Andrew J. Kassianos’s research while affiliated with Royal Brisbane Hospital and other places

Background: Metastatic renal cell carcinoma (RCC) is often treated with a combination of immunotherapy and tyrosine kinase inhibitors (TKIs). Patient-derived RCC cells were cultured and inter-individual differences to treatment with a panel of TKIs were evaluated. Methods: Tumor tissue was collected during nephrectomy. Cells were cultured and treated with a panel of clinically relevant TKIs (sunitinib, cabozantinib, pazopanib, axitinib) at concentrations of 5 µM for 48-72 hours. Cell viability was evaluated using MTT assays. One-sided T-tests were used to evaluate results. Results: Patient-derived cancer cells were able to be grown beyond 10 passages from 12/38 samples collected (27%). Four patient-derived samples were tested against the TKI panel. No substantial difference between drugs was seen for two samples. In one sample, there was a clear superior response to sunitinib (48% mean viability, vs >75% for the other drugs). For the final sample, sunitinib, cabozantinib, and axitinib demonstrated a superior response compared with pazopanib (71%, 77%, 70%, and 85% mean viability, respectively). Conclusions: Inter-individual variability in the responses of patient-derived RCC cultures to TKIs was seen, which may have biological and clinical significance. Future directions could build on this work to develop personalized cancer susceptibility profiles, with potential for translation into a clinical trial.

Tubulointerstitial fibrosis is a characteristic hallmark of chronic kidney disease (CKD). Metabolic perturbations in cellular energy metabolism contribute to the pathogenesis of CKD, but the chemical contributors remain unclear. The aim of this investigation was to use two dimensional ¹H‐nuclear magnetic resonance (2D‐COSY) metabolomics to identify the chemical changes of kidney fibrogenesis. An in vitro transforming growth factor‐β1 (TGF‐β1)‐induced model of kidney fibrogenesis with human kidney‐2 (HK‐2) proximal tubular epithelial cells (PTEC) was used. The model was validated by assaying for various pro‐fibrotic molecules, using quantitative PCR and Western blotting. 2D‐COSY was performed on treated cells. Morphological and functional changes characteristic of tubulointerstitial fibrosis were confirmed in the model; expression of fibronectin, collagen type IV, smooth muscle actin, oxidative stress enzymes increased (p < 0.05). NMR metabolomics provided evidence of altered metabolite signatures associated with glycolysis and glutamine metabolism, with decreased myo‐inositol and choline, and metabolites of the oxidative phase of the pentose phosphate pathway with increased glucose and glucuronic acid. The altered PTEC cellular metabolism likely supports the rapid fibrogenic energy demands. These results, using 2D‐COSY metabolomics, support development of a biomarker panel of fibrosis detectable using clinical magnetic resonance spectroscopy to diagnose and manage CKD.

Heterozygous familial hypercholesterolaemia is one of the most common genetic conditions leading to premature atherosclerotic cardiovascular disease. It can be diagnosed using a combination of clinical, biochemical, and genetic tools. Most guidelines recommend screening during childhood and treatment from the age of 8–10 years. However, screening remains sporadic in most countries and the majority of individuals remain undiagnosed. Registry studies have highlighted the ongoing delayed and low percentage of detection of FH in children. Universal early childhood screening models utilising a combination of biomarker-based and genetic testing have been trialled and are in practice in some countries. Newborn screening is a public health success story and one of the most effective public health measures. It offers universal screening for conditions that can result in significant morbidity or even death if left untreated. There has been renewed interest in including familial hypercholesterolaemia in newborn screening programmes. Using cord blood to identify familial hypercholesterolaemia has not yielded convincing results. However, novel screening approaches on dried blood spots that include biomarker-based lipid profile testing alone, in combination with confirmatory genetic testing, or first-line genetic testing have shown promising results. This provides the opportunity of early diagnosis and treatment of infants and their extended families. However, challenges are associated with the inclusion of familial hypercholesterolaemia in newborn screening programmes with significant impacts on the newborn, family members, and public health.

Hypoxia and interleukin (IL)-1β are independent mediators of tubulointerstitial fibrosis, the histological hallmark of chronic kidney disease (CKD). Here, we examine how hypoxia and IL-1β act in synergy to augment maladaptive proximal tubular epithelial cell (PTEC) repair in human CKD. Ex vivo patient-derived PTECs were cultured under normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions in the absence or presence of IL-1β and examined for maladaptive repair signatures. Hypoxic PTECs incubated with IL-1β displayed a discrete transcriptomic profile distinct from PTECs cultured under hypoxia alone, IL-1β alone or under normoxia. Hypoxia+IL-1β-treated PTECs had 692 ‘unique’ differentially expressed genes (DEGs) compared to normoxic PTECs, with ‘cell cycle’ the most significantly enriched KEGG pathway based on ‘unique’ down-regulated DEGs (including CCNA2 , CCNB1 and CCNB2 ). Hypoxia+IL-1β-treated PTECs displayed signatures of cellular senescence, with reduced proliferation, G2/M cell cycle arrest, increased p21 expression, elevated senescence-associated β-galactosidase (SA-β-gal) activity and increased production of pro-inflammatory/fibrotic senescence-associated secretory phenotype (SASP) factors compared to normoxic conditions. Treatment of Hypoxia+IL-1β-treated PTECs with either a type I IL-1 receptor (IL-1RI) neutralizing antibody or a senolytic drug combination, quercetin+dasatinib, attenuated senescent cell burden. In vitro findings were validated in human CKD bio-specimens (kidney tissue, urine), with elevated PTEC IL-1RI expression and senescence (SA-β-gal activity) detected in fibrotic kidneys and numbers of senescent (SA-β-gal ⁺ ) urinary PTECs correlating with urinary IL-1β levels and severity of interstitial fibrosis. Our data identify a mechanism whereby hypoxia in combination with IL-1β/IL-1RI signalling trigger PTEC senescence, providing novel therapeutic and diagnostic check-points for restoring tubular regeneration in human CKD.

Kidney aging is the loss of tissue and organ function over time. Cellular senescence is a state of permanent growth arrest and cessation of cell division. Although aging and senescence share some features, the two processes differ. In contrast to aging, senescence occurs when a cell irreversibly exits the cell cycle and assumes a distinct phenotype. This chapter on nephron senescence and mechanisms explores the proposition that senescence in the kidney is more than just normal organ aging but instead encompasses pathobiological pathways that are activated by injury and result in pathologic alterations in kidney tissue and function. We discuss the role of cellular senescence in a variety of kidney diseases and consider the potential therapeutic benefits of targeting senescent cells in diseased and aged kidneys.

Introduction Fabry disease (FD) results from pathogenic GLA variants, leading to a deficiency in lysosomal α-galactosidase A (α-Gal A) and accumulation of the sphingolipid globotriaosylceramide (Gb3). This leads to severe renal and cardiovascular complications, primarily affecting kidney podocytes. As a multisystemic disorder, FD initiates at the cellular level; however, the mechanism(s) underlying Gb3-induced cell dysfunction remain largely unknown. This study aimed to identify potential drivers of FD and explore the underlying cell pathology in induced pluripotent stem cell (iPSC)–derived podocytes from patients with FD. Methods iPSCs were derived from patients with FD with GLAc.851T>C or GLAc.1193_1196del variants and compared with controls or CRISPR-Cas9–corrected cell lines. iPSCs were differentiated into podocytes; and α-Gal A activity, Gb3 accumulation, and cell morphology were assessed. Label-free mass spectrometry identified the top, differentially expressed proteins which were validated by using western blot. Results Podocytes derived from patients with FD exhibited expression of podocyte-specific markers and morphological features of FD. Reduced α-Gal A activity was observed in FD iPSC-derived podocytes along with the accumulation of Gb3. Proteomic profiling revealed distinct proteomic signatures between control and iPSC-derived podocytes from a patient with FD, with apparent variations among FD lines, highlighting GLA variant–specific proteomic alterations. Notably, the ferroptosis-associated protein, arachidonate 15-lipoxygenase (ALOX15), was the most upregulated protein in FD podocytes and ferroptosis was the most enriched pathway. Western blot analysis confirmed the upregulation of ALOX15 in FD podocytes, with validation of other markers implicating ferroptosis in FD pathology. Conclusion These findings underscore the heterogeneity of FD and, for the first time, implicate ferroptosis as a potential common pathway driving its pathology.

Chronic kidney disease (CKD) is the progressive loss of kidney function/structure over a period of at least 3 months. It is characterised histologically by the triad of cell loss, inflammation and fibrosis. This literature review focuses on the forms of cell death that trigger downstream inflammation and fibrosis, collectively called regulated cell death (RCD) pathways. Discrete forms of RCD have emerged as central mediators of CKD pathology. In particular, pathways of regulated necrosis – including mitochondrial permeability transition pore (mPTP)-mediated necrosis, necroptosis, ferroptosis and pyroptosis – have been shown to mediate kidney pathology directly or through the release of danger signals that trigger a pro-inflammatory response, further amplifying tissue injury in a cellular process called necroinflammation. Despite accumulating evidence in pre-clinical models, no clinical studies have yet targeted these RCD modes in human CKD. The review summarizes recent advances in our understanding of RCD pathways in CKD, looks at inter-relations between the pathways (with the emphasis on propagation of death signals) and the evidence for therapeutic targeting of molecules in the RCD pathways to prevent or treat CKD.