From infancy to adulthood: A molecular look at kidney malformations
By Nadia Boachie
Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common form of malformation at birth, identified in over 1% of overall live births and accounting for up to 23% of overall birth defects. CAKUT represent the cause of 40 – 50% of pediatric end-stage renal disease worldwide.1 While some CAKUT are treatable-albeit with long term health consequences-others are extremely life threatening.2 UofT researchers are using genetically modified mice and human kidney organoids grown from patients’ urine samples to understand the molecular mechanisms underlying kidney disease. Their goal is to one day develop individualized treatments that target these molecular pathways in pediatric patients.
The causes of kidney failure vary from infancy to adulthood. Before age five, congenital malformations of the kidney and urinary tract are the leading causes. From ages five to 14, it is most commonly caused by nephrotic syndrome, systemic diseases, and hereditary diseases such as autosomal dominant polycystic kidney disease. At around 15 years to early adulthood, diseases that affect the glomeruli-tiny ball-shaped structures in the kidney composed of capillary blood vessels that are involved in the filtration of the blood to form urine-become the leading cause of kidney failure.3
With so many different contributors and causes of kidney failure in the pediatric population, researchers are trying to better understand the molecular mechanisms of kidney development that occur prenatally and postnatally, with a goal to treat patients. The IMS Magazine had the pleasure of sitting down with Pediatric Nephrologist and Senior Scientist at The Hospital for Sick Children, Dr. Norman Rosenblum. He completed his MD at Dalhousie University and postgraduate clinical and research training at Children’s Hospital and Harvard Medical School, and has been conducting research in nephrology for his entire career as a clinician-scientist. Dr. Rosenblum set up his lab in 1993 and his first graduate unit appointment in 1995 was with The Institute of Medical Sciences (IMS).
Dr. Rosenblum began using mouse models as well as other experimental methods to ask three important questions; ‘How is formation of the kidney accomplished?’, ‘What are the cellular and molecular mechanisms underlying kidney malformation?’, and ‘How can we use human genetics and experimental nephrology to inform our understanding of kidney development and human disease?’
To address these questions, Dr. Rosenblum’s lab “focuses on different signalling pathways, both alone and together during the development of the kidney in the mouse” he explained. Specifically, the lab looks at pathways controlled by Bone Morphogenetic Proteins (BMPs), Hedgehogs (Hh), and Wnt proteins during normal kidney formation and renal malformation.4 Hedgehog signaling has been of particular interest, as hedgehog proteins are important for tissue patterning and cell differentiation. They also transduce cellular signals via the GLI protein family of transcription factors. “Everything we seem to touch, from how progenitor cells are differentiated to specific kidney elements, variation in nephron number, or interactions between different tissue elements – in whatever we inquire about, there seems to be a story about how hedgehog signaling is involved” says Dr. Rosenblum.
In a 2018 paper published in Journal of American Society for Nephrology, Dr. Rosenblum and his collaborators used patients’ specimens and mouse models to better understand the pathogenesis of ureteropelvic junction obstruction, the most common form of congenital urinary tract obstruction. The mice in this study were genetically modified to be deficient for the Hedgehog target, Ptch1. Rosenblum and collaborators analyzed obstructive ureteric tissue obtained from children with congenital intrinsic ureteropelvic junction obstruction and discovered that molecularly, these human samples were similar to what was observed in Ptch1-deficient mice. Their results demonstrate a Hedgehog-dependent mechanism underlying urinary tract blockage. In another recent 2018 publication in Development, they found a mechanism by which Hedgehog-GLI signaling in Foxd1-positive stromal cells, via TGFβ signaling, controls the number of nephrons formed in the kidney.5 “We keep working at [hedgehog] because the story continues to unfold, in a very interesting way” he explains.
Dr. Rosenblum clarifies that experiments in mice are vital but are not the entire picture. “There are important differences in what happens in mice and what happens in humans. Same gene, same mutation, doesn’t look the same in terms of what the phenotype is in humans versus mice” he explains, “We really wonder why that is. So, we are using human organoids as a way to model these things and inquire further into mechanisms of human disease” he adds.
Recently, the Rosenblum lab and collaborators were able to isolate human urine cells (UCs) from pediatric urine specimens. These urine samples were reprogrammed into urinary induced pluripotent stem cells from which human kidney organoids were generated.6 “When you take urine, even small amounts, and put it on a plate, epithelial cells grow. What you then can do is treat those cells with plasmids that contain four factors that were described in Nobel winning work7 to convert the cells from a differentiated cell to a pluripotent cell. Once you have that pluripotent cell, then you can direct that cell to differentiate in various ways using other factors” Dr. Rosenblum explained. He further elaborated that “even though [the organoid] is a very immature kidney tissue, it is human tissue, and [it is] from the very patients that we would be interested in.” This is a big step in the right direction for individualized kidney treatments.
There are still major challenges in this field of research. A healthy and functioning kidney requires formation of a critical number of nephrons; they are necessary to drain urinary filtrate into the ureter towards the bladder. A large body of epidemiological studies have shown that the number of nephrons that you are born with varies tremendously in the population.8 There may be as much as a six-fold difference between individuals. Some may be born with 200,000 nephron elements, and others with about 1.8 million. “Clinically, we don’t even have a method to measure the number of nephrons elements an individual is born with” explains Dr. Rosenblum. This poses an issue when trying to identify infants that may have potential issues with kidney development in the future. Dr. Rosenblum’s lab is trying to find non-invasive ways to determine nephron number. “We are aiming to determine biomarkers that tell us this information, for example, based on their number of nephrons we’ll be able to say ‘well this person may be at higher risk [for kidney disease/malfunction] so we should be aware of it,’” he explains.
Dr. Rosenblum’s impressive list of publications and his successful career as a clinician-scientist seems to be made possible because of his well-roundedness. Outside of the lab, Dr. Rosenblum is a seasoned cello player, an instrument he has played since the age of 14. His passion for classical music drives him to continue to practice and play with other musicians to this day. He wants students and young researchers to know that in their professional careers, they should go after what they are passionate about. He also advised that researchers “find other things to do which cater to other parts of you,” and to spend time on that too. Lastly, he added that “The management of time and energy is a big skill and maybe one of the most important skills that people have. Just because you work long doesn’t mean you work well.” These pieces of advice seem to be how Dr. Rosenblum continues to thrive in his scientific endeavours.
1. Sanna-Cherchi, Simone, Pietro Ravani, Valentina Corbani, Stefano Parodi, Riccardo Haupt, Giorgio Piaggio, Maria L. Degli Innocenti et al. “Renal outcome in patients with congenital anomalies of the kidney and urinary tract.” Kidney international 76, no. 5 (2009): 528-533.
2. Kidney Disease in Children [homepage on the internet] National Institute of Diabetes and Digestive and Kidney Diseases, U.S. Department of Health and Human Services, 1 Mar. 2014, Available from: http//www.niddk.nih.gov/health-information/kidney-disease/children.
3. National Institutes of Health. National Institute of Diabetes and Digestive and Kidney Diseases. United States Renal Data System. 1993 Annual Data Report. 1993 Mar: 55-67.
4. Norman Rosenblum Lab [homepage on internet]” Rosenblum Lab, Available from: http//lab.research.sickkids.ca/rosenblum/.
5. Rowan CJ, Li W, Martirosyan H, Erwood S, Hu D, Kim YK, Sheybani-Deloui S, Mulder J, Blake J, Chen L, Rosenblum ND. Hedgehog-GLI signaling in Foxd1-positive stromal cells promotes murine nephrogenesis via TGFβ signaling. Development. 2018 Jul 1;145(13):dev159947.
6. Mulder J, Sharmin S, Chow T et al. Generation of infant-and pediatric-derived urinary induced pluripotent stem cells competent to form kidney organoids. Pediatric Research. 2019 Oct 19:1-0.
7. Takahashi K, Okita K, Nakagawa M, Yamanaka S. Induction of pluripotent stem cells from fibroblast cultures. Nature protocols. 2007 Dec;2(12):3081.
8. Wang X, Garrett MR. Nephron number, hypertension, and CKD: physiological and genetic insight from humans and animal models. Physiological genomics. 2017 Jan 27;49(3):180-92.