Citations

Key Points Robotic ultrasound performed favorably compared with magnetic resonance imaging for evaluating total kidney volume. Collagen evaluation by two novel methods of picrosirius red imaging were more informative than the standard method by brightfield imaging. Findings can improve research by increasing speed and access to total kidney volume determination and sensitivity of collagen assessment. Background 3D imaging and histology are critical tools for assessing polycystic kidney disease (PKD) in patients and animal models. Magnetic resonance (MR) imaging provides micron resolution but is time consuming and expensive, and access to equipment and expertise is limited. Robotic ultrasound (US) imaging has lower spatial resolution but is faster, more cost-effective, and accessible. Similarly, picrosirius red (PSR) staining and brightfield microscopy are commonly used to assess fibrosis; however, alternative methods have been shown in non-kidney tissues to provide greater sensitivity and more detailed structural characterization. Methods In this study, we evaluated the utility of robotic US and alternative methods of quantifying PSR staining for PKD research. We compared longitudinal total kidney volume measurements using US and MR imaging. We additionally compared PSR imaging and quantification using standard brightfield microscopy with that by circularly polarized light with hue analysis and fluorescence imaging analyzed using curvelet transform fiber extraction software for automatic detection of individual collagen fibers. Results Increased total kidney volume was detected by US in Pkd1 RC/RC versus wild-type (WT) at time points spanning from early to established disease. US interobserver variability was greater but allowed scanning in 2–5 minutes/mouse, whereas MR imaging required 20–30 minutes/mouse. While no change in fibrotic index was detected in this cohort of relatively mild disease using brightfield microscopy, polarized light showed fibers skewed thinner in Pkd1 RC/RC versus WT. Fluorescence imaging showed a higher density of collagen fibers in Pkd1 RC/RC versus WT, and fibers were thinner and curvier with no change in length. In addition, fiber density was higher in both glomeruli and tubules in Pkd1 RC/RC , and glomeruli had a higher fiber density than tubules in Pkd1 RC/RC and trended higher in WT. Conclusions These studies show robotic US is a rigorous imaging tool for preclinical PKD research. In addition, they demonstrate the increased sensitivity of polarized and fluorescence analysis of PSR-stained collagen.

Maintaining pH is critical for proper cellular and organ function. To measure intracellular pH, we made a novel genetically encoded protein, pHire, that increases red fluorescence with increasing pH. Thus, pHire allows for the coupling of multiple fluorescent tags without spectral overlap. We have expressed pHire in mammalian epithelial cells, Drosophila, and mice. In this study, our goal was to characterize pHire’s spectral properties by expression in human cells (HEK293) and in E.coli. To biophysically characterize pHire, we made a maltose binding protein (MBP)-pHire fusion protein to purify from E.coli. Pelleting individual colony growths revealed pink bacterial pellets which fluoresced with mCherry filters. Rather than purifying the MBP-pHire protein, equal volumes of bacteria were permeabilized with nigericin and high K+ solution for pH 5 to 9. These aliquots were then used to gather the fluorescence excitation and emission spectra of pHire, allowing us to determine the excitation and emission peaks of pHire as a function of pH. Our results showed peaks at Ex579 nm and Em611 nm, or roughly mCherry. Pelleted bacteria could also be fixed in perfusion chambers, revealing the same calibration curve as in mammalian cells and flies. Next, stable HEK293-pHire cells were selected to assay acid-base transport using NH4Cl or CO2/HCO3− in perfusion experiments. These HEK-pHire cells have less fluorescence than MBP-pHire bacteria but show the same Ex/Em peaks. However, an in-frame ATG 5’ to pHire in a mouse (adding 23 amino acids), removed pHire pH-dependence and decreased bacterial fluorescent output. Together these results indicate that pHire is biophysically similar in cells tested but that unstructured N-terminal additions block pH-responses. Funding: R25-DK101405, R21-DK129897. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Key Points Drosophila can be a model for Dent Disease type 1. Drosophila Clc-C mutations function similar to human CLC-5 Dent 1 mutations. Background Drosophila serve as exceptional alternative models for in vivo and ex vivo research and may provide an avenue for in-depth investigation for human ClC-5 and Dent disease type 1 (DD1). The Drosophila ClC-c (CG5284) has sequence homology with human ClC-5 and is hypothesized to encompass similar functional and phenotypical roles with ClC-5 and variants that cause DD1. Methods Ion transport function and activity of Drosophila ClC-c and homologous DD1 variants were assessed by voltage clamp electrophysiology. Membrane localization was demonstrated in Drosophila expressing a GFP-labeled construct of ClC-c. Genetic expression of an RNAi against ClC-c mRNA was used to generate a knockdown fly that serves as a DD1 disease model. Tubule secretion of cations and protein were assessed, as well as the crystal formation in the Malpighian tubules. Results Voltage clamp experiments demonstrate that ClC-c is voltage-gated with Cl − -dependent and pH-sensitive currents. Inclusion of homologous DD1 mutations pathogenic variants (S393L, R494W, and Q777X) impairs ClC-c ion transport activity. In vivo expression of ClC-c-eGFP in Malpighian tubules reveals that the membrane transporter localizes to the apical membrane and nearby cytosolic regions. RNAi knockdown of ClC-c (48% decreased mRNA expression) causes increased secretion of both urinary protein and Ca 2+ and increased occurrence of spontaneous tubule crystals. Conclusions Drosophila ClC-c shows orthologous function and localization to human ClC-5. Thus, Drosophila and ClC-c regulation may be useful for future investigations of Cl − transport, Ca 2+ homeostasis, and urinary protein loss in DD1.