Macura, Slobodan

Combined histopathological and metabolomic analysis of the same tissue specimen can reduce biological variability compared to independent analyses of similar samples. However, conventional workflows are incompatible: tissue fixation leads to metabolite loss through dilution and chemical reactions with fixative, while metabolomic extraction typically destroys the specimen. We propose a method that reconciles these conflicting requirements by enabling metabolite recovery from fixative while preserving tissue for histopathology. In this approach, tissue is initially fixed in a small volume of fixative; an aliquot of the fixative is then collected for nuclear magnetic resonance-based metabolomic analysis before additional fixative is added for standard processing. As a proof-of-concept, mouse kidneys from the same subject were analyzed using two protocols: standard perchloric acid extraction (ensuring complete metabolite recovery but destroying tissue) and metabolite extraction from fixative. Several metabolites were fully recoverable from the fixative, others partially, while some were lost due to chemical reactions. Despite some limitations, this strategy may be useful for analyzing precious specimens, such as human biopsies, that must remain intact for further studies. Moreover, using the same specimen for dual analyses reduces the number of animals required and enhances statistical robustness.

Key Points Experimental autosomal dominant polycystic kidney disease is associated with longitudinal changes in the kidney micro-RNA and mRNA profiles. Alterations in cellular transport, a feature of CKD, and a common biological process within polycystic kidney disease models and human autosomal dominant polycystic kidney disease, is present from early stages. Early versus late micro-RNA expression changes in autosomal dominant polycystic kidney disease could be targeted for a more tailored therapeutic intervention in the disease. Background Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the progressive development and enlargement of bilateral kidney cysts, which often leads to kidney failure. This study comprehensively characterized the expression profile of micro-RNAs (miRNAs) and their target genes in Pkd1 RC/RC mice and further compared them with other murine models of polycystic kidney disease (PKD) and a model of CKD and individuals with ADPKD. Methods Pkd1 RC/RC and wild-type (WT) mice ( n =10 each, five males and five females) were studied at 1, 6, and 12 months. At each time point, kidney volume was determined in vivo (magnetic resonance imaging), followed by ex vivo histomorphometric analyses. In randomly selected Pkd1 RC/RC and WT mouse kidneys ( n =5/genotype, at 1 and 12 months), miRNA-sequencing (seq) was performed, followed by mRNA-seq, integrated (miRNA-seq/mRNA-seq analysis), and functional analysis of miRNA target genes. Venn diagrams were constructed to identify overlapping and novel differentially expressed (DE) miRNAs in Pkd1 RC/RC and other commonly used murine models of PKD, diabetic kidney disease, and individuals with ADPKD. Results miRNA-seq analysis identified 41 and 181 miRNAs DE in Pkd1 RC/RC versus WT kidneys at 1 and 12 months, respectively, which were confirmed by quantitative PCR. Target genes of miRNAs DE in Pkd1 RC/RC at early stages encoded for proteins mainly implicated in cell proliferation and α -ketoglutarate ( α -KG) transport, whereas those DE at late stages encoded for transport proteins involved in proinflammatory and metabolic processes. Urine α -KG concentration ( 1 H nuclear magnetic resonance spectroscopy), its fractional excretion, and tissue levels were higher in Pkd1 RC/RC during the entire course of the disease and associated with decreased protein expression of α -KG transporters sodium-coupled dicarboxylate transporter 3 and organic anion transporter 1. We further identified common DE miRNAs among murine models of PKD, diabetic kidney disease, and previous reports in individuals with ADPKD, as well as several novel miRNAs DE in early and late Pkd1 RC/RC kidneys, which have not been previously reported in either other murine models of PKD or patients with ADPKD. Conclusions Our study demonstrates that the renal miRNA expression profile changes longitudinally with the progression of the disease and might suggest that the post-transcriptional regulation of α -KG transport could represent a novel early feature of the disease.

Abstract Despite recent approval of monoclonal antibodies that reduce amyloid (Aβ) accumulation, the development of disease-modifying strategies targeting the underlying mechanisms of Alzheimer’s disease (AD) is urgently needed. We demonstrate that mitochondrial complex I (mtCI) represents a druggable target, where its weak inhibition activates neuroprotective signaling, benefiting AD mouse models with Aβ and p-Tau pathologies. Rational design and structure‒activity relationship studies yielded novel mtCI inhibitors profiled in a drug discovery funnel designed to address their safety, selectivity, and efficacy. The new lead compound C458 is highly protective against Aβ toxicity, has favorable pharmacokinetics, and has minimal off-target effects. C458 exhibited excellent brain penetrance, activating neuroprotective pathways with a single dose. Preclinical studies in APP/PS1 mice were conducted via functional tests, metabolic assessment, in vivo 31 P- NMR spectroscopy, blood cytokine panels, ex vivo electrophysiology, and Western blotting. Chronic oral administration improved long-term potentiation, reduced oxidative stress and inflammation, and enhanced mitochondrial biogenesis, antioxidant signaling, and cellular energetics. These studies provide further evidence that the restoration of mitochondrial function and brain energetics in response to mild energetic stress represents a promising disease- modifying strategy for AD.

Our study shows that a brief harvesting-freezing delay associated with organ collection and freezing can significantly alter the kidney metabolic profile of both male and female wild-type and a genetic model of chronic kidney disease. Importantly, given that the effect of this delay differs among genotypes, it is not safe to assume that equally delaying harvesting-freezing in wild-type and polycystic kidney disease kidneys adequately controls this effect, ultimately leading to false positive and negative results among different renal diseases.

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.

Background Autosomal Dominant Polycystic Kidney Disease (PKD) is the most frequent hereditary renal disease and the fourth leading cause of end‐stage renal disease. However, the exact mechanisms of cystogenesis and disease progression remain to be elucidated. Recent evidence suggests that a metabolic reprograming may underlie the dysregulation of several signaling pathways known to be affected in PKD. To identify the metabolic pathways associated with cystogenesis in the PKD kidney, we characterized the urinary, plasma, and tissue metabolome of PCK (a well established model of PKD) compared to Sprague‐Dawley (SD) wild‐type rats. Methods Male and female PCK and SD rats were treated with dDAVP (PCK‐D, n=16 or SD‐D, n=12) to aggravate the cystic phenotype and achieve animals with different degrees of disease severity for a given age, or saline solution (PCK‐S, n=16 or SD‐S, n=12) as controls. Total kidney volume (TKV) gold standard to assess disease severity, were measured by MRI, and urinary, plasma, and tissue metabolites by proton nuclear magnetic resonance, 1H‐NMR. Kidney metabolites identified from renal tissues collected at euthanasia were correlated with TKV. Mitochondrial structure was assessed by electron microscopy in proximal tubules (PT), medullary thick ascending limb (mTAL), distal tubules (DT), and collecting ducts (CD). Results Consistent with previously published data, administration of dDAVP significantly aggravated the disease in PCK rats (TKV) without inducing a cystic phenotype in SD rats (). Analysis of PCK and SD metabolomes by Partial least squares‐discriminant analysis (PLS‐DA) score plots showed a significant separation between the groups (). Integration of tissue, urine and plasma analysis revealed 5 metabolites (α‐ketoglutarate, fumarate, malate, citrate, and hippurate) consistently differentially expressed, among which 4 participate in the mitochondrial tricarboxylic acid (TCA) cycle (). Moreover, tissue TCA cycle metabolites correlated directly with TKV (). Mitochondrial area and matrix density were preserved in PT, mTAL, and DT of PCK rats, yet disruption of cristae membranes, swelling, and decreased matrix density were observed in their CD cystic cells (). Conclusions Comprehensive multilevel metabolomics analysis showed significant dysregulations of several mitochondrial TCA cycle metabolites in PCK rats, which further aggravated with the more severe phenotype. Notably, these changes were associated with mitochondrial structural abnormalities in the CD. Our observations suggest that mitochondrial damage and metabolic dysregulations may be implicated in cystogenesis, and position mitochondria as important therapeutic targets in PKD. Support or Funding Information This study was supported by the Mayo Translational Polycystic Kidney Disease Center (NIDDK P30 DK090728) and an endowment from Robert M. and Billie J. Pirnie for Kidney Research

AbstractBackgroundConsistent failure of clinical trials for Alzheimer Disease (AD) emphasizes the necessity to identify novel therapeutic targets for this debilitating condition. We recently identified the mitochondrial complex I (MCI) as a small molecule druggable therapeutic target for AD.MethodUsing rational design and extensive structure‐activity relationship studies, we developed novel classes of partial MCI inhibitors with high selectivity, specificity, safety, and drug‐like properties. Target engagement, selectivity and specificity of new compounds were established using multiple in vitro assays, including surface plasmon resonance (SPR), competitive and direct binding, proteomics and pull down experiments. Using these techniques, we developed new lead compound C458 that was interrogated in in vivo efficacy studies in APP/PS1 mice.ResultWe demonstrated that the new lead C458 directly binds to MCI. A single dose administration of C458 to mice or treatment of primary mouse neurons activates AMPK‐a, Akt, autophagy, Sirtuins 1 and 3, and upregulates PGC‐1a leading to enhanced mitochondrial biogenesis. C458 has excellent oral bioavailability, blood–brain‐barrier penetrance, and was remarkably clean in the 250 kinase and CEREP 44 safety panels. Chronic administration of C458 (25 mg/kg/day in drinking water ad lib) to APP/PS1 mice for 6 months starting at pre‐symptomatic stage did not cause detectable side effects, and resulted in cognitive protection, improved long‐term potentiation, and decreased oxidative stress and inflammation. Furthermore, APP/PS1 mice treated with C458 showed a significant increase in glucose uptake and utilization in the brain detected with a translational in vivo biomarker FDG‐PET, as well as an enhancement of ATP production measured in vivo using 31P NMR.ConclusionMechanistic studies confirmed that C458 treatment triggered mitochondrial stress response augmenting mitochondrial biogenesis, anti‐oxidant signaling, and cellular energetics that ultimately restored energy brain homeostasis, leading to cognitive protection in a mouse model of AD. Our studies demonstrate that modulation of MCI activity using novel specific MCI inhibitors improves bioenergetics in the brain and averts cognitive decline representing novel therapeutic approach for AD.

AbstractSummary1H nuclear magnetic resonance (NMR) spectroscopy is an established bioanalytical technology for metabolic profiling of biofluids in both clinical and large-scale population screening applications. Recently, urinary protein quantification has been demonstrated using the same 1D 1H NMR experimental data captured for metabolic profiling. Here, we introduce NMRpQuant, a freely available platform that builds on these findings with both novel and further optimized computational NMR approaches for rigorous, automated protein urine quantification. The results are validated by interlaboratory comparisons, demonstrating agreement with clinical/biochemical methodologies, pointing at a ready-to-use tool for routine protein urinalyses.Availability and implementationNMRpQuant was developed on MATLAB programming environment. Source code and Windows/macOS compiled applications are available at https://github.com/pantakis/NMRpQuant, and working examples are available at https://doi.org/10.6084/m9.figshare.18737189.v1.Supplementary informationSupplementary data are available at Bioinformatics online.