Single-cell transcriptomics identifies aberrant glomerular angiogenic signalling in the early stages of WT1 kidney disease.

WT1 encodes a podocyte transcription factor whose variants can cause an untreatable glomerular disease in early childhood. Although WT1 regulates many podocyte genes, it is poorly understood which of them are initiators in disease and how they subsequently influence other cell-types in the glomerulus. We hypothesised that this could be resolved using single-cell RNA sequencing (scRNA-seq) and ligand-receptor analysis to profile glomerular cell-cell communication during the early stages of disease in mice harbouring an orthologous human mutation in WT1 (Wt1R394W/+). Podocytes were the most dysregulated cell-type in the early stages of Wt1R394W/+ disease, with disrupted angiogenic signalling between podocytes and the endothelium, including the significant downregulation of transcripts for the vascular factors Vegfa and Nrp1. These signalling changes preceded glomerular endothelial cell loss in advancing disease, a feature also observed in biopsy samples from human WT1 glomerulopathies. Addition of conditioned medium from murine Wt1R394W/+ primary podocytes to wild-type glomerular endothelial cells resulted in impaired endothelial looping and reduced vascular complexity. Despite the loss of key angiogenic molecules in Wt1R394W/+ podocytes, the pro-vascular molecule adrenomedullin was upregulated in Wt1R394W/+ podocytes and plasma and its further administration was able to rescue the impaired looping observed when glomerular endothelium was exposed to Wt1R394W/+ podocyte medium. In comparative analyses, adrenomedullin upregulation was part of a common injury signature across multiple murine and human glomerular disease datasets, whilst other gene changes were unique to WT1 disease. Collectively, our study describes a novel role for altered angiogenic signalling in the initiation of WT1 glomerulopathy. We also identify adrenomedullin as a proangiogenic factor, which despite being upregulated in early injury, offers an insufficient protective response due to the wider milieu of dampened vascular signalling that results in endothelial cell loss in later disease. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

SGLT1 contributes to glucose-mediated exacerbation of ischemia-reperfusion injury in ex vivo rat heart.

Hyperglycaemia is common during acute coronary syndromes (ACS) irrespective of diabetic status and portends excess infarct size and mortality, but the mechanisms underlying this effect are poorly understood. We hypothesized that sodium/glucose linked transporter-1 (SGLT1) might contribute to the effect of high-glucose during ACS and examined this using an ex-vivo rodent heart model of ischaemia-reperfusion injury. Langendorff-perfused rat hearts were subjected to 35 min ischemia and 2 h reperfusion, with variable glucose and reciprocal mannitol given during reperfusion in the presence of pharmacological inhibitors of SGLT1. Myocardial SGLT1 expression was determined in rat by rtPCR, RNAscope and immunohistochemistry, as well as in human by single-cell transcriptomic analysis. High glucose in non-diabetic rat heart exacerbated reperfusion injury, significantly increasing infarct size from 45 ± 3 to 65 ± 4% at 11-22 mmol/L glucose, respectively (p < 0.01), an association absent in diabetic heart (32 ± 1-37 ± 5%, p = NS). Rat heart expressed SGLT1 RNA and protein in vascular endothelium and cardiomyocytes, with similar expression found in human myocardium by single-nucleus RNA-sequencing. Rat SGLT1 expression was significantly reduced in diabetic versus non-diabetic heart (0.608 ± 0.08 compared with 1.116 ± 0.13 probe/nuclei, p < 0.01). Pharmacological inhibitors phlorizin, canagliflozin or mizagliflozoin in non-diabetic heart revealed that blockade of SGLT1 but not SGLT2, abrogated glucose-mediated excess reperfusion injury. Elevated glucose is injurious to the rat heart during reperfusion, exacerbating myocardial infarction in non-diabetic heart, whereas the diabetic heart is resistant to raised glucose, a finding which may be explained by lower myocardial SGLT1 expression. SGLT1 is expressed in vascular endothelium and cardiomyocytes and inhibiting SGLT1 abrogates excess glucose-mediated infarction. These data highlight SGLT1 as a potential clinical translational target to improve morbidity/mortality outcomes in hyperglycemic ACS patients.

Cardiotrophin-1 therapy reduces disease severity in a murine model of glomerular disease.

Cardiotrophin-1 (CT-1), a member of the interleukin (IL)-6 cytokine family, has renoprotective effects in mouse models of acute kidney disease and tubulointerstitial fibrosis, but its role in glomerular disease is unknown. To address this, we used the mouse model of nephrotoxic nephritis to test the hypothesis that CT-1 also has a protective role in immune-mediated glomerular disease. Using immunohistochemistry and analysis of single-cell RNA-sequencing data of isolated glomeruli, we demonstrate that CT-1 is expressed in the glomerulus in male mice, predominantly in parietal epithelial cells and is downregulated in mice with nephrotoxic nephritis. Furthermore, analysis of data from patients revealed that human glomerular disease is also associated with reduced glomerular CT-1 transcript levels. In male mice with nephrotoxic nephritis and established proteinuria, administration of CT-1 resulted in reduced albuminuria, prevented podocyte loss, and sustained plasma creatinine, compared with mice administered saline. CT-1 treatment also reduced fibrosis in the kidney cortex, peri-glomerular macrophage accumulation and the kidney levels of the pro-inflammatory mediator complement component 5a. In conclusion, CT-1 intervention therapy delays the progression of glomerular disease in mice by preserving kidney function and inhibiting renal inflammation and fibrosis.

Single-modulator, dual comb serrodyne spectroscopy.

Dual optical frequency comb spectroscopy allows for high speed, broadband measurements without any moving parts. Here, we combine differential chirp downconversion to probe large spectral bandwidths and serrodyne modulation to separate the positive and negative sidebands in a single modulator. As an initial demonstration, we apply this approach to measure a sharp cavity resonance to illustrate the system performance. We then measure methane transitions in the near-infrared and compare the resulting spectra to models based upon the current spectroscopic databases. The serrodyne method has lower hardware requirements compared to many existing approaches, and its simplicity enables a high degree of mutual coherence between the two combs. Further, this method is readily amenable to chip-scale photonic integration.

A microfluidic platform integrating functional vascularized organoids-on-chip.

The development of vascular networks in microfluidic chips is crucial for the long-term culture of three-dimensional cell aggregates such as spheroids, organoids, tumoroids, or tissue explants. Despite rapid advancement in microvascular network systems and organoid technologies, vascularizing organoids-on-chips remains a challenge in tissue engineering. Most existing microfluidic devices poorly reflect the complexity of in vivo flows and require complex technical set-ups. Considering these constraints, we develop a platform to establish and monitor the formation of endothelial networks around mesenchymal and pancreatic islet spheroids, as well as blood vessel organoids generated from pluripotent stem cells, cultured for up to 30 days on-chip. We show that these networks establish functional connections with the endothelium-rich spheroids and vascular organoids, as they successfully provide intravascular perfusion to these structures. We find that organoid growth, maturation, and function are enhanced when cultured on-chip using our vascularization method. This microphysiological system represents a viable organ-on-chip model to vascularize diverse biological 3D tissues and sets the stage to establish organoid perfusions using advanced microfluidics.

Micro to macro scale analysis of the intact human renal arterial tree with Synchrotron Tomography.

Background: The kidney vasculature is exquisitely structured to orchestrate renal function. Structural profiling of the vasculature in intact rodent kidneys, has provided insights into renal haemodynamics and oxygenation, but has never been extended to the human kidney beyond a few vascular generations. We hypothesised that synchrotron-based imaging of a human kidney would enable assessment of vasculature across the whole organ. Methods: An intact kidney from a 63-year-old male was scanned using hierarchical phase-contrast tomography (HiP-CT), followed by semi-automated vessel segmentation and quantitative analysis. These data were compared to published micro-CT data of whole rat kidney. Results: The intact human kidney vascular network was imaged with HiP-CT at 25 µm voxels, representing a 20-fold increase in resolution compared to clinical CT scanners. Our comparative quantitative analysis revealed the number of vessel generations, vascular asymmetry and a structural organisation optimised for minimal resistance to flow, are conserved between species, whereas the normalised radii are not. We further demonstrate regional heterogeneity in vessel geometry between renal cortex, medulla, and hilum, showing how the distance between vessels provides a structural basis for renal oxygenation and hypoxia. Conclusions: Through the application of HiP-CT, we have provided the first quantification of the human renal arterial network, with a resolution comparable to that of light microscopy yet at a scale several orders of magnitude larger than that of a renal punch biopsy. Our findings bridge anatomical scales, profiling blood vessels across the intact human kidney, with implications for renal physiology, biophysical modelling, and tissue engineering.

Intrinsically accurate sensing with an optomechanical accelerometer.

We demonstrate a microfabricated optomechanical accelerometer that is capable of percent-level accuracy without external calibration. To achieve this capability, we use a mechanical model of the device behavior that can be characterized by the thermal noise response along with an optical frequency comb readout method that enables high sensitivity, high bandwidth, high dynamic range, and SI-traceable displacement measurements. The resulting intrinsic accuracy was evaluated over a wide frequency range by comparing to a primary vibration calibration system and local gravity. The average agreement was found to be 2.1 % for the calibration system between 0.1 kHz and 15 kHz and better than 0.2 % for the static acceleration. This capability has the potential to replace costly external calibrations and improve the accuracy of inertial guidance systems and remotely deployed accelerometers. Due to the fundamental nature of the intrinsic accuracy approach, it could be extended to other optomechanical transducers, including force and pressure sensors.

Systemic gene therapy with thymosin ß4 alleviates glomerular injury in mice.

Plasma ultrafiltration in the kidney occurs across glomerular capillaries, which are surrounded by epithelial cells called podocytes. Podocytes have a unique shape maintained by a complex cytoskeleton, which becomes disrupted in glomerular disease resulting in defective filtration and albuminuria. Lack of endogenous thymosin ß4 (TB4), an actin sequestering peptide, exacerbates glomerular injury and disrupts the organisation of the podocyte actin cytoskeleton, however, the potential of exogenous TB4 therapy to improve podocyte injury is unknown. Here, we have used Adriamycin (ADR), a toxin which injures podocytes and damages the glomerular filtration barrier leading to albuminuria in mice. Through interrogating single-cell RNA-sequencing data of isolated glomeruli we demonstrate that ADR injury results in reduced levels of podocyte TB4. Administration of an adeno-associated viral vector encoding TB4 increased the circulating level of TB4 and prevented ADR-induced podocyte loss and albuminuria. ADR injury was associated with disorganisation of the podocyte actin cytoskeleton in vitro, which was ameliorated by treatment with exogenous TB4. Collectively, we propose that systemic gene therapy with TB4 prevents podocyte injury and maintains glomerular filtration via protection of the podocyte cytoskeleton thus presenting a novel treatment strategy for glomerular disease.

The timing of auditory sensory deficits in Norrie disease has implications for therapeutic intervention.

Norrie disease is caused by mutation of the NDP gene, presenting as congenital blindness followed by later onset of hearing loss. Protecting patients from hearing loss is critical for maintaining their quality of life. This study aimed to understand the onset of pathology in cochlear structure and function. By investigating patients and juvenile Ndp-mutant mice, we elucidated the sequence of onset of physiological changes (in auditory brainstem responses, distortion product otoacoustic emissions, endocochlear potential, blood-labyrinth barrier integrity) and determined the cellular, histological, and ultrastructural events leading to hearing loss. We found that cochlear vascular pathology occurs earlier than previously reported and precedes sensorineural hearing loss. The work defines a disease mechanism whereby early malformation of the cochlear microvasculature precedes loss of vessel integrity and decline of endocochlear potential, leading to hearing loss and hair cell death while sparing spiral ganglion cells. This provides essential information on events defining the optimal therapeutic window and indicates that early intervention is needed. In an era of advancing gene therapy and small-molecule technologies, this study establishes Ndp-mutant mice as a platform to test such interventions and has important implications for understanding the progression of hearing loss in Norrie disease.

Kidney organoids recapitulate human basement membrane assembly in health and disease.

Basement membranes (BMs) are complex macromolecular networks underlying all continuous layers of cells. Essential components include collagen IV and laminins, which are affected by human genetic variants leading to a range of debilitating conditions including kidney, muscle, and cerebrovascular phenotypes. We investigated the dynamics of BM assembly in human pluripotent stem cell-derived kidney organoids. We resolved their global BM composition and discovered a conserved temporal sequence in BM assembly that paralleled mammalian fetal kidneys. We identified the emergence of key BM isoforms, which were altered by a pathogenic variant in COL4A5. Integrating organoid, fetal, and adult kidney proteomes, we found dynamic regulation of BM composition through development to adulthood, and with single-cell transcriptomic analysis we mapped the cellular origins of BM components. Overall, we define the complex and dynamic nature of kidney organoid BM assembly and provide a platform for understanding its wider relevance in human development and disease.

Experimental long-term diabetes mellitus alters the transcriptome and biomechanical properties of the rat urinary bladder.

Diabetes mellitus (DM) is the leading cause of chronic kidney disease and diabetic nephropathy is widely studied. In contrast, the pathobiology of diabetic urinary bladder disease is less understood despite dysfunctional voiding being common in DM. We hypothesised that diabetic cystopathy has a characteristic molecular signature. We therefore studied bladders of hyperglycaemic and polyuric rats with streptozotocin (STZ)-induced DM. Sixteen weeks after induction of DM, as assessed by RNA arrays, wide-ranging changes of gene expression occurred in DM bladders over and above those induced in bladders of non-hyperglycaemic rats with sucrose-induced polyuria. The altered transcripts included those coding for extracellular matrix regulators and neural molecules. Changes in key genes deregulated in DM rat bladders were also detected in db/db mouse bladders. In DM rat bladders there was reduced birefringent collagen between detrusor muscle bundles, and atomic force microscopy showed a significant reduction in tissue stiffness; neither change was found in bladders of sucrose-treated rats. Thus, altered extracellular matrix with reduced tissue rigidity may contribute to voiding dysfunction in people with long-term DM. These results serve as an informative stepping stone towards understanding the complex pathobiology of diabetic cystopathy.

Identification of an Altered Matrix Signature in Kidney Aging and Disease.

BACKGROUND: Accumulation of extracellular matrix in organs and tissues is a feature of both aging and disease. In the kidney, glomerulosclerosis and tubulointerstitial fibrosis accompany the decline in function, which current therapies cannot address, leading to organ failure. Although histologic and ultrastructural patterns of excess matrix form the basis of human disease classifications, a comprehensive molecular resolution of abnormal matrix is lacking. METHODS: Using mass spectrometry-based proteomics, we resolved matrix composition over age in mouse models of kidney disease. We compared the changes in mice with a global characterization of human kidneymatrix during aging and to existing kidney disease datasets to identify common molecular features. RESULTS: Ultrastructural changes in basement membranes are associated with altered cell adhesion and metabolic processes and with distinct matrix proteomes during aging and kidney disease progression in mice. Within the altered matrix, basement membrane components (laminins, type IV collagen, type XVIII collagen) were reduced and interstitial matrix proteins (collagens I, III, VI, and XV; fibrinogens; and nephronectin) were increased, a pattern also seen in human kidney aging. Indeed, this signature of matrix proteins was consistently modulated across all age and disease comparisons, and the increase in interstitial matrix was also observed in human kidney disease datasets. CONCLUSIONS: This study provides deep molecular resolution of matrix accumulation in kidney aging and disease, and identifies a common signature of proteins that provides insight into mechanisms of response to kidney injury and repair.

Mechanisms and cell lineages in lymphatic vascular development.

Lymphatic vessels have critical roles in both health and disease and their study is a rapidly evolving area of vascular biology. The consensus on how the first lymphatic vessels arise in the developing embryo has recently shifted. Originally, they were thought to solely derive by sprouting from veins. Since then, several studies have uncovered novel cellular mechanisms and a diversity of contributing cell lineages in the formation of organ lymphatic vasculature. Here, we review the key mechanisms and cell lineages contributing to lymphatic development, discuss the advantages and limitations of experimental techniques used for their study and highlight remaining knowledge gaps that require urgent attention. Emerging technologies should accelerate our understanding of how lymphatic vessels develop normally and how they contribute to disease.

The Biological Significance and Implications of Planar Cell Polarity for Nephrology.

The orientation of cells in two-dimensional and three-dimensional space underpins how the kidney develops and responds to disease. The process by which cells orientate themselves within the plane of a tissue is termed planar cell polarity. In this Review, we discuss how planar cell polarity and the proteins that underpin it govern kidney organogenesis and pathology. The importance of planar cell polarity and its constituent proteins in multiple facets of kidney development is emphasised, including ureteric bud branching, tubular morphogenesis and nephron maturation. An overview is given of the relevance of planar cell polarity and its proteins for inherited human renal diseases, including congenital malformations with unknown aetiology and polycystic kidney disease. Finally, recent work is described outlining the influence of planar cell polarity proteins on glomerular diseases and highlight how this fundamental pathway could yield a new treatment paradigm for nephrology.

Near-infrared cavity ring-down spectroscopy measurements of nitrous oxide in the (4200)←(0000) and (5000)←(0000) bands.

Using frequency-agile rapid scanning cavity ring-down spectroscopy, we measured line intensities and line shape parameters of 14N2 16O in air in the (4200)←(0000) and (5000)←(0000) bands near 1.6 µm. The absorption spectra were modeled with multi-spectrum fits of Voigt and speed-dependent Voigt profiles. The measured line intensities and air-broadening parameters exhibit deviations of several percent relative to values provided in HITRAN 2016. Our measured intensities for these two bands have relative combined standard uncertainties of ∼1% which is approximately five times smaller than literature values. Comparison of the present air-broadening and speed-dependent broadening parameters to experimental literature values for other rotation-vibration bands of N2O indicates significant differences in magnitude and J-dependence. For applications requiring high spectral fidelity, these results suggest that the assumption of band-independent line shape parameters is not appropriate.

Improvement of the spectroscopic parameters of the air- and self-broadened N2O and CO lines for the HITRAN2020 database applications.

This paper outlines the major updates of the line-shape parameters that were performed for the nitrous oxide (N2O) and carbon monoxide (CO) molecules listed in the HITRAN2020 database. We reviewed the collected measurements for the air- and self-broadened N2O and CO spectra to determine proper values for the spectroscopic parameters. Careful comparisons of broadening parameters using the Voigt and speed-dependent Voigt line-shape profiles were performed among various published results for both N2O and CO. Selected data allowed for developing semi-empirical models, which were used to extrapolate/interpolate existing data to update broadening parameters of all the lines of these molecules in the HITRAN database. In addition to the line broadening parameters (and their temperature dependences), the pressure shift values were revised for N2O and CO broadened by air and self for all the bands. The air and self speed-dependence of the broadening parameter for these two molecules were added for every transition as well. Furthermore, we determined the first-order line-mixing parameters using the Exponential Power Gap (EPG) scaling law. These new parameters are now available at HITRAN online.

Air-broadening in near-infrared carbon dioxide line shapes: Quantifying contributions from O2, N2, and Ar.

We measured air broadening in the (30012) ← (00001) carbon dioxide (CO2) band up to J″=50 using frequency-agile rapid scanning cavity ring-down spectroscopy. By using synthetic air samples with varying levels of nitrogen, oxygen, and argon, multi-spectrum fitting allowed for the collisional broadening terms of each major air component to be simultaneously determined in addition to advanced line shape parameters at atmospherically relevant CO2 mixing ratios. These values were compared to broadener-specific line shape parameters from the literature. Fits to measured spectra were also constrained with results from requantized classical molecular dynamic simulations. We show that this approach enables differentiation between narrowing mechanisms in advanced line shape parameters retrieved from experimental spectra of limited signal-to-noise ratio.

Beyond a Passive Conduit: Implications of Lymphatic Biology for Kidney Diseases.

The kidney contains a network of lymphatic vessels that clear fluid, small molecules, and cells from the renal interstitium. Through modulating immune responses and via crosstalk with surrounding renal cells, lymphatic vessels have been implicated in the progression and maintenance of kidney disease. In this Review, we provide an overview of the development, structure, and function of lymphatic vessels in the healthy adult kidney. We then highlight the contributions of lymphatic vessels to multiple forms of renal pathology, emphasizing CKD, transplant rejection, and polycystic kidney disease and discuss strategies to target renal lymphatics using genetic and pharmacologic approaches. Overall, we argue the case for lymphatics playing a fundamental role in renal physiology and pathology and treatments modulating these vessels having therapeutic potential across the spectrum of kidney disease.

Endothelial-epithelial communication in polycystic kidney disease: Role of vascular endothelial growth factor signalling.

Whereas targeting the cyst epithelium and its molecular machinery has been the prevailing clinical strategy for polycystic kidney disease, the endothelium, including blood vasculature and lymphatics, is emerging as an important player in this disorder. In this Review, we provide an overview of the structural and functional alterations to blood vasculature and lymphatic vessels in the polycystic kidney. We also discuss evidence for vascular endothelial growth factor signalling, otherwise critical for endothelial cell development and maintenance, as being a fundamental molecular pathway in polycystic kidney disease and a potential therapeutic target for modulating cyst expansion.

Free 25-hydroxyvitamin-D concentrations are lower in children with renal transplant compared with chronic kidney disease.

BACKGROUND: Total serum 25-hydroxyvitamin D [25(OH)D] is considered the best marker of vitamin D status and used routinely in clinical practice. However, 25(OH)D is predominantly bound to vitamin D-binding protein (VDBP), and it has been reported that the free-25(OH)D and 25(OH)D loosely bound to albumin fraction correlates better with clinical outcomes. METHODS: We assessed total-25(OH)D, measured free-25(OH)D, and calculated free-25(OH)D and their relationship with VDBP and biomarkers of mineral metabolism in 61 children (22 CKD 2-3, 18 dialysis, and 21 post-transplant). RESULTS: Total-25(OH)D concentrations were comparable across the three groups (p = 0.09), but free- and bioavailable-25(OH)D (free- and albumin-25(OH)D) were significantly lower in the transplant group (both: p = 0.01). Compared to CKD and dialysis patients, the transplant group had significantly higher VDBP concentrations (p = 0.03). In all three groups, total-25(OH)D concentrations were positively associated with measured free-, calculated free-, and bioavailable-25(OH)D. Multivariable regression analysis showed that total-25(OH)D was the only predictor of measured free-25(OH)D concentrations in the dialysis group (ß = 0.9; R2 = 90%). In the transplant group, measured free-25(OH)D concentrations were predicted by both total-25(OH)D and VDBP concentrations (ß = 0.6, - 0.6, respectively; R2 = 80%). Correlations between parathyroid hormone with total-25(OH)D and measured and calculated free-25(OH)D were only observed in the transplant group (all: p < 0.001). CONCLUSIONS: In transplanted patients, VDBP concentrations were significantly higher compared to CKD and dialysis patients, and consequently, free-25(OH)D concentrations were lower, despite a comparable total-25(OH)D concentration. We suggest that free-25(OH)D measures may be required in children with CKD, dialysis, and transplant, with further research required to understand its association with markers of mineral metabolism.