The study of kidney disease is complicated, because the pathogenesis is often undetected, the disease may be acute or chronic in nature, the genetic composition of the host leads to different clinical syndromes, and multiple organs are often involved at the same time. In order to study disease susceptibility, mechanism, prognosis and potential therapies, the use of animal experimental models has proven to be invaluable.
Although cells grown in vitro (that is, in culture) cannot fully replicate the in vivo environment, they have several major advantages. These include the ability to directly study mechanical events, control the environment so that specific hypotheses can be tested, and can perform multiple experiments to verify initial observations. Although it may be obvious, the use of podocytes is essential to study podocyte-related events in culture. Therefore, although the information is abundant, the use of other cells such as human embryonic kidney cell lines, COS, and mouse embryonic fibroblasts (MEF) to study podocytes is not completely representative. It is necessary to confirm the functional relevance of these new interactions in podocytes in order to generalize these findings to podocytes.
The differentiation of primary human and rat podocytes leads to rapid growth arrest. Although this reflects a mature in vivo counterpart, it limits cell culture capacity because passage cells that do not increase in number are technically problematic. In short, unlicensed conditions cause the growth of most podocytes to stagnate within 6 days and induce many characteristics of differentiated podocytes. Both proliferating and differentiated podocytes express WT-1. During the differentiation process, the orderly arrangement of actin fibers and microtubules begins to extend into the process of forming cells, reminiscent of the podocyte process in the body. Similar to the primary culture, cytoskeleton rearrangement and process formation are accompanied by the onset of synaptopod protein expression. In addition, electrophysiological studies have shown that differentiated murine podocytes respond to bradykinin through changes in intracellular calcium concentration.
Podocytes in culture are now widely used to study almost all aspects of podocyte biology in health and disease. These innovative research by laboratories around the world include the regulation of the cytoskeleton, cell cycle, and podocyte channel physiology. Cultured podocytes have been successfully used to gain mechanical insight into the role of podocytes in the pathogenesis of diabetes and the study of human immunodeficiency virus-related nephropathy.
Gene expression and gene silencing are valuable tools for understanding podocyte biology. However, like other post-mitotic cells (such as neurons or cardiomyocytes), the transfection efficiency of podocytes is relatively low, and the transfection efficiency of cells grown under permissible conditions is usually between 10% and 20%. In the past few years, a series of new transfection reagents have been introduced, which can successfully transfect podocytes, especially green fluorescent protein or other marker proteins, to display transfected cells. Due to the loss of expression in daughter cells after mitosis, transient transfection is not ideal for podocytes under growth permitting conditions. However, transient transfection is easier to perform and less time-consuming than stable transfection or viral transfection.
Viral transduction is becoming an ideal method to change gene expression in cultured podocytes. Viral transduction is achieved by transfecting an expression vector into a cell line that produces viral particles. The virus particles are shed into the culture medium and then transferred to the podocytes for infection. Viral transduction combines the advantages of stable transfection, including continuous expression and selective ability, to introduce cells better than transient transfection. In addition, the media containing virus particles can be frozen for later use. Most importantly, viral transduction of cDNA or shRNA, for example, through retrovirus, adenovirus, or lentiviral vectors, can also be achieved in growth-stagnated and differentiated podocytes maintained under non-licensed conditions.
With the rapid discovery and exciting pace of new podocyte genes, several transgenic mice have been produced. Therefore, it is not surprising that there are more and more wild-type and knockout mice, and wild-type and genetically modified human podocyte cell lines that can be used for cell culture research. Several other new podocyte cultures are now available, including co-culture studies of podocytes and glomerular endothelial cells in an attempt to better study permeability, podocytes cultured in Matrigel to obtain three-dimensional effects, or culture podocytes isolated from urine.