Cystinosis (ctns) zebrafish mutant shows pronephric glomerular and tubular dysfunction

Abstract

The human ubiquitous protein cystinosin is responsible for transporting the disulphide amino acid cystine from the lysosomal compartment into the cytosol. In humans, Pathogenic mutations of CTNS lead to defective cystinosin function, intralysosomal cystine accumulation and the development of cystinosis. Kidneys are initially affected with generalized proximal tubular dysfunction (renal Fanconi syndrome), then the disease rapidly affects glomeruli and progresses towards end stage renal failure and multiple organ dysfunction. Animal models of cystinosis are limited, with only a Ctns knockout mouse reported, showing cystine accumulation and late signs of tubular dysfunction but lacking the glomerular phenotype. We established and characterized a mutant zebrafish model with a homozygous nonsense mutation (c.706 C > T; p.Q236X) in exon 8 of ctns. Cystinotic mutant larvae showed cystine accumulation, delayed development, and signs of pronephric glomerular and tubular dysfunction mimicking the early phenotype of human cystinotic patients. Furthermore, cystinotic larvae showed a significantly increased rate of apoptosis that could be ameliorated with cysteamine, the human cystine depleting therapy. Our data demonstrate that, ctns gene is essential for zebrafish pronephric podocyte and proximal tubular function and that the ctns-mutant can be used for studying the disease pathogenic mechanisms and for testing novel therapies for cystinosis.

Figure 1. Alignment of zebrafish Ctns protein and human cystinosin.

(a) Amino-acid sequence alignment of the zebrafish Ctns protein and human cystinosin. The site of the genetic zebrafish model truncating mutation (c.706 C > T; p.Q236X) is marked in red. Identical amino-acids are denoted by asterisks and similar amino acids by double dots. The seven transmembrane domains are highlighted in grey and the two lysosomal targeting motifs in black. (b) Exon 8 of the zebrafish ctns gene showing the wild-type (wt), the heterozygous (het) and the homozygous (hom) sequences for the c. 706 C > T mutation. Typical base sequence is marked above each electrophoretogram, while altered sequence is marked below.

Figure 2. Morphology and cystine measurements.

(a) Morphology of wild-type and ctns^−/− larvae at 4 dpf. Wild-type larva shows normal morphology, while mutant ctns^−/− larvae show various degrees of developmental delay and deformity: upper larva show signs of growth retardation in the form of slightly bigger yolk, bulging heart and bent-down head, while the middle and lower larvae show mild and severe deformity, respectively (bars = 1 mm). (b) Cystine content in homogenates of 6 dpf wt or ctns^−/− zebrafish larvae. ctns^−/− larvae were either free of treatment (N = 133) or subjected to 0.1 or 1.0 mM of cysteamine in the swimming water (N = 111 and 121 larvae, respectively). Comparison was performed with wt larvae (N = 191). (c) Oxidized glutathione (GSSG) content in homogenates of 6 dpf wt or ctns^−/− zebrafish larvae (same conditions and larval numbers as cystine). (d) Total glutathione (GSH) content in homogenates of 6 dpf wt or ctns^−/− zebrafish larvae. ctns^−/− larvae were either free of treatment (N = 80) or subjected to 0.1 or 1.0 mM of cysteamine in the swimming water (N = 108 and 104 larvae, respectively). Comparison was performed with wt larvae (N = 158). (e) Free cysteine content in homogenates of 6 dpf wt or ctns^−/− zebrafish larvae (same conditions and larval numbers as GSH). (f–i) Cystine content in homogenates of 8-month-old adults (Kidney, brain, heart and liver, respectively) (N = 3 of each genotype). Concentrations of cystine and other thiol compounds were expressed as nmol/mg protein. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. Early developmental stages of zebrafish ctns^−/− embryos and response to cysteamine therapy.

Embryonic development was monitored over the first 3 days of life at predetermined time points: (a) 3 hpf, (b) 6 hpf, (c) 24 hpf, (d) 48 hpf and (e) 72 hpf. The outcomes of four different mating settings, 16 females and eight males from each genotype were used (363 ctns^−/− and 322 wt embryos). Percentages of different developmental stages at each time point were calculated per the total number of living embryos for each genotype at each time point. *P < 0.05, ***P < 0.001 against wild-type percentages using Pearson chi-square test. (f) Effect of different doses of cysteamine therapy on mortality rates of ctns^−/− larvae during the first 96 hpf. *P < 0.05, **P < 0.01, ***P < 0.001 against untreated ctns^−/− larvae using student’s t test.

Figure 4. Apoptosis in ctns^−/− larvae.

(a–d) Acridine orange: Five dpf wt larvae and ctns^−/− larvae, naïve to treatment or treated with 0.1 mM of cysteamine (N = 10 for each group), were incubated with Acridine Orange (AO). Fluorescent spots (white arrows) were delineated in high magnification mode and quantified by ImageJ software. (a) A representative tail segment of 5 dpf wt larva (bar = 200 μm). (b) A representative tail segment of 5 dpf ctns^−/− untreated larva (bar = 200 μm). (c) A representative tail segment of 5 dpf ctns^−/− larva treated with 0.1 mM cysteamine (bar = 200 μm). (d) Quantitation of the relative fluorescence intensity of apoptotic spots. Average intensity of untreated ctns^−/− larvae was set at 100%. *** P < 0.001 against untreated ctns^−/− larvae. (e,f) Caspase-3 immunohistochemistry. (e) Representative images showing increased apoptotic signal over the proximal tubule in 5 dpf ctns^−/− larva (left) compared to the negative control (right), bar = 10 μm. pt, proximal tubule. (f) Representative images showing increased apoptotic signal over the liver in 5 dpf ctns^−/− larva (left) compared to the negative control (right), bar = 30 μm. Rabbit serum was used for the negative control sections instead of 1ry Ab. (g) Caspase-3/7 enzyme activity. Quantitation of Caspase-3/7 enzyme activity by a luciferase based assay in the homogenates of 5 dpf wt and ctns^‒/‒ larvae (On average 60 larvae over 3 separate homogenates for each genotype were used). Results were expressed in luminescence units (RLU)/μg protein of each homogenate. ***P < 0.001.

Figure 5. Morphology of the pronephros of ctns^−/− larvae compared to the wt.

(a) H&E stained cut-section of a 6 dpf wt larva at the level of the glomerulus and proximal tubules (bar = 50 μm). (b) H&E stained cut-section of a 6 dpf ctns^−/− larva at the level of the glomerulus and proximal tubules showing no apparent abnormality (bar = 50 μm). (c) Block face scanning EM image of the proximal tubule of a 4 dpf wt larva (bar = 5 μm). Demarcated area was magnified (right) to show size and distribution of lysosomes (asterisks) in the wt (bar = 2 μm). (d) Block face scanning EM image of the proximal tubule of a 4 dpf ctns^−/− larva showing intact brush border (bar = 5 μm). Demarcated area was magnified (right) to show larger number of lysosomes (asterisks) many of which were significantly enlarged in size compared to the wt (bar = 2 μm). (e) Quantitation of the number and surface area of lysosomes in cut sections at the level of proximal tubules in both genotypes. (f) Transmission EM image of the glomerulus of a 6 dpf wt larva showing normal foot processes (bar = 2 μm). A magnified EM image (right) of podocytes of 6 dpf wt larva showing preserved podocytes slit diaphragms (bar = 1 μm). (g) Transmission EM image of the glomerulus of a 6 dpf ctns^−/− larva showing partial foot process effacement (black arrows) (bar = 2 μm). A magnified EM image (right) of podocytes of 6 dpf ctns^−/− larva showing narrowed podocyte slit diaphragmatic spaces (white arrows) (bar = 1 μm). (h) Quantitation of podocyte foot process width (FPW) in cut sections at the level of the glomerulus in both genotypes. bb, brush border; bs, Bowman’s space; g, glomerulus; n, nucleus; pt, proximal tubule. *P < 0.05, ***P < 0.001 between the 2 genotypes using student’s t test.

Figure 6. Functional evaluation of glomerular permeability and tubular reabsorption of ctns^−/− larvae.

(a–c) Eye fluorescence assay: peak fluorescence intensity in the retinal vascular bed of ctns^−/− zebrafish larvae and wild-type larvae (N = 20 each). Fluorescence intensities were evaluated using fixed diameter circles by the ImageJ software. (a) A representative wild-type 4 dpf larva (24 h post-injection) (bar = 200 μm). (b) A representative ctns^−/− 4 pdf larva (24 h post-injection) (bar = 200 μm). (c) Quantitation of peak fluorescence intensities in the retinal vascular bed of both genotypes. (d–i) Histopathological functional evaluation: (d) A representative proximal tubule of wt larva injected with the 70-kDa labelled dextran (bar = 10 μm). (e) A representative proximal tubule of wt larva injected with the 4-kDa labelled dextran (bar = 10 μm). (f) A representative proximal tubule of ctns^−/− larva injected with the 70-kDa labelled dextran (bar = 10 μm). (g) A representative proximal tubule of ctns^−/− larva injected with the 4-kDa labelled dextran (bar = 10 μm). (h) A higher magnification of the proximal tubules of both genotypes showing internalized 70-kDa dextran within cytosolic puncta that likely correspond to endocytic compartments (marked areas in panels d and f) (bars = 5 μm). (i) Quantitation of the number of dextran puncta in both high and low molecular weight dextran injections in both genotypes (N = 10 for each genotype and each condition). *P < 0.05, ***P < 0.001.

Figure 7. Megalin expression in proximal tubular cells.

(a) Transverse fluorescent image of the proximal pronephric region of wt 5 dpf larva labelled with anti-megalin antibody (bar = 10 μm). (b) Higher magnification image of wt proximal tubule (square in panel a) showing mainly the diffuse distribution of megalin at the cellular brush border (bar = 3 μm). (c) The proximal pronephric region of ctns^−/− 5 dpf larva labelled with anti-megalin antibody (bar = 10 μm). (d) Higher magnification image of ctns^−/− proximal tubule (square in panel c) showing majority of megalin staining in sub-apical intracytoplasmic vacuoles (white arrows) (bar = 3 μm). Outer boundaries of proximal tubules were delineated with green, lumen with red, and nuclear boundaries were delineated with white. (e) Quantitation of megalin protein abundance in proximal tubules of wt and ctns^−/− larvae (N = 5 for each genotype). (f) Quantitation of the megalin encoding lrp2a RNA expression in homogenized larvae of 6 dpf wt vs ctns^−/− larvae (N = 5 individually separated RNA samples for each genotype). *P < 0.05.