Inhibition of Centrosome Clustering Reduces Cystogenesis and Improves Kidney Function in Autosomal Dominant Polycystic Kidney Disease
Preprint posted on 17 November 2022 https://www.biorxiv.org/content/10.1101/2022.11.16.516801v1
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an inherited disease caused by mutations in either the PKD1 or PKD2 genes. ADPKD is characterised by the formation and growth of fluid-filled kidney cysts in the renal epithelia, hypertrophy of the kidney, inflammation, and fibrosis ultimately leading to kidney failure. Cyst formation does not occur in every renal epithelial cell, which has led to the “two-hit” hypothesis of cyst formation in which one allele contains an inherited mutation in a PKD gene, and the second PKD allele undergoes a somatic mutation leading to ciliary dysfunction, dysregulation of cell signalling, hyperproliferation, and cyst development. Cilia are organelles that protrude from the cell and are involved in chemical and mechanosensory signalling. In ADPKD, the cilia are disrupted and ADPKD is therefore described as a ciliopathy (Bergmann et al., 2018).
The centrosome regulates mitosis, acting as the main microtubule-organising centre of the cell. Typically, renal epithelial cells have one cilia and one centrosome. Centrosome amplification has been observed in cystic renal epithelia, leading to genomic instability, disruption of cilia assembly, and pro-inflammatory cytokine secretion; together these factors lead to hyperproliferation (Battini et al., 2008). Cells with excess centrosomes, as observed in ADPKD and in cancer, avoid apoptosis via a process called centrosome clustering, which allows the cell to survive and divide despite centrosome amplification. In this preprint, the authors target centrosome clustering with inhibitors to enhance apoptosis of cells with centrosome amplification.
(i) Centrosome amplification accelerates cystic disease
To assess the effect of centrosome amplification in ADPKD in vivo, a Pkd1 mutant mouse model of ADPKD (Pkd1RC/RC) in which the disease progresses slowly was crossed with a transgenic model of centrosome amplification via conditional expression of Plk4. Mice with induced Plk4 expression displayed increased centrosome amplification, accompanied by larger kidney volume, increased kidney cystic area and elevated markers of renal damage when compared to Pkd1 mutant mice without Plk4 induction. This indicates that centrosome amplification is sufficient to accelerate cystic disease progression.
(ii) Centrosome clustering inhibitors activate the spindle assembly checkpoint and block proliferation in cellular models of ADPKD
To determine whether inhibition of centrosome clustering could prevent proliferation, centrosome clustering inhibitors (CCB02 or PJ34) were applied to WT and PKD1-null kidney cells. These centrosome clustering inhibitors have previously been shown to block proliferation in cancer cells. Cells with a normal centrosome number were unaffected by the centrosome clustering inhibitors. The centrosome clustering inhibitors decluster the centrosomes, resulting in disorganised multipolar spindles during mitosis, a delay in mitosis, and induced activation of the spindle assembly checkpoint only in cells that contain excess centrosomes.
(iii) Inhibition of centrosome clustering delays cystic disease progression
Next, to test whether inhibition of centrosome clustering translated to reduced cyst expansion in vivo, CCB02 and PJ34 were administered to Pkd1RC/RC mice. Interestingly, when compared to vehicle controls, centrosome clustering inhibitors resulted in significantly decreased kidney size, fibrosis, and reduced both kidney cyst formation and cyst expansion, as measured by cyst number and cystic index of the kidney, respectively. Alongside reduced cyst expansion, mice treated with either CCB02 and PJ34 also exhibited reduced blood urea nitrogen and serum creatinine levels compared to their vehicle-treated counterparts, indicating that centrosome clustering inhibitors effectively protect renal function. Furthermore, when compared to the only approved drug that delays progression of ADPKD (tolvaptan), CCB02 performs just as well, and in some cases better than tolvaptan to reduce cystogenesis and protect renal function.
(iv) Centrosome clustering inhibitors block proliferation, promote apoptosis, and reduce inflammation
Finally, to determine whether inhibition of centrosome clustering impacts apoptosis of cells with excess centrosomes, Pkd1RC/RC mouse kidneys were immunostained for p53, a mediator of cell death. Mice treated with CCB02 or PJ34 had higher levels of p53 nuclear accumulation and cell death compared to untreated controls, which coincides with reduced DNA damage response in daughter cells after division. Together, this suggests that inhibition of centrosome clustering removes cells with excess centrosomes via apoptosis. As centrosome amplification has previously been shown to have paracrine effects on neighbouring cells via secretion of cytokines and other proinflammatory and proproliferative factors, the authors suggest that removal of cells with centrosome amplification through apoptosis could have additional benefits through the reduction of these factors that may contribute toward pathogenesis in ADPKD. In CCB02-treated mice, there was a downregulation of a number of profibrotic and proinflammatory cytokines e.g TNF-ɑ, IL-1ꞵ, IL-6, as well as proproliferative signalling molecules e.g. JAK/STAT, MAPK/ERK. Thus, inhibition of centrosome clustering can have additional therapeutic benefits by reducing inflammation and proliferation to further protect against renal injury.
Why I find this interesting
There is currently only one drug on the market for the treatment of ADPKD (tolvaptan) that delays progression of disease. There is a need to explore new therapeutic targets as not all patients with ADPKD are eligible or can tolerate tolvaptan. The authors of this preprint provide important new insight into the mechanism whereby centrosome clustering accelerates disease, which can be exploited as a therapeutic target. This is then taken one step further by demonstrating the ability of centrosome clustering inhibitors to prevent disease progression, offering an exciting potential for a new avenue for treatment.
Questions for the authors
- What are the potential off target effects of centrosome clustering inhibitors? As these inhibitors are specific to cells with excess centrosomes, are there healthy cells within the body that have excess centrosomes that may also be targeted?
- Could centrosome clustering inhibitors have an additive benefit on disease progression as part of a combination therapy?
BERGMANN C., GUAY-WOODFORD L.M., HARRIS P.C., HORIE S., PETERS D.J.M. & TORRES V.E. 2018. Polycystic kidney disease. Nature Reviews Disease Primers, 4, 50.
Battini L. ,Macip S., E. Fedorova E., S. Dikman S., S. Somlo S, C. Montagna C., G. L. Gusella G. L. 2008. Loss of polycystin-1 causes centrosome amplification and genomic instability. Human molecular genetics 17, 2819-2833.
Posted on: 24 April 2023Read preprint