Assessing cardio‐renal function in zebrafish larvae

Abstract

The larva of the Zebrafish (Danio rerio) has become a leading model in various fields of physiology (e.g. Panula, 2010; Leong et al. 2010) and in developmental physiology (Grunwald & Eisen, 2002; Zetterberg, 2011). It is now possible to track gene expression with imaging methods and thereby gain detailed understanding of organ function in an intact vertebrate. In a recent issue of The Journal of Physiology, Rider et al. (2012) demonstrate this physiological application to better assess renal function. Previous measures of renal clearance in the zebrafish larvae have been made in the absence of flow changes and disregarding trapped fluorescence (Hentschel et al. 2007). In these new studies assays are established to determine renal clearance based on cardiovascular erythrocyte flow and extinction of injected fluorescein isothiocyanate (FITC)-labelled inulin. The method was evaluated in two models of kidney damage: using the nephrotoxic gentamicin or by giving a salt challenge. No matter how well we are acquainted with graphed or tabulated data, imaging is often more convincing. Seeing is believing. However, invasive visualization perturbs the steady-state and it is often not possible to image physiological processes over extended time periods. The zebrafish model provides a way around these limitations because the transparency of the embryo allows long-term monitoring. The development of the zebrafish embryo is established and the specific development of the kidney and the vasculature are well known (Kimmel et al. 1995; Ackermann & Paw, 2003). The zebrafish kidney is genetically and morphologically related to that of mammals, although the pronephric kidney of zebrafish larvae consist of only a fused glomerulus with two nephrons (Drummond & Davidson, 2010). The metanephros is the most complex kind of kidney, and is found only in birds and mammals; it develops out of the simpler pronephric and mesonephric kidneys. Although zebrafish lack the metanephros, they have pronephric (embryonic) and mesonephric (adult) kidneys. Three and half days after fertilization, the pronephros is functional and exhibits many features of the metanephros. On the other hand, important features of adult mammalian kidney function are not seen in the zebrafish pronephros. For instance, as described in the highlighted article by Rider et al., renal blood flow is not uncoupled from systemic blood flow. Thus, renal autoregulation, a key feature of renal haemodynamics, is not functional. Renal filtration is the surrogate parameter used for overall renal function. The gold standard for assessing glomerular filtration rate (GFR) is the inulin method. Inulin is a polysaccharide which is freely filtered through the glomerular filtration barrier into the tubules, where it cannot be reabsorbed or secreted. Therefore, provided the urine flow rate can be assessed, the clearance of inulin can be taken as a measure for GFR. Unfortunately, zebrafish urine flow rate is not yet assessable, but two measures were used by Rider et al. for determining renal clearance in the mature pronephric kidney: FITC intensity changes in the caudal region of the larvae and, reciprocally, the excretion of FITC inulin in water, determined over a 4 h period. The values of FITC intensity change and venous erythrocyte velocity were compared to the development of oedema in the yolk sac and pericardium. In this study it was also shown that renal clearance does not adjust in response to salt loading. This is different from the mature human kidney, where high salt decreases GFR via tubuloglomerular feedback (Schnermann et al. 1976). These new techniques for measuring renal clearance in the zebrafish are milestones in the study of the development of kidney haemodynamics. Before these techniques became available, the zebrafish renal model was restricted to more morphological studies, of, for example, cilia and the filtration barrier (Hentschel et al. 2007; Swanhart et al. 2011). Even determining GFR may become possible, once a method is established to determine zebrafish urine flow rate.