Development of an in vitro 3D model of the proximal tubule interstitial interface as a high throughput renal fibrosis assay platform

Abstract

Chronic kidney disease (CKD) is a progressive, irreversible disease with a worldwide prevalence of 10-13 %. Hypertension and diabetes are the main causes of CKD, and they seem to be determined by epigenetic factors as well as socioeconomical conditions. This condition is asymptomatic until generic symptoms arise in patients with CKD, such as fatigue and itchiness, therefore the disease can be assessed only via specific functional and laboratory tests, the most indicative being the measurement of glomerular filtration rate (GFR). When such parameter falls beneath 60 mL/min per 1·73 m2 for longer than 3 months, the patient is considered to have developed chronic kidney disease that will eventually lead to end stage renal disease (ESRD) (Moll et al., 2013). Regardless of its aetiology, the underlying pathophysiological mechanism of CKD is renal fibrosis which is characterised by progressive scarring of the renal parenchyma which, in turn, leads to loss of basic kidney functions such as reabsorption, secretion, and excretion of solutes. Current pharmacological treatments for CKD are limited to blood pressure management via angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs), or management of glycemia in diabetes. Therefore, such therapies do not treat the underlying causes of CKD but components of the metabolic syndrome associated with it. There is urgent need for development of compounds that may target directly known mechanisms associated with the condition such as fibrosis and oxidisation (Turner et al., 2012). The call for the development of novel treatments combined with a global shift of interest towards replacing, refining, and reusing in vivo animal models of disease has led to an evolution and expansion within the in vitro disease modelling field. Therefore, we developed a 3D in vitro renal fibrosis model that recapitulates the key cellular events underlying tubule-interstitial fibrosis in the proximal tubule interstitial interface. The model is generated via treatment of primary human proximal tubule cells (hPTC) and renal fibroblasts (HRF) in co-culture from autologous donor kidney with key endogenous pro-fibrotic cytokines and hormones, and it is used as an assay platform to generate dose-response assays where the magnitude of maladaptive molecular mechanisms such as cell dedifferentiation and trans-differentiation, and cell cycle dysregulation are measured via high content imaging and high throughput flow cytometry and are put in relationship with the dose of pro-fibrotic compound used. The model is suitable for the high throughput screening of the in vitro efficacy of anti-fibrotic compounds and for the investigation of unknown molecular mechanisms of fibrosis.