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The potential of human kidney stem/progenitor cells for use in drug discovery and regenerative therapy programmes

Final Report Summary - NEPHROTOOLS (The potential of human kidney stem/progenitor cells for use in drug discovery and regenerative therapy programmes.)

The overarching goal of the NephroTools ITN was to provide state-of-the-art multidisciplinary training for a cohort of 15 young researchers in order to equip them with the skills required to make a significant impact in renal medicine. The Network had the following research objectives:-

1. To generate and characterise human kidney stem/progenitor cell (KSPC) lines. We generated KSPC lines from fetal, infant and adult human kidneys and characterised them using a range of techniques. The most promising lines were selected for entry into subsequent parts of the research programme.
2. To design biomaterials capable of directing the differentiation of the KSPCs to specialised renal cells. Using a range of interdisciplinary bio- and chemical-engineering approaches, we generated novel biomimetic and biomaterial culture substrates that were screened for their ability to direct the differentiation of the KSPCs to important renal cell types, such as podocytes and proximal tubule cells.
3. To determine which KSCP lines have greatest potential for use in drug discovery. The physiological properties of the KSCP derivatives and their pharmacological responses to nephrotoxic and renoprotective drugs were assessed using established in vitro assays.
4. To determine which KSCP lines have greatest potential for the development of cellular-based therapies to treat chronic renal disease. We developed an electronic device for accurately measuring the glomerular filtration rate (GFR) in rodents. We then established which KSPC lines were able to improve renal function following transplantation in rodent models of chronic renal disease.

Training activites
All fellows developed a personal career development plan and received training in transferrable skills that was either provided by their host institution, during secondments, or during the NephroTools training events (workshops and conferences). Each fellow undertook at least one secondment in order for them to meet their project objectives and learn new skills. The fellows benefitted from participating in four NephroTools interdisciplinary workshops that provided training and knowledge in the key research fields of the programme. The fellows also participated in the three NephroTools international conferences, which gave them the opportunity to network and present their work to internationally renowned scientists in the fields of stem cells, pharmacology, biomaterials and nephrology. Each fellow gave at least one oral presentation at the conferences.

Research activities
1. We generated and characterised 22 fetal KSPC lines, 5 infant KSPC lines and 30 adult KSPC lines. Furthermore, strategies for reprogramming mature human kidney cells to a progenitor-like state were used to develop 4 novel reprogrammed KSPC lines. The nephrogenic potential of the different lines was investigated by incorporating the cells into mouse kidney rudiments ex vivo and assessing their ability to integrate into developing renal structures. Selected KSPCs were assessed for their ability to ameliorate injury in a rat model of cisplatin-induced renal disease and a mouse model of adriamycin-induced nephropathy.

2. We designed the following types of biomaterials and assessed their physico-chemical properties using a range of techniques: (i) StarPEG-heparin gels; (ii) polyacrylate biomimetic substrates modelled on moieties present in the extra-cellular matrix protein, fibronectin; (iii) plasma-functionalised substrates comprising gradients of specific chemical functional groups; (iv) 3-D macroporous scaffolds. We investigated the ability of these biomaterials to regulate the behaviour of the different KSPCs.

3. We used ciPTECs and conditionally immortalised podocyte cell lines to establish a range of physiological and pharmacological assays. We compared the properties of KSPC-derived podocyte- and proximal tubule-like cells with the conditionally immortalised cells. We also established in vitro protocols for inducing reproducible renal cell damage using the nephrotoxic drug, cisplatin, and assessed the ability of KSPC-derived microvesicles (MVs) to prevent such damage.

4. We developed a miniaturised electronic device for measuring the glomerular filtration rate (GFR) in awake mice. This device was used to monitor disease progression in a mouse model of adriamycin-induced renal injury. We identified an FDA-approved carbohydrate entity (X) and simultaneously developed near infrared (NIR) cyanine dyes that were used to label ‘X’. We found that one of our NIR-X fluorescent compounds is specifically filtered by the kidneys with a faster half-life than sinistrin, the current gold standard (patent filed). We also established the cisplatin-induced kidney injury model in immune-compromised rats and used the electronic device to assess the ability of the 4 different types of KPSCs to improve renal function and ameliorate histological damage

Main results achieved
We generated KSPCs from various sources of human renal tissues and found that in a mouse model of glycerol-induced renal injury, adult KSPCs could integrate into the kidney, reduce fibrosis and generate erythropoietin. In a mouse model of adriamycin-induced injury, infant KSPCs appeared to ameliorate injury, but did not integrate in the kidney. Furthermore, we isolated KSPCs from preterm neonatal urine and found that these cells had long-term self-renewing capacity and were able to generate functional podocytes.

We generated matrix metalloproteinase- degradable starPEG-heparin hydrogels and found that they provided a synthetic, tunable system that promoted in vivo-like human renal tubulogenesis. We developed bio-functionalised polyethylene terephthalate porous membranes and showed that they supported the differentiation and polarisation of proximal tubule cells. We fabricated a range of polyacrylate-based biomimetic substrates and identified substrates that promoted either proximal tubule cell or podocyte differentiation.

Differentiating fetal KSPCs showed similar functional activities as ciPTECs, suggesting their maturation towards a renal tubular epithelial phenotype. MVs harvested from adult KSPCs derived from urine could protect primary kidney cells from cisplatin-induced injury but following knockdown of CD133, kidney cells showed a lower capacity to recover from cisplatin-induced injury. A novel KSPC population isolated from neonatal urine (nKSPCs) differentiated into functional podocytes and proximal tubule cells and thus have great potential to be used for drug discovery and cell-based therapies.

We developed a miniaturised electronic device for measuring the GFR in awake mice and a novel near infra-red carbohydrate compound with improved physico-chemical and functional properties than the current gold-standard, FITC-sinistrin. We assessed the efficacy of fetal, infant and reprogrammed KSPCs in a rat model of cisplatin-induced renal injury and found that all cell types could improve renal function and in some cases improve histological damage, despite the fact that they did not localize to the kidney.

Potential impact
• We found that KSPCs have therapeutic potential and could therefore be appropriate for clinical translation. If so, this could lead to a novel therapy for patients with kidney disease, and in future, could potentially reduce the number of patients requiring renal replacement therapies.
• We found that ciPTECs, fetal KSPCs and KSPCs derived from the urine of pre-term neonates could have potential in drug discovery programmes. If so, this could expedite the discovery of new renoprotective drugs, which would have a positive impact on renal patients, and furthermore, could help identify potential nephrotoxicants at an early stage in the drug development process, thus preventing valuable resources being wasted on the development of inappropriate drugs.
• The novel biomaterials and fluorescence compounds we developed have the potential to become marketable products and could thus create wealth and employment.
• The novel fluorescent compound we developed for use in the electronic device could potentially be used in the clinic to measure GFR in patients, enabling early diagnosis of patients with kidney disease.

Contact: NephroTools coordinator, Dr Patricia Murray,