Periodic Reporting for period 1 - EPIORGABOLISM (Diabetic nephropathy modelling in hESC-derived 3D kidney organoids) Okres sprawozdawczy: 2019-04-18 do 2021-04-17 Podsumowanie kontekstu i ogólnych celów projektu With more than 422 million people worldwide with diabetes and 60 million in the European region, diabetes and, especially the complications associated, are becoming a major public health problem with significant social and economic burden. Among all the complications, Diabetic kidney disease (DKD) can explain most excess mortality associated with diabetes. DKD is the most common cause of the end-stage renal disease (ESRD). Regardless of intensive treatments with hyperglycaemic control, blood pressure control and the use of renin-angiotensin system blockades the prevalence of DKD remains high and more than 40% of diabetic patients develop the disease. For this reason, better diagnostic tools, predictive markers, and treatment options are still urgently needed. Although over the last years the understanding of DKD has been improved, the development of new therapies in DKD is facing three major challenges; First, it is still unknown when a diabetic patient is prone to develop DKD. We do not have sensitive and specific biomarkers to identify high risk patients and genetic factors with strong association have not been identified. Secondly, it is unknown when DKD is initiated. A metabolic disease which normally appears in adulthood lives nonetheless may have its origin in an abnormal development due to maternal metabolic disturbances. And lastly, there is the lack of a good preclinical model that can recapitulate the disease. Several studies have described epigenetic changes in DKD. However, none of these studies have been able to proof that metabolic alterations during kidney development can give rise to changes in the landscape of the kidney epigenetic profile that may influence the diabetic outcome.Although human epidemiological studies have shown that hyperglycaemia during pregnancy may be a prevailing factor that contributes to the develop of DKD of the offspring in adulthood, little is known about the molecular basis of this link and how the hyperglycaemic state may drive epigenetic alterations that result in DKD. Taking this into consideration, EPIORGABOLISM proposed to test if DKD is promoted by the metabolic alterations occurring in development due to hyperglycaemia episodes which could affect the DNA methylation profile, using the innovative in vitro system of 3 dimensional (3D) cultures of kidney organ-like structures, called kidney organoids.The findings of EPIORGABOLISM enlighten for the first time the link epigenetic-metabolism in the context of DKD. The action shows that human Embryonic Stem Cell derived kidney organoids cultured in intermittent hyperglycaemia condition can recapitulate important functional and structural characteristic of DKD. Moreover, the intermittent glucose model developed in EPIORGABOLISM can offer improved insight into the mechanism underlying the metabolic memory in DKD. We found that changes in TET1 expression in tubular renal cells from hESC kidney organoids and patients might suggest a link between metabolic changes and DNA methylation. Thus, our model may also serve as a tool for drug discovery to identify therapeutic targets for DKD. Prace wykonane od początku projektu do końca okresu sprawozdawczego oraz najważniejsze dotychczasowe rezultaty In EPIORGABOLISM we were able to demonstrate that human embryonic stem cells (hESC)- derived kidney organoids cultured in intermittent hyperglycaemia condition are able to recapitulate important functional and structural characteristic of DKD. Furthermore, in collaboration with Hospital Clinic we discovered that tubular renal cells from diabetic patients have higher maximal respiration capacity than control patients. This metabolic feature was erased by treating the cells with 5’Azytidine, a drug which remove methyl groups from DNA and has been used mainly in the treatment of myelodysplastic syndrome. Interestingly, when we looked at mRNA expression of the main DNA methyl enzymes, diabetic patients cells show a lower expression of TET1, which suggest a first possible causal link between metabolism and DNA methylation in diabetic patients. Regarding kidney organoids, the particular relevance was that tubular renal cells sorted from kidney organoids under intermittent hyperglycaemic condition show similar oxygen consumption profile than tubular renal cell from diabetic patients and the expression of TET1 was downregulated in the hyperglycaemic condition. To explore if the metabolic alterations by hyperglycaemia could be erased in hESC kidney organoids by upregulation of Peroxisome Proliferator-Activated Receptor γ Coactivator 1α (PGC1a), a master regulator in mitochondria biogenesis, we developed an inducible CRISPR/Cas9 engineered line. Tubular renal cells from PGC1a inducible doxycycline CRISPR/Cas9 engineered line kidney organoids under intermittent high glucose rescued the maximal respiratory capacity of the tubular cells. In summary, the results from EPIORGABOLISM enlighten for the first time the epigenetic-metabolism link in the context of DKD. The information gained by using hESC kidney organoids in intermittent hyperglycaemic conditions will offer improved insights into the mechanism underlying the metabolic memory in DKD. Changes in TET1 expression suggest a link between metabolic changes and DNA methylation in our system. Our model, it may also serve as a tool for drug discovery to identify therapeutic targets for DKD.The scientific results of EPIORGABOLISM were presented at EASD 2019 (Barcelona) and EASD 2020 (On line) and were highlighted at the Sociedad Valenciana de Nefrología website and twiter (https://www.svnefrologia.es/noticias/2019/9/23/highlights-european-association-for-the-study-of-diabetes-easd-congress-barcelona-2019). Innowacyjność oraz oczekiwany potencjalny wpływ (w tym dotychczasowe znaczenie społeczno-gospodarcze i szersze implikacje społeczne projektu) EPIORGABOLISM aimed to understand the mechanism by which DKD is originated during kidney development by using an innovative in vitro system of hESC-kidney organoids. Since the epidemiologist David Barker proposed that intrauterine growth retardation, low birth weight, and premature birth have a causal relationship with the origins of hypertension, coronary heart disease, and non-insulin-dependent diabetes, in adulthood, many epidemiological studies in humans and mice have proved a correlation between nutritional environment in utero and metabolic diseases in adulthood. However, the molecular mechanisms behind DKD are still unknown. Thus, no therapy is available to treat the disease. The results in EPIORGABOLISM might contribute to iidentify genetic factors of DKD that can help to discern between diabetic patients that develop DKD and iidentify a possible molecular target like TET1 which may provide significant proof of concept that dysregulation of the epigenetic landscape may be consequential rather than casual in the development of pathology. Overall, the findings in EPIORGABOLISM will help to understand the molecular and the epigenetic mechanisms of DKD and might help in the development of new therapies to treat kidney failure. Confocal microscopy image of the hESC derived kidney organoid
