Investigating the Role of Angptl4 in Proteinuria associated with Nephrotic Syndrome caused by Minimal Change Disease

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.

(National Institute of Diabetes and Digestive Kidney Diseases)
(National Institute of Diabetes and Digestive Kidney Diseases)

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.



What role, if any, does Angptl4 play in proteinuria associated with nephrotic syndrome?



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.



1. Mansur, A., Georgescu, F., Lew, S. (2015). Minimal-Change Disease. Medscape.

Exploring Ciliary Mechanosensation as a Means to Understanding Polycystic Kidney Disease

Summary of:

Low, S. H., Vasanth, S., Larson, C. H., Mukherjee, S., Sharma, N., Kinter, M. T., Kane, M. E., Obara, T. & Weimbs, T. (2006). Polycystin-1, STAT6, and P100 Function in a Pathway that Transduces Ciliary Mechanosensation and Is Activated in Polycystic Kidney Disease


By: Lizz Reese and JT Stoner


What is ADPKD?

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common forms of polycystic kidney disease. It is known to occur in individuals and families of all different races and is estimated to currently impact the lives of 400,000+ people in the US. ADPKD usually presents in adulthood with about 50% of patients developing end-stage kidney disease by the age of 60.

This disease is characterized by fluid-filled cyst growth in both kidneys, which begin to replace much of the normal kidney mass. As a result, reduced function and organ failure may occur, requiring the patient to undergo dialysis and/or kidney transplant. PKD can also cause cysts in the liver as well as additional problems in the heart and blood vessels of the brain.

While there is no definitive treatment for ADPKD, treatments have been tailored to mitigate  non-kidney symptoms (e.g. blood pressure) and pain (e.g. treated with painkillers and antidepressants). Kidney-related treatments typically aim to control buildup of acid and to prevent elevated phosphate levels.


What causes ADPKD?

ADPKD is genetically inherited in an  autosomal dominant fashion. The mutated gene (either PKD1 or PKD2) causes renal cells to proliferate abnormally, resulting in the formation of fluid-filled cysts which eventually replace most of the normal renal tissue and lead to renal failure.


What is known?

Mutations in PKD1 or PKD2 are recognized as the underlying causes of ADPKD, with PKD1 being mutated in 85% of the cases. The function of the gene product, PC1, is poorly understood.  PC1 is a large, integral membrane protein which is believed to have extracytoplasmic ligand binding domains, although ligands have yet to be identified.  The C-terminal tail has been implicated in signal transduction pathways such as the wnt pathway, a pathway leading to AP-1 transcription factor activation, G protein signaling, calcium signaling, and activation of STAT1. It is unknown, however, if any of these pathways are altered in ADPKD.

In a process that is thought to incorporate PC1, primary cilia of renal epithelial cells act as mechanosensors that respond to changes in lumenal fluid and flow (see figure below). Moreover, it has been shown in a number of case that defects in cilia proteins can lead to renal cystic diseases in humans and animals, though PC1’s role remains unclear.


Blog Post Picture V. Singla et al., Science (2006) Published by AAAS.


Article Summary

Autosomal dominant polycystic kidney disease (ADPKD) results from polycystin-1 (PC1) defects, which are poorly understood but known to implicate primary cilia. This paper identifies a novel mechanism of cilia function that leads to changes in gene expression via PC1 and shows that this pathway is inappropriately activated in ADPKD.  Under normal conditions, the PC1 cytoplasmic tail interacts with transcription factor STAT6 and the coactivator P100 to stimulate STAT6-dependent gene expression. Termination of apical fluid flow results in nuclear translocation of STAT6. Under ADPKD conditions cyst-lining cells exhibit higher levels of nuclear STAT6, P100, and the PC1 tail. Exogenous expression of human PC1 in zebrafish embryos results in cyst formation.



The signaling pathway that transduces a mechanical signal from primary cilia to changes in gene expression is inappropriately activated in ADPKD by the proteolytic cleavage and nuclear translocation of polycystin-1 (PC1).


Methods & Models

A number of different cells lines and models were used throughout the experiment: MDCK renal epithelial cell line (derived from a canine), COS-7 cells (derived from monkey kidney tissue), and HEK293T cells (human embryonic kidney cells) as well as normal and diseased human and mouse kidneys and zebrafish embryos. The MDCK and COS-7 cells were transfected with FLS-PC1 or CTM-PC1 to demonstrate that the cytoplasmic tail of PC1 localizes to the nucleus and to show that the C-terminal half of the PC1 tail is cleaved, released from the membrane, and targeted to the nucleus. Results were viewed via Western blots and immunostaining. Additionally, MDCK cells were utilized to demonstrate that the PC1 tail interacts with P100 via CTM-PC1 or NTM-PC1 transfection, Coomassie staining, and confocal/immunofluorescence microscopy. MDKC and HEK293T cells were used in experiments to show that the C-terminal half of PC1 tail interacts with STAT6 and activates STAT6-dependent transcription by transfection with FLS-PC1 or luciferase reporter constructs, respectively, followed by Western blotting and reporter assays. MDCK cells continued to be used for experimentation to determine that STAT6 localized to cilia and translocated to the nucleus under ‘no-flow’ conditions via immunofluorescence microscopy with tagged STAT6. Immunohistochemistry was used to detect STAT6 in human ADPKD and normal kidneys. Primary cilia were detected with H&E staining. Zebrafish embryos were utilized to show that the human PC1 tail causes pronephric cysts by injecting one population with FLS-PC1 and monitoring human PC1 mRNA via RT-PCR at 3 days post fertilization.


Key Findings

Because the function of PC1 is poorly understood, these authors aimed to explore and better understand how its binding to transcription factor STAT6 and coactivator P100 affects the relationship between ciliary mechanosensation and the onset of autosomal dominant polycystic kidney disease.  The most significant discoveries in this study are outlined below:

  • Full length PC1 is vulnerable to rapid proteasomal degradation, localizes to the nucleoplasm, and is overexpressed in ADPKD conditions.
  • In a diseased kidney,  the C-terminal half of the PC1 tail is cleaved not only at a GPS domain, but another site as well, which leads to its release from the membrane.
  • Different constructs of PC1 localize to either the cytoplasm (NTS-PC1) or the nucleus (CTS-PC1 and CTSP-PC1), the latter of which are mediated by the C-terminal half of the PC1 tail, suggesting that the tail undergoes nuclear shuttling.
  • P100, which is a coactivator to transcription factor STAT6, binds to the PC1 tail. It  localizes to the basal body and primary cilia in polarized MDCK cells and is overexpressed in ADPKD renal tissue, specifically in cyst-lining epithelial cells.
  • In addition to PC1 binding with P100, it also binds with transcription factor STAT6 to stimulate STAT6-dependent transcription. More specifically, the C-terminal half of the cytoplasmic tail positively regulates STAT6-dependent transcription.
  • STAT6 was found to be moderately expressed in renal epithelial cells of normal human kidneys, overexpressed in cyst-lining epithelial cells of ADPKD kidneys, and is involved in transduction of mechanical signal originating at primary cilia. It is activated under no-flow conditions, translocating from primary cilia to nuclei of renal epithelial cells. Further experimentation suggests that STAT6-dependent gene expression is highly upregulated in ADPKD.
  • Using a zebrafish model, the authors were able to demonstrate that exogenous overexpression of soluble PC1 tail alone can stimulate renal cyst formation.


Broader Context

The primary findings outlined above have allowed the authors to construct the following signaling mechanism, which demonstrates how PC1 modulates STAT6-dependent transcription. Upon initial cleavage of the cytoplasmic tail of PC1,  the membrane-bound C-terminal half of the tail is freed from the membrane. It can then bind to STAT6 and the transcriptional coactivator P100 and translocate into the nucleus where it induces STAT6-dependent transcription. Evidence demonstrating that, in the absence of apical fluid flow, STAT6 translocates from primary cilia to the nucleus allowed the authors to hypothesize that the PC1/STAT6/P100 pathway may have greater implications in the relationship between mechanical signal transduction and transcriptional response. Moreover, because this pathway is highly upregulated in human ADPKD cysts, it is thought to have a significant role in the progression of this human disease. With a better understanding of how PC1/STAT6/P100 pathway is regulated, researchers can now focus their attention on specific components of this pathway that may serve as therapeutic targets for the treatment of ADPKD.




Polycystins and mechanosensation: