Nephrin Trafficking beyond the Kidney—Role in Glucose–Stimulated Insulin Secretion in β Cells
Authors: Sanna Lehtonen, Hannu Jalanko
Journal: Journal of the American Society of Nephrology (2015)

The Ig superfamily member nephrin is the key structural component of the slit diaphragm, which interconnects neighboring podocyte foot processes in kidney glomerulus. Mutations in the nephrin gene (Nephrotic Syndrome Gene 1 [NPHS1]) cause the congenital nephrotic syndrome of the Finnish type (NPHS1) characterized by severe proteinuria during the first weeks of life.1 The disease is most prominent in Finland, with an incidence of one in 8200 births, and >90% of the Finnish patients have two truncating mutations, Finn-major and Finn-minor,1 which lead to total lack of nephrin protein expression.2 NPHS1 caused by Finn-major or Finn-minor mutations is a progressive disease, with renal transplantation providing the only curative therapy. Worldwide, >200 NPHS1 mutations have been identified, and most non-Finns have individual mutations spanning over the whole gene. Although most NPHS1 mutations lead to congenital nephrotic syndrome, it has become clear that some mild mutations can lead to nephrotic syndrome manifesting in older children or even adults, expanding the phenotype caused by this gene defect. The mutations include, among others, numerous missense mutations, which lead to altered post–translational modification and defective trafficking of nephrin to the plasma membrane,3 the main localization site of nephrin in podocytes. Nephrin has multiple functions in podocytes. Together with other membrane proteins, such as podocin, Neph proteins, and cadherin superfamily members, it forms the structural core of the slit diaphragm.4 In addition, nephrin possesses signaling capacities through multiple phosphorylation sites in its intracellular domain and thus, regulates pathways controlling endocytosis, dynamics of the actin cytoskeleton, cytosolic calcium levels, and metabolic and cell survival pathways associated with phosphoinositide-3 kinase (PI3K)/Akt activity.4 Nephrin has also been linked to vesicle trafficking. Specifically, through its interaction with vesicle–associated membrane protein 2, nephrin functions in the fusion of glucose transporter 4 storage vesicles with the plasma membrane to regulate glucose uptake into podocytes.5 In addition to kidney podocytes, nephrin has been reported to be expressed in other tissues, although the data have been controversial. The native murine nephrin gene and a reporter gene driven by nephrin promoter were found to be expressed in mouse podocytes, brain, and pancreas.6,7 The expression in pancreas was confirmed by immunofluorescence staining localizing nephrin to β cells.8 Another study using immunofluorescence, immunoblotting, and in situ hybridization did not report detectable levels of nephrin outside of kidney glomerulus in pigs and humans.9 Clinically, patients with NPHS1 do not have extrarenal manifestations, with the exception of reversible cardiac hypertrophy and muscular hypotonia caused by the severe hypoalbuminemia.2 In an elegant series of works culminating in the study published in this issue of the Journal of the American Society of Nephrology, the group of Fornoni and colleagues10–12 revealed a novel function for nephrin outside podocytes and coupled nephrin to insulin secretion in β cells. Fornoni and colleagues10–12 first confirmed that nephrin is expressed in β cells in human pancreas and showed that nephrin localizes to both the plasma membrane and the surface of insulin-containing vesicles in these cells. Furthermore, nephrin was shown to promote glucose–stimulated insulin release in insulinoma cells and dissociated human islet cells.10 The group describes generation of a mouse model with β cell–specific knockout of nephrin and shows that nephrin signaling is linked to autocrine insulin signaling and the function of β cells. Mice depleted of nephrin in β cells showed no change in body weight and random glycemia or random plasma insulin levels but presented glucose intolerance in vivo and impaired glucose–stimulated release of insulin ex vivo.12 The absence of pancreatic nephrin had no effect on the size or number of the islets or the β cell mass, and the mice did not develop diabetes.12 Interestingly, Fornoni and colleagues12 observed that eight patients with mutations in NPHS1 showed glucose intolerance compared with nine healthy controls and three patients with other genetic forms of nephrotic syndrome. This is in contrast to a previous observation by Kuusniemi et al.,9 who addressed the function of β cells by oral glucose tolerance test in patients with NPHS1 who had received a kidney transplant 1–5 years earlier. In this study, the glucose and insulin response was not different in 36 children with NPSH1 and 25 other patients with kidney transplants.9 It is also known that the patients with NPHS1 do not develop diabetes during the nephrotic stage or after transplantation when diabetogenic drugs, such as tacrolimus, cyclosporin A, and glucocorticoids, are used.13 How can one explain the different results in the pancreatic manifestations when nephrin is mutated in the two study populations? The apparently different mutations in nephrin of the Finnish and German patient groups do not provide a plausible explanation. The Finnish mutations lead to total absence of nephrin protein and the most severe disease manifestation as opposed to the many other mutations, prevailing outside Finland, that often lead to milder outcome. Villareal et al.12 performed glucose tolerance tests in the nephrotic stage when the patients showed heavy proteinuria and renal failure, whereas the previous studies were performed after transplantation in patients with NPHS1 and relatively normal graft function. Because glucose intolerance has been described in patients with proteinuria and impaired renal function for various reasons,14,15 it is important to compare the results between different groups of patients with renal disease. In the study by Villarreal et al.,12 there were eight patients with NPHS1 and three control patients with other proteinuric disorders. Thus, it is obvious that new studies with larger patient populations are needed, which was pointed out by Villarreal et al.12 An interesting observation in the work by Villarreal et al.12 is the finding that nephrin participates in the control of autocrine regulation of insulin secretion by binding to insulin receptor and directly regulating the downstream targets of the cascade. Insulin receptor has two isoforms, insulin receptor A (IRA) and insulin receptor B (IRB), which regulate cellular metabolism through different pathways.16 Insulin stimulates the promoter of insulin gene through IRA and a signaling pathway involving PI3K class Ia and p70 s6 kinase (p70S6K). Insulin-stimulated transcription of glucokinase, a glucose sensor functioning in the maintenance of glucose homeostasis, occurs through IRB and involves PI3K class II and protein kinase B/Akt.16 Nephrin was found to form a complex with IRB but not with IRA, and the complex formation required phosphorylation of IRB.12 Interestingly, overexpression of nephrin in insulinoma cells led to phosphorylation of p70S6K without insulin stimulation, and the increase in p70S6K phosphorylation by nephrin was PI3K/Akt/mammalian target of rapamycin–dependent. Inhibition of the function or activity of the insulin receptor further increased phosphorylation of p70S6K by nephrin, suggesting that complex formation between nephrin and IRB regulates the signaling activity of nephrin by p70S6K. The expression of glucokinase was reduced in the pancreas in the absence of nephrin, further indicating that deficiency of nephrin affects glucose sensing. Numerous in vitro studies have proposed a direct autocrine effect of insulin on β cells, but the significance of this in vivo has been questioned.17 The findings by Villarreal et al.12 suggesting that, during fasting, nephrin can, independent of insulin receptor, stimulate p70S6K-dependent transcription of insulin are intriguing and provide novel mechanistic insights into the regulation of β cell function. The changes observed in the expression and localization of nephrin in diabetes raise an interesting question of whether nephrin could serve as a drug target in diabetes through either modulation of its expression or signaling activity. In patients with diabetes, nephrin is downregulated in the pancreas10 and either downregulated or dislocated from the plasma membrane to the cytoplasm in podocytes.4 Interestingly, glucose seems to play a role in regulating the expression and function of nephrin in both the kidney and pancreas by phosphorylating nephrin on specific residues. In the kidney, high glucose phosphorylates nephrin on threonine residues 1120 and 1125 through protein kinase C-α. This enhances endocytosis of nephrin and contributes to increased albuminuria, apparently because of reduced nephrin at the slit diaphragm.18 In the insulinoma cells, high glucose phosphorylates nephrin on tyrosine residues 1176 and 1193 in a dynamin-dependent manner and enhances nephrin endocytosis and glucose–stimulated insulin release.10,11 Chronic exposure of insulinoma cells to high glucose downregulates nephrin.10 Modulation of nephrin signaling as a treatment would require great care, because nephrin has multiple phosphorylation sites with various effects in different tissues, leading to degradation of nephrin as occurs after phosphorylation of tyrosine 1217 in insulinoma cells.11 The fact that nephrin expression is mainly restricted to glomerular podocytes and pancreatic β cells would allow targeting of these two cell types in a specific manner through nephrin. Additional work is still warranted to fully understand the seemingly complex signaling pathways in β cells that nephrin is involved in. Nevertheless, specific targeting of nephrin or molecules regulating its expression, localization, and signaling in podocytes and β cells would be welcome as new treatment options for diabetes and its kidney complication. Disclosures None. S.L. is supported by Academy of Finland Grant 277485 and grants from the Sigrid Jusélius Foundation, the Diabetes Research Foundation, the Päivikki and Sakari Sohlberg Foundation, and the Faculty of Medicine, University of Helsinki.