Inactivation of mediator complex protein 22 in podocytes results in intracellular vacuole formation, podocyte loss and premature death

www.nature.com/scientificreports OPEN Inactivation of mediator complex protein 22 in podocytes results in intracellular vacuole formation, podocyte loss and premature death Patricia Q. Rodriguez1, David Unnersjö‑Jess2, Sonia S. Zambrano1, Jing Guo3,4, Katja Möller‑Hackbarth1, Hans Blom2, Timo Jahnukainen5, Lwaki Ebarasi1 & Jaakko Patrakka1* Podocytes are critical for the maintenance of kidney ultrafiltration barrier and play a key role in the progression of glomerular diseases. Although mediator complex proteins have been shown to be important for many physiological and pathological processes, their role in kidney tissue has not been studied. In this study, we identified a mediator complex protein 22 (Med22) as a renal podocyte cellenriched molecule. Podocyte-specific Med22 knockout mouse showed that Med22 was not needed for normal podocyte maturation. However, it was critical for the maintenance of podocyte health as the mice developed progressive glomerular disease and died due to renal failure. Detailed morphological analyses showed that Med22-deficiency in podocytes resulted in intracellular vacuole formation followed by podocyte loss. Moreover, Med22-deficiency in younger mice promoted the progression of glomerular disease, suggesting Med22-mediated processes may have a role in the development of glomerulopathies. This study shows for the first time that mediator complex has a critical role in kidney physiology. Renal podocyte cells are terminally differentiated epithelial cells of the glomerulus in where they form the final ultrafiltration barrier of the k idney1. Podocytes are morphologically divided into three compartments: cell bodies, major and foot p rocesses2. Large cell bodies are located in the urinary space from which they extend large cellular protrusion, major processes, towards glomerular capillaries. Close to the capillary wall, the cellular extensions divide to finely interdigitating foot processes that wrap around capillaries. The foot processes are interconnected by a specialized cell–cell junction called the slit diaphragm. In most glomerular diseases, the slit diaphragm and/ or foot processes of podocytes are i njured1. Morphologically, this is observed as foot process e ffacement2. This is considered to be a key event in the development of a lbuminuria2, which, on the other hand, is a cardinal sign of kidney disease. Foot process effacement is a reversible process mediated by actin-rich cytoskeleton of foot processes, and it is a common down-stream effect of various pathological stimuli2. However, in progressive renal diseases, the effacement may lead to loss of p odocytes3. Overwhelming evidence pinpoints podocytes as central players in the pathogenesis of glomerulopathies and chronic kidney disease (CKD)1. One of the key reasons for this is that podocytes are terminally differentiated and cannot proliferate1. Studies in human diseases have shown that podocyte loss correlates with the progression rate of glomerular d isease4,5. Studies in mouse have validated the key pathogenic role of podocytes as overexpression or inactivation of genes specifically in podocytes can either aggravate or slow the development of CKD6,7. Obviously, understanding mechanisms that govern podocyte homeostasis is of crucial importance. The Mediator complex (MED) is a multi-protein assembly that acts as a transcriptional co-activator in all eukaryotes8. The complex is composed of at least 26 subunits in mammals, and its function is to communicate signals from DNA-bound transcription factors to the RNA polymerase II enzyme. Different transcription 1 Integrated Cardio Metabolic Center, Division of Pathology, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden. 2Science for Life Laboratory, Department of Applied Physics, Royal Institute of Technology, Solna, Sweden. 3Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. 4Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School, Singapore, Singapore. 5Department of Pediatric Nephrology and Transplantation, New Children’s Hospital, Helsinki University Hospital, University of Helsinki, Helsinki, Finland. *email: Scientific Reports | (2020) 10:20037 | https://doi.org/10.1038/s41598-020-76870-0 1 Vol.:(0123456789) www.nature.com/scientificreports/ factors bind to different mediator subunits and many transcription factors can simultaneously bind to MED8. An important feature of MED is that its subunit composition can v ary8. Consequently, the lack of individual MED subunits is associated with silencing of specific transcription pathways. Studies in knockout animals have unravelled roles of MED subunits in cellular differentiation and studies in human genetics have linked them to a number of human diseases. However, to date, no studies on MED in kidney tissue have been performed. In this study, we explored MED subunits in renal podocyte cells. We show that MED subunit 22 (Med22) is highly enriched in podocytes and although it is not needed for the normal development of podocytes, it is essential for the maintenance of glomerular homeostasis as mice lacking Med22 in podocytes develop progressive renal disease and die prematurely. Results Human Protein Atlas suggests Med22 as a podocyte‑associated MED subunit. As the role of MED in kidney tissue is unknown, we analysed Human Protein Atlas (www.proteinatlas.org) to identify podocyte-associated MED subunits. We found immunohistochemical data in kidney tissue for 34 MED subunits (Suppl. Figure 1). Med21 and Med22 showed strong glomerular staining with only low signal in tubuli. As Med22 mRNA seemed to give more glomerulus-specific staining (Suppl. Figure 1), we focused on Med22 in further studies. Med22 is enriched in podocytes and localizes to major processes. In the kidney, the expression of Med22 was enriched in the glomerulus in comparison to the kidney fraction devoid of glomeruli as shown by qPCR human kidney tissue (Fig. 1A). The expression of podocyte-specific gene p odocin9 was analysed to control the purity of glomerulus fractions (Fig. 1A). In immunofluorescence of human kidney tissue a strong reactivity for Med22 was detected in glomeruli with clearly weaker signal in rest of kidney tissue (Fig. 1B). In double stainings, Med22 co-localized partially with podocyte major process marker vimentin (Fig. 1C). No significant overlap was detected with podocyte foot process marker nephrin, mesangial cell expressed protein alpha-smooth muscle actin and endothelial marker CD31 (Fig. 1C). Moreover, Med22 did not co-localize significantly with a podocyte nucleus marker Wt1 (Suppl. Figure 2A). Taken together, Med22 staining was in the glomerulus detected in podocytes in which it located to major processes. The specificity of anti-Med22 antibody was validated by immunostaining and Western blotting of cultured podocytes transfected with full-length human (...truncated)