Systemic gene therapy with thymosin β4 alleviates glomerular injury in mice

Abstract

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 β4 (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 Pathophysiological Role of Thymosin β4 in the Kidney Glomerulus

“…In addition, the expression of another member of the beta-thymosin family which has been previously described in podocytes [29], Tmsb10, was also downregulated in injured compared with healthy podocytes. Similar observations were made in vitro where injury of mouse immortalised podocytes with Adriamycin resulted in a reduction in the mRNA levels of Tmsb4x and Tmsb10 in a dose-dependent manner [33]. These findings highlight that podocyte injury is associated with reduced expression of Tmsb4x across multiple mouse models of glomerular disease and that these changes are not accurately reflected when assessing the expression of Tmsb4x in glomerular or whole kidney extracts.…”

“…The mRNA transcript for Tmsb4x was detected in developing glomeruli in embryonic day 16 [29], embryonic day 18, and in mature glomeruli in kidneys from 8-week-old mice by in situ hybridisation [30] with a localisation pattern indicative of expression in podocyte cells. These findings were corroborated by microarray analysis that identified Tmsb4x as a transcript enriched in adult mouse podocyte cells [31] and more recently by analysis of single-cell RNA sequencing (scRNAseq) datasets [32,33]. Tβ4 expression has also been demonstrated at the protein level in developing and mature mouse glomeruli by immunohistochemistry, and the localisation of Tβ4 peptide in podocytes has been identified by co-localisation with the podocyte markers nephrin and nestin [29].…”

“…Tβ4 protein levels were assessed five weeks later, and it was shown that gene therapy with Tβ4 resulted in transduction of liver cells followed by secretion of Tβ4 and a twofold increase in Tβ4 plasma levels in mice injected with AAV-Tmsb4x compared with controls injected with AAV-LacZ. Validation experiments in Tmsb4x -/y mice showed that AAV-delivered Tβ4 was internalised by podocyte cells, demonstrating that this is an effective strategy to systemically deliver Tβ4 to podocyte cells over a prolonged period [33]. A preventative strategy was used, where mice were intravenously injected with Adriamycin (10 mg/kg) three weeks after AAV administration.…”

“…A study by Chung et al generated singlecell transcriptomic profiles of the glomerulus from healthy mice and mice with different types of glomerular injury, including mice with nephrotoxic nephritis; mice injured with Adriamycin, a chemotherapeutic drug which replicates some of the features of human focal segmental glomerulosclerosis; and BTBR ob/ob (Lepr Ob/Ob ) mice with diabetic kidney disease [32]. Analysis of these scRNAseq datasets revealed that the podocyte expression of Tmsb4x was significantly reduced in all these disease models compared with Tmsb4x levels in the podocytes of healthy control mice [33]. In addition, the expression of another member of the beta-thymosin family which has been previously described in podocytes [29], Tmsb10, was also downregulated in injured compared with healthy podocytes.…”

“…Subsequently, the mouse model of Adriamycin nephropathy, which primarily targets podocytes, was used to assess the effect of exogenous Tβ4 on podocyte injury [33] (Figure 2). A particular strength of this study was using gene therapy to achieve sustained systemic upregulation of Tβ4.…”