Mike Aimino & Lisa Freeman
Summary of:
Clement, L.C., Avila-Casado, C., Macé, C., Soria, E., Bakker, W.W., Kersten, S. & Chugh, S.S. (2011). Podocyte-secreted angiopoietin-like-4 mediates proteinuria in glucocorticoid-sensitive nephrotic syndrome. Nature Medicine, 17(1), 117-122. doi: 10.1038/nm.2261.
What is Nephrotic Syndrome?
Nephrotic syndrome is a condition that causes proteinuria, hypoalbuminemia, edema, hyperlipidemia, and lipiduria in individuals that have it. Proetinuria and lipiduria are when there are proteins and lipids in the urine, respectively. Hypoalbuminemia is when there is a low amount of albumin in the blood while hyperlipidemia is when there are high amounts of lipids in the blood. It can be caused by diabetic nephropathy, minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS), or membranous nephropathy. MCD is the primary cause of nephrotic syndrome in pre-adolescents, making up 85-95% of the cases. About 15 in 100,000 children have MCD with 2-7 new cases annually in 100,000 children. The prevalence of MCD is much lower in adults, making up only 10-15% of the cases. MCD is sensitive to glucocorticoid treatment, while the other diseases show a varied response, making it a good target to study.
Other complications of nephrotic syndrome include foot process effacement of podocytes. Normally, podocytes extend primary processes to the glomerular basement membrane (GBM) of the capillaries. Foot processes extend from the primary processes and lie on the GBM. Adjacent foot processes then interdigitate, which looks similar to locking your fingers together (pictured below). In individuals with necrotic syndrome, the foot processes disappear, making it look like the cell membrane is continuous. This leaves spaces between podocytes which allows proteins to leave and enter the urine.
All angioproietin-like proteins (Angptl) are glycoproteins that are sensitive to glucocorticoids. Angiopoietin-like proteins have been found to play a role in the development of hypertriglyceridemia and tumor metastasis. They have many different effects on cells depending on the part of the body in which they are found. Angptl4 is an inhibitor of lipoprotein lipase and has an effect on triglyceride levels in the blood. There has been no previous research to show that Angptl4 plays a role in proteinuria.
Question
What role, if any, does Angptl4 play in proteinuria associated with nephrotic syndrome?
Approach
The researchers employed a large arsenal of experiments and analysis techniques, so we will only give an overview of the experimental approach here. They started by evaluating four different nephropathy models, each model simulating a different disease that causes nephrotic syndrome. They identified the model that yielded the greatest increase in Angptl4 expression (puromycin nephrosis, or PAN), and used this model in later experimentation. They then studied a previously established Angptl4 transgenic mouse model and developed two new transgenic rat models. The NPHS2-Angptl4 model is characterized by upregulated Angptl4 in podocytes, while the aP2-Angptl4 is characterized by an upregulation of circulating Angptl4, secreted from adipose tissue. They evaluated Angptl4 expression and morphological changes for each model.
Although data for the aP2-Angptl4 is presented in supplemental materials, the remainder of the paper is focused on continuing experimentation with the NPHS2-Angptl4 model. With this model, they measured albuminuria in rats at varying ages. They then induced PAN, a model for minimal change disease (MCD), and measured albuminuria again. To analyze an additional variable affecting protein expression, they treated the rats with glucocorticoids after inducing PAN and measured resulting proteinuria and Angptl4 expression. Finally, the researchers conducted an in vitro study in two different cell lines of the sialylation of Angptl4 on its electrophoretic migration and the level of proteinuria occurring in the transgenic models.
A variety of analysis techniques were utilized throughout the experiments described above. Light microscopy and electron microscopy were used to analyze morphological changes at the glomerular and cellular levels, respectively. Immunohistochemistry with confocal microscopy and immunogold electron microscopy were used to localize and quantify Angptl4 expression. The researchers used SDS-PAGE to detect urinary protein and a combination of 2-D gel electrophoresis and western blotting to differentiate between forms of Angptl4.
Main Findings
A key result from these experiments was that increased expression of podocyte-secreted Angptl4 (NPHS2-Angptl4 transgenic model) caused increased proteinuria, similarly to proteinuria observed in rat models and human MCD patients. Angptl4 expression was also associated with morphological changes characteristic of MCD. More specifically, proteinuria was induced when the glomerular basement membrane (GBM) showed the presence of Angptl4, even though the podocytes did not yet show morphological changes. The researchers interpreted these results as an indication that Angptl4 causes a defect in the GBM that ultimately leads to proteinuria. They also found that Angptl4 decreased after glucocorticoid treatment. Finally, their results showed that sialylation of Angptl4 was associated with decreased proteinuria.
Broader Context
The results show that in the PAN model there is a 60-80 fold upregulation of glomerular Angptl4 expression in the PAN model. This is close to the 120 fold increase found in the NPHS2-Angptl4 heterozygous, male rats, meaning that it is a good model for studying nephrotic syndrome. This model is the first demonstration of the important role Angptl4 plays in proteinuria. Because the NPHS2-Angptl4 rats had reduced albuminuria when fed with ManNac and had an increase in the sialylation of glomerular Angptl4, it suggests that hyposialylation could be a mechanism by which Anglt4 overexpression causes proteinuria. Therefore, treatment with sialic acid precursors could be a potential therapy for individuals with some forms of nephrotic syndrome, particularly minimal change disease.
References
2. National Institute of Diabetes and Digestive and Kidney Diseases. Glomerular Disease Primer: The Normal Kidney. http://www.niddk.nih.gov/research-funding/at-niddk/labs-branches/kidney-disease-branch/kidney-diseases-section/glomerular-disease-primer/-normal-kidney/Pages/normal-kidneys.aspx
I find this article very interesting, especially as I had not learned about podocyte function previously. I would like to learn more about how the secretion of high-pI Angpt14 can facilitate its tethering to the GBM and its role in binding heparan sulfate proteoglycans.
I was totally with this article until they started talking about sialylation and the charge on the protein. I wasn’t really sure how to interpret the data shown in figure four, although I understand a 2D gel electrophoresis in concept. I couldn’t tell what each band was. It is interesting that the authors started their research pretty broadly, first testing Angptl4 mRNA levels across several disease causing conditions. This makes me think that this research was very novel, and that there is room to explore the topic beyond the scope of this paper.
I found this article to be very informative in a lot of ways. I do think that the format was a little strange and made the paper more difficult to follow because of the lack of subheadings. There were a few things that stuck out to me in the paper as I read. One of which was that in Figure 3, they briefly mentioned that heterozygous females were not albuminuric. I wonder if this was something they expected or was a new finding. I did find Figure 4 more confusing than the rest and am looking forward to your breakdown of the figure. I also found the idea of hyposialylation as a factor that causes overexpression of Angptl4 leading to proteinuria to be an interesting idea, and I do think they have the beginnings of supporting evidence for this. The last thing I wanted to mention that I did not care for with the paper was the large amount of supplemental figures that were referenced. They seemed to have a great deal of valuable evidence through which they gathered a lot of conclusions and it may have been more beneficial for that to be present within the paper rather than as supplements in some cases.
I thought this paper was engaging. I felt all of the different models, causes of disease, and experiments they used were great. I was intrigued by the order in which their experiments were conducted. They first used mouse models and ended with in vitro work–why would they conduct experiments in mouse models before using cell-lines? Additionally, I was a bit confused by figure 4. I could not concur with their interpretations of the results.
I am interested in the sialylation and Angptl4 mechanism. Moreover, how the increase or decrease in sialylation can cause Angptl4 overexpression which contributes to proteinuria. I am curious how increased amounts of sialic acid precursors decrease podocyte-secreted Angptl4 that is a contributor in proteinuria in variations of nephrotic syndrome.
This article is very interesting and definitely introduces some new concepts that I have not studied previously! I am also a little bit confused by the concept of sialylation. I don’t really understand how sailic acid precursors could be therapeutic targets. On a different note, I really did not like the organization of this article. I found it pretty difficult to navigate without subsections or structure.
Why would this disease be more prevalent in children than adults? Would this disease be related to issues of kidney development in childhood and adolescence? Additionally, I think that the information given in this paper is kind of scattered which makes it hard to read but overall, the results were clearly explained so I understand what was learned from this study. However, the experimental approach could have been better explained in regards to the various mouse models.
I think the findings of this paper are definitely really interesting, but the way this paper was designed/written really makes it hard to comprehend. I have been warned by previous professors that Nature papers are incredibly dense, though. Like others, I was a bit confused by this idea of sialylation and therefore am not entire sure how treatment with sialic acid precursors could be therapeutic. I look forward to hearing how this is explained!
The feature of podocyte function was really interesting and I enjoyed being able to apply my (very limited) previous knowledge from Bio 120 regarding their role in renal physiology. The authors touch on a number of different models and look at things pretty broadly which lends themselves to further and deeper explanation on this topic. Though the breadth of their paper was interesting to see, some of their data was confusing, specifically for me – Figure 4. Toward the end the authors point out different facets of Angpt14 expression, and I’d love to know more about how this information can be applied to different facets for AD as well as other organs and functions.
It appears as if over-expression of Angptl4 leads to hyposialyation therefore preventing it from interacting with the GBM appropriately leading to problems with podocyte interaction and protein leakage into the urine. I can imagine that the glycosylation process is being overwhelmed by the overproduction of this protein, but why can’t the fraction that gets properly sialyated function in the GBM? Figure 4b shows an increase in both sialyated and non-sialyated protein in the PAN model of disease.
I like the fact that they investigated Angptl4 gene in vitro and in vivo with ManNAc. I also like the fact that not only they looked at Angptl4, they also looked at NPHS2. However, I was confused how to interpret figure 4. Other than doing western blot with blurry images, I think they could have done other methods to test the hypothesis. I also think the method section could be divided more (where they could have separate western blot section). The result and discussion were combined together from this Nature Medicine article. I think this was little bit different type of paper unlike we had in past. Also, not only test transgenic mice in PAN, maybe they could have test the similar effect in different cell types. They could have drawn the model systems in order to help readers to understand what they are talking about. They talked about the glomerular expression of Angptl 4 was glucocorticoid sensitive and was highly unregulated in the serum from the models of MCD.
I found the connections shown in figure 3 to be of particular interest. The ability to create a precise transgenic model for this disease was key for the research and findings seen in this study, like the selectivity of proteins in the filtrate of the urine in transgenic animals. I would be interested to learn more (and understand) the background information regarding figure 4. it seems like there is a large volume of data present, but is a bit difficult to interpret.
In this experiment they make the general claim that the effectiveness of the two models are interchangeable as the PAN model shows a 60 to 80 fold increase in the up-regulation of glomerular Angptl-4 in comparison to the 120 fold increase seen in NPHS2-Angptl-4, heterozygous, male rats; what is it that makes a 60-80 fold increase close enough to be considered a “good model”? Also I am curious to at this stage in the research how implementable are these treatment and therapy ideas? I would also be interested in investigating more about the specific mechanism in which sialic acid inhibits the proteinuria… Overall I found this article very interesting and unique because of the specific content that it is covering.