Biology subjects

Russell, L. G.

Publications and source records attributed to Russell, L. G..

3 recordsLinked to original sources

bioRxiv2024

Reversal of elevated Gli3 in Autosomal Recessive Polycystic Kidney Disease does not alter cystogenesis

Polycystic kidney diseases (PKD) are genetic disorders characterised by the formation of fluid-filled cysts, which disrupt kidney architecture and function. Autosomal recessive PKD (ARPKD) is a rare form of PKD, caused by mutations in PKHD1, and clinically more severe than the more common autosomal dominant PKD (ADPKD). Prior studies have implicated the ciliary-located Hedgehog (Hh) pathway in ADPKD, with increased levels of Hh components in experimental ADPKD models, and reduced cystogenesis following pharmacological Hh inhibition. In contrast, the role of the Hh pathway in ARPKD is poorly understood. We hypothesised that Hh pathway activity would be elevated during ARPKD pathogenesis, and its modulation may inhibit cystogenesis, akin to prior findings in ADPKD. To test this, we utilised Cpk mice, a model which replicates the pathophysiology of ARPKD, and generated a human cellular ARPKD 3-dimensional cystogenesis model by mutating PKHD1 in human collecting duct cells through CRISPR-Cas9 technology. We found significantly elevated levels of the Hh transcriptional effector Gli3 in the Cpk mouse, a finding replicated in our human cellular ARPKD model. In the Cpk mouse, we also observed an increase in total GLI3 and GLI3 repressor protein levels. However, reduction of increased Gli3 levels via genetic deletion in the Cpk mouse did not affect cyst formation. Similarly, lowering GLI3 transcript to wildtype levels, did not influence cyst size in our human cellular ARPKD model. Collectively, these data show that elevated Gli3 does not modulate cyst progression in the context of ARPKD, highlighting the complexity of the Hh pathway in PKD. New and NoteworthyThe role of the Hedgehog pathway in autosomal recessive polycystic kidney disease (ARPKD) is poorly understood. Here, we describe elevated levels of Gli3, the Hedgehog transcriptional effector, in murine and human ARPKD models. However, reversal of the increase in Gli3 did not significantly affect cystogenesis in a human cell model of ARPKD or disease progression in a mouse model which replicates ARPKD pathophysiology. Collectively, our data indicates that Gli3 does not modulate ARPKD progression.

physiology↗

bioRxiv2024

Nanobody immunolabelling and three-dimensional imaging reveals spatially restricted LYVE1 expression by kidney lymphatic vessels in mice

Lymphatic vessels are complex three-dimensional (3D) structures that facilitate tissue fluid clearance and regulate immune responses in health and inflammatory contexts. Recent advances in wholemount immunolabelling and 3D imaging have provided insights into organ-specific heterogeneity of lymphatic structure and function. However, the visualisation of lymphatic vessels deep within an intact organ remains a challenge. We hypothesised that nanobodies, single-domain antibodies raised in camelid species, would result in improved labelling of lymphatics in intact mouse organs, without loss of information due to organ sectioning or inadequate penetration. We generated and characterised nanobody clones targeting lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), a marker of lymphatic capillaries. Nanobodies were superior at penetrating whole mouse organs and enhanced labelling of lymphatics compared with a conventional anti-LYVE1 polyclonal antibody. Utilising this new tool, we found that kidney lymphatics; an organ in which labelling of lymphatics is challenging, have spatially restricted LYVE1 expression compared with lymphatics of skin, heart, and lung. The timing of this LYVE1 spatial restriction coincides with the early postnatal period in mice. Our findings highlight an unexpected, organ-specific characteristic of kidney lymphatic vessels, whilst providing a novel experimental tool for characterisation, isolation, or perturbation of lymphatic vessels in health and disease.

immunology↗

bioRxiv2022

Three-dimensional imaging and single-cell transcriptomics of the human kidney implicate perturbation of lymphatics in alloimmunity

Studies of the structural and molecular features of the lymphatic vasculature, which clears fluid, macromolecules and leukocytes from the tissue microenvironment, have largely relied on animal models, with limited information in human organs beyond traditional immunohistochemical assessment. Here, we use three-dimensional imaging and single-cell RNA-sequencing to study lymphatics in the human kidney. We found a hierarchical arrangement of lymphatic vessels within human kidneys, initiating along specialised nephron epithelium in the renal cortex and displaying a distinct, kidney-specific transcriptional profile. In chronic transplant rejection we found kidney allograft lymphatic expansion alongside a loss of structural hierarchy, with human leukocyte antigen-expressing lymphatic vessels infiltrating the medulla, presenting a putative target for alloreactive antibodies. This occurred concurrently with lymphatic vessels invading and interconnecting tertiary lymphoid structures at early stages of lymphocyte colonisation. Analysis of intercellular signalling revealed upregulation of co-inhibitory molecule-mediated CD4+ T cell-lymphatic crosstalk in rejecting kidneys, potentially acting to limit local alloimmune responses. Overall, we delineate novel structural and molecular features of human kidney lymphatics and reveal perturbations to their phenotype and transcriptome in the context of alloimmunity. SUMMARYLymphatics regulate fluid balance and immune cell accumulation but are under-studied in human organs such as the kidney. Jafree and colleagues profiled human kidney lymphatics using three-dimensional imaging and single-cell RNA-sequencing, revealing structural and transcriptional perturbations in rejecting kidney transplants.

immunology↗