microscopy-based in vivo analysis of the integrity of the glomerular filtration barrier. However, whereas routine assays of fluorescent dextran clearance have been established for the mouse (12), the same degree of reliability has yet to be reached working with zebrafish larvae.In most studies, defects of the filtration barrier in...

BDNF: mRNA expression in urine cells of patients with chronic kidney disease and its role in kidney function

“…bdnf KD larvae developed pericardial oedema as an indicator for an impairment of the glomerular filtration barrier. This finding could be confirmed by a decrease in the intravascular eGFP intensity in bdnfMO‐treated CADE larvae, also indicating a leaky filtration barrier . We also found a reduced expression of the podocyte markers podocin and nephrin, and a disrupted F‐actin structure in bdnfMO‐treated larvae, which are suggestive of podocyte loss.…”

“…Furthermore, bdnf KD larvae showed reduced expression of nphs2 (podocin) in RT‐PCR analysis (Figure C), which was confirmed by qRT‐PCR (Figure D). To study the glomerular morphology, we stained cryosections of zebrafish larvae, utilizing the ET strain that expresses eGFP specifically in podocytes . After staining the F‐actin cytoskeleton with Alexa‐546 phalloidin, we observed significant changes in the morphology of the glomeruli in response to the KD of bdnf .…”

“…To study the glomerular morphology, we stained cryosections of zebrafish larvae, utilizing the ET strain that expresses eGFP specifically in podocytes. 28,30 After staining the F-actin cytoskeleton with Alexa-546 phalloidin, we observed significant changes in the morphology of the glomeruli in response to the KD of bdnf. In addition to an enlargement of the glomerular tuft and Bowman's space, we observed a reduced number of podocytes in bdnf KD larvae (3 dpf) in contrast to CtrlMO-treated zebrafish larvae ( Figure 6A).…”

“…The following zebrafish strains were used: ET (Tg( wt1a :eGFP); mitfa w2/w2 ; roy a9/a9 ), CADE (Tg( fabp10a :DBP‐eGFP); mitfa w2/w2 ; roy a9/a9 ) . All zebrafish strains were raised, mated and maintained in E3 medium at 28.5°C, as previously described …”

“…38 All zebrafish strains were raised, mated and maintained in E3 medium at 28.5°C, as previously described. 30,39…”