Biology subjects

Khalid, U.

Publications and source records attributed to Khalid, U..

4 recordsLinked to original sources

bioRxiv2024

The Hyaluronan Synthase isoforms direct distinct patterns of fibroblast activation linked to fibrosis progression versus resolution following renal ischaemia

BackgroundThe stroma plays a key role during renal development and in regeneration after injury. However, following injury, the stroma expands driving progressive fibrosis. Hyaluronan (HA) is a glycosaminoglycan that is absent in healthy kidney stroma but highly expressed in disease. To understand strategies to modulate stromal HA towards therapeutic advantage, this study compares HA and HA Synthase (HAS) enzyme expression in kidney development, health, disease, and recovery. MethodsRats underwent ischaemia reperfusion injury (IRI) with/without ischaemic preconditioning (IPC) and kidneys histologically analysed. Kidneys from C57BL/6 embryos and HAS1/3-/- mice were also analysed and parallel mechanistic cell studies performed using primary human fibroblasts. ResultsIn health, stromal HA was absent from the renal cortex. HAS1 was expressed in some epithelial cells, whilst HAS2 was not expressed. Following IRI there was increased stromal HA in areas of chronic fibrosis, alongside increased HAS2 (but not HAS1) expression. In contrast, during development prominent stromal HA matrices were evident in areas of tubular generation, with strong HAS1 (not HAS2) expression. Following IPC+IRI, stromal HA and HAS2 were attenuated; whilst HAS1+ cells expanded but were distinct from -SMA+ myofibroblasts. Cell studies demonstrated that HAS1+ fibroblasts had a functionally distinct phenotype, with enhanced migration and FAP expression but attenuated -SMA, EDA-FN and COL1A1 expression, whereas HAS2+ fibroblasts demonstrated a classic -SMA+ contractile myofibroblast phenotype with high EDA-FN and COL1A1. ConclusionsHA is a key regulator of stromal fibroblast heterogeneity, with HAS1 and HAS2 defining phenotypically distinct populations that may influence divergent renal outcomes following injury. SIGNIFICANCE STATEMENTHyaluronan (HA) is a matrix glycosaminoglycans that is absent in healthy kidney cortex but demonstrates increased expression in the renal stroma during progressive fibrosis. This study makes comparisons of HA accumulation, localisation, and HA Synthase (HAS) protein expression during kidney development, in health, following ischaemic kidney injury and during renal recovery. The study identifies that different HAS isoenzymes (HAS1 and HAS2) mediate distinct functional fibroblast phenotypes in vitro and are localised in distinct stromal localisations and cell sub-populations in vivo. The data provides interesting insights into HA dependent regulation of fibroblast stromal heterogeneity and identifies the novel finding that HAS1 defines cell populations that are associated with kidney recovery following ischaemic injury and are protective against progressive renal fibrosis.

pathology↗

bioRxiv2023

Ischaemic Preconditioning attenuates Chronic Renal Damage following Ischaemia Reperfusion Injury

Acute Kidney Injury (AKI) is a common cause of Chronic Kidney Disease (CKD). The leading cause of AKI worldwide is Ischaemia Reperfusion Injury (IRI), seen most commonly in the clinical setting as a result of sepsis-driven hypotension. We are increasingly recognising, however, that AKI and CKD are one closely associated continuum of disease, rather than distinct entities. Ischaemic Preconditioning (IPC) is a strategy aimed at reducing the deleterious effects of IRI. This study demonstrates an efficacious model of kidney IRI, and the protective influence of IPC in attenuating renal injury/fibrosis. A rat model of bilateral kidney IRI was used: Male Lewis rats (n=84) were assigned to IRI, sham or IPC. In IRI, renal pedicles were clamped for 45 minutes. IPC groups underwent pulsatile IPC prior to IRI. Kidneys were retrieved at 24-hours, 48-hours, 7-days, 14-days and 28-days, and assessed histologically. IRI led to marked histological damage and renal fibrosis development by 28 days. Histological injury scores and degree of fibrosis were significantly increased following IRI and attenuated with IPC. IPC resulted in a 66% reduction in renal fibrosis at 28 days (p<0.001). IRI also led to a significant increase in serum creatinine acutely, which was attenuated by IPC (p<0.0001). Interestingly at 14-days, there was limited histological damage and differentiation between IRI and IPC kidneys was difficult. IPC can protect from both acute and chronic kidney damage. 14-days post IRI represents a transitional phase, which maybe a timepoint for commitment to either fibrosis or recovery, and hence offers potential for therapeutic intervention.

pathology↗

bioRxiv2021

A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis

Chromothripsis is a form of genomic instability characterized by the occurrence of tens to hundreds of clustered DNA double-strand breaks in a one-off catastrophic event. Rearrangements associated with chromothripsis are detectable in numerous tumor entities and linked with poor prognosis in some of these, such as Sonic Hedgehog medulloblastoma, neuroblastoma and osteosarcoma. Hence, there is a need for therapeutic strategies eliminating tumor cells with chromothripsis. Defects in DNA double-strand break repair, and in particular homologous recombination repair, have been linked with chromothripsis. Targeting DNA repair deficiencies by synthetic lethality approaches, we performed a synergy screen using drug libraries (n = 375 compounds, 15 models) combined with either a PARP inhibitor or cisplatin. This revealed a synergistic interaction between the HDAC inhibitor romidepsin and PARP inhibition. Functional assays, transcriptome analyses, and in vivo validation in patient-derived xenograft mouse models confirmed the efficacy of the combinatorial treatment.

cancer biology↗

bioRxiv2020

A versatile system to introduce clusters of genomic double-strand breaks in large cell populations

In vitro assays for clustered DNA lesions will facilitate the analysis of the mechanisms underlying complex genome rearrangements such as chromothripsis, including the recruitment of repair factors to sites of DNA double-strand breaks. We present a novel method generating localized DNA double-strand breaks using UV-irradiation with photomasks. The size of the damage foci and the spacing between lesions are fully adjustable, making the assay suitable for different cell types and targeted areas. We validated this set-up with genomically stable epithelial cells, normal fibroblasts, pluripotent stem cells and patient-derived primary cultures. Our method does not require a specialized device such as a laser, making it accessible to a broad range of users. Sensitization by BrdU incorporation is not required, which enables analyzing the DNA damage response in post-mitotic cells. Irradiated cells can be cultivated further, followed by time-lapse imaging or used for downstream biochemical analyses, thanks to the high-throughput of the system. Importantly, we showed genome rearrangements in the irradiated cells, providing a proof of principle for the induction of structural variants by localized DNA lesions.

genomics↗