A novel nanoluciferase transgenic reporter measures proteinuria in zebrafish.

The zebrafish is an important animal system for modeling human diseases. This includes kidney dysfunction as the embryonic kidney (pronephros) shares considerable molecular and morphological homology with the human nephron. A key clinical indicator of kidney disease is proteinuria, but a high-throughput readout of proteinuria in the zebrafish is currently lacking. To remedy this, we used the Tol2 transposon system to generate a transgenic zebrafish line that uses the fabp10a liver-specific promoter to over-express a nanoluciferase molecule fused with the D3 domain of Receptor-Associated Protein (a type of molecular chaperone) which we term NL-D3. Using a luminometer, we quantified proteinuria in NL-D3 zebrafish larvae by measuring the intensity of luminescence in the embryo medium. In the healthy state, NL-D3 is not excreted, but when embryos were treated with chemicals that affected either proximal tubular reabsorption (cisplatin, gentamicin) or glomerular filtration (angiotensin II, Hanks Balanced Salt Solution, Bovine Serum Albumin), NL-D3 is detected in fish medium. Similarly, depletion of several gene products associated with kidney disease (nphs1, nphs2, lrp2a, ocrl, col4a3, and col4a4) also induced NL-D3 proteinuria. Treating col4a4 depleted zebrafish larvae (a model of Alport syndrome) with captopril reduced proteinuria in this system. Thus, our findings validate the use of the NL-D3 transgenic zebrafish as a robust and quantifiable proteinuria reporter. Hence, given the feasibility of high-throughput assays in zebrafish, this novel reporter will permit screening for drugs that ameliorate proteinuria, thereby prioritizing candidates for further translational studies.

Basement membrane defects in CD151-associated glomerular disease.

BACKGROUND: CD151 is a cell-surface molecule of the tetraspanin family. Its lateral interaction with laminin-binding integrin ɑ3ß1 is important for podocyte adhesion to the glomerular basement membrane (GBM). Deletion of Cd151 in mice induces glomerular dysfunction, with proteinuria and associated focal glomerulosclerosis, disorganisation of GBM and tubular cystic dilation. Despite this, CD151 is not routinely screened for in patients with nephrotic-range proteinuria. We aimed to better understand the relevance of CD151 in human kidney disease. METHODS: Next-generation sequencing (NGS) was used to detect the variant in CD151. Electron and light microscopy were used to visualise the filtration barrier in the patient kidney biopsy, and immunoreactivity of patient red blood cells to anti-CD151/MER2 antibodies was performed. Further validation of the CD151 variant as disease-causing was performed in zebrafish using CRISPR-Cas9. RESULTS: We report a young child with nail dystrophy and persistent urinary tract infections who was incidentally found to have nephrotic-range proteinuria. Through targeted NGS, a novel, homozygous truncating variant was identified in CD151, a gene rarely reported in patients with nephrotic syndrome. Electron microscopy imaging of patient kidney tissue showed thickening of GBM and podocyte effacement. Immunofluorescence of patient kidney tissue demonstrated that CD151 was significantly reduced, and we did not detect immunoreactivity to CD151/MER2 on patient red blood cells. CRISPR-Cas9 depletion of cd151 in zebrafish caused proteinuria, which was rescued by injection of wild-type CD151 mRNA, but not CD151 mRNA containing the variant sequence. CONCLUSIONS: Our results indicate that a novel variant in CD151 is associated with nephrotic-range proteinuria and microscopic haematuria and provides further evidence for a role of CD151 in glomerular disease. Our work highlights a functional testing pipeline for future analysis of patient genetic variants. A higher resolution version of the Graphical abstract is available as Supplementary information.

Post-Myocardial Infarction T-tubules Form Enlarged Branched Structures With Dysregulation of Junctophilin-2 and Bridging Integrator 1 (BIN-1).

BACKGROUND: Heart failure is a common secondary complication following a myocardial infarction (MI), characterized by impaired cardiac contraction and t-tubule (t-t) loss. However, post-MI nano-scale morphological changes to the remaining t-ts are poorly understood. METHOD AND RESULTS: We utilized a porcine model of MI, using a nonlethal microembolization method to generate controlled microinfarcts. Using serial block face scanning electron microscopy, we report that post-MI, after mild left-ventricular dysfunction has developed, t-ts are not only lost in the peri-infarct region, but also the remnant t-ts form enlarged, highly branched disordered structures, containing a dense intricate inner membrane. Biochemical and proteomics analyses showed that the calcium release channel, ryanodine receptor 2 (RyR2), abundance is unchanged, but junctophilin-2 (JP2), important for maintaining t-t trajectory, is depressed (-0.5×) in keeping with the t-ts being disorganized. However, immunolabeling shows that populations of RyR2 and JP2 remain associated with the remodeled t-ts. The bridging integrator 1 protein (BIN-1), a regulator of tubulogensis, is upregulated (+5.4×), consistent with an overdeveloped internal membrane system, a feature not present in control t-ts. Importantly, we have determined that t-ts, in the remote region, are narrowed and also contain dense membrane folds (BIN-1 is up-regulated +3.4×), whereas the t-ts have a radial organization comparable to control JP2 is upregulated +1.7×. CONCLUSIONS: This study reveals previously unidentified remodeling of the t-t nano-architecture in the post-MI heart that extends to the remote region. Our findings highlight that targeting JP2 may be beneficial for preserving the orientation of the t-ts, attenuating the development of hypocontractility post-MI.

Three-dimensional structure of the intercalated disc reveals plicate domain and gap junction remodeling in heart failure.

The intercalated disc (ICD) orchestrates electrochemical and mechanical communication between neighboring cardiac myocytes, properties that are perturbed in heart failure (HF). Although structural data from transmission electron microscopy two-dimensional images have provided valuable insights into the domains forming the ICD, there are currently no three-dimensional (3D) reconstructions for an entire ICD in healthy or diseased hearts. Here, we aimed to understand the link between changes in protein expression in an ovine tachypacing-induced HF model and ultrastructural remodeling of the ICD by determining the 3D intercalated disc architecture using serial block face scanning electron microscopy. In the failing myocardium there is no change to the number of ICDs within the left ventricle, but there is an almost doubling of the number of discs with a surface area of <1.0 × 10(8)µm(2) in comparison to control. The 3D reconstructions further revealed that there is remodeling of the plicate domains and gap junctions with vacuole formation around and between the contributing membranes that form the ICDs in HF. Biochemical analysis revealed upregulation of proteins involved in stabilizing the adhesive and mechanical properties consistent with the morphological changes. Our studies here have shown that in tachypacing-induced HF mechanical stresses are associated with both structural and molecular alterations. To our knowledge, these data together provide novel, to our knowledge, insights as to how remodeling at the molecular and structural levels leads to impaired intercellular communication.

Three-dimensional reconstruction of cardiac sarcoplasmic reticulum reveals a continuous network linking transverse-tubules: this organization is perturbed in heart failure.

RATIONALE: The organization of the transverse-tubular (t-t) system and relationship to the sarcoplasmic reticulum (SR) underpins cardiac excitation-contraction coupling. The architecture of the SR, and relationship with the t-ts, is not well characterized at the whole-cell level. Furthermore, little is known regarding changes to SR ultrastructure in heart failure. OBJECTIVE: The aim of this study was to unravel interspecies differences and commonalities between the relationship of SR and t-t networks within cardiac myocytes, as well as the modifications that occur in heart failure, using a novel high-resolution 3-dimensional (3D) imaging technique. METHODS AND RESULTS: Using serial block face imaging coupled with scanning electron microscopy and image analysis, we have generated 3D reconstructions of whole cardiomyocytes from sheep and rat left ventricle, revealing that the SR forms a continuous network linking t-ts throughout the cell in both species. In sheep, but not rat, the SR has an intimate relationship with the sarcolemma forming junctional domains. 3D reconstructions also reveal details of the sheep t-t system. Using a model of tachypacing-induced heart failure, we show that there are populations of swollen and collapsed t-ts, patches of SR tangling, and disorder with rearrangement of the mitochondria. CONCLUSIONS: We provide the first high-resolution 3D structure of the SR network showing that it forms a cell-wide communication pipeline facilitating Ca(2+) diffusion, buffering, and synchronicity. The distribution of the SR within the cell is related to interspecies differences in excitation-contraction coupling, and we report the first detailed analysis of SR remodeling as a result of heart failure.