Advertisement
Commentary Free access | 10.1172/JCI32482
The Samuel Lunenfeld Research Institute, Mount Sinai Hospital, and Division of Nephrology, St. Michael’s Hospital, University of Toronto, Toronto, Ontario, Canada.
Address correspondence to: Susan E. Quaggin, The Samuel Lunenfeld Research Institute, Room 855Q, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada. Phone: (416) 586-4800 ext. 2859; Fax: (416) 586-8588; E-mail: [email protected].
Find articles by Quaggin, S. in: JCI | PubMed | Google Scholar
Published June 1, 2007 - More info
Mutations in the key enzyme of sialic acid biosynthesis, uridine diphospho–N-acetylglucosamine 2-epimerase/N-acetylmannosamine (ManNAc) kinase (GNE/MNK), result in hereditary inclusion body myopathy (HIBM), an adult-onset, progressive neuromuscular disorder. We created knockin mice harboring the M712T Gne/Mnk mutation. Homozygous mutant (GneM712T/M712T) mice did not survive beyond P3. At P2, significantly decreased Gne-epimerase activity was observed in GneM712T/M712T muscle, but no myopathic features were apparent. Rather, homozygous mutant mice had glomerular hematuria, proteinuria, and podocytopathy. Renal findings included segmental splitting of the glomerular basement membrane, effacement of podocyte foot processes, and reduced sialylation of the major podocyte sialoprotein, podocalyxin. ManNAc administration yielded survival beyond P3 in 43% of the GneM712T/M712T pups. Survivors exhibited improved renal histology, increased sialylation of podocalyxin, and increased Gne/Mnk protein expression and Gne-epimerase activities. These findings establish this GneM712T/M712T knockin mouse as what we believe to be the first genetic model of podocyte injury and segmental glomerular basement membrane splitting due to hyposialylation. The results also support evaluation of ManNAc as a treatment not only for HIBM but also for renal disorders involving proteinuria and hematuria due to podocytopathy and/or segmental splitting of the glomerular basement membrane.
Belinda Galeano, Riko Klootwijk, Irini Manoli, MaoSen Sun, Carla Ciccone, Daniel Darvish, Matthew F. Starost, Patricia M. Zerfas, Victoria J. Hoffmann, Shelley Hoogstraten-Miller, Donna M. Krasnewich, William A. Gahl, Marjan Huizing
A new study by Galeano and colleagues in this issue of the JCI reports the first glomerular disease caused by a genetic defect in sialic acid biosynthesis (see the related article beginning on page 1585). Mice that harbor mutations in the Gne/Mnk gene produce lower amounts of sialic acid, suffer from hematuria, proteinuria, and structural defects in the glomerulus and die within days after birth. Remarkably, the lesion can be reversed through dietary addition of N-acetylmannosamine, a sialic acid precursor, raising the intriguing possibility that this approach might have therapeutic benefit in patients with glomerular disease.
In this issue of the JCI, Galeano, Huizing, and colleagues (1) describe kidney defects in knockin mice that harbor the M712T mutation in the gene encoding the key bifunctional enzyme of sialic acid biosynthesis — uridine diphospho–N-acetylglucosamine 2-epimerase/N-acetylmannosamine (ManNAc) kinase (GNE/MNK) (GneM712T/M712T mice) (2, 3). In patients, mutations in the GNE gene result in the autosomal recessive neuromuscular disorder, hereditary inclusion body myopathy (HIBM; MIM 600737) that presents late in life as a slowly progressive myopathy (4, 5). The N-acetylglucosamine/ManNAc enzyme is ubiquitously expressed and catalyzes the first rate-limiting steps in the biosynthesis of sialic acid (Figure 1). 5-N-acetylneuraminic acid (Neu5Ac) is the most plentiful mammalian sialic acid and is the terminal sugar on glycoconjugates, where it functions in cellular interactions and signaling. Muscle fibers from patients with HIBM exhibit reduced sialylation of proteins, which is believed to underlie disease pathogenesis. Currently, there is no effective therapy for this disorder.
The biochemical pathway of sialic acid formation. CMP, cytidine monophosphate; MaNAc-6-P, MaNAc-6-phosphate; NeuAc-9-P, N-acetylneuraminic acid–9-phosphate; sialyl-OGS, sialylated oligosaccharides; PEP, phosphoenolpyruvate. Reproduced from Galeano et al. (1). CTP, cytidine triphosphate.
In the current study (1), the investigators sought to develop a mouse model to test whether dietary supplementation of sialic acid or its precursor, ManNAc, could reverse the hyposialylation defect observed in patients. Standard Gne knockout mice die in utero (6). To overcome this early mortality, the authors generated a mouse carrying one of the most common non-lethal mutations observed in patients with HIBM. Surprisingly, mice homozygous for the GneM712T allele died within days of birth with major defects in the glomerulus of the kidney. They did not survive long enough to determine whether they would develop any muscle disease late in life. Patients with HIBM are not reported to have glomerular disease or abnormalities in renal function. Given the discrepancy in phenotype between species, the most obvious first question is whether sialylation defects in the kidney can explain the glomerular defect.
The glomerular filtration barrier separates the blood from the urinary space and is comprised of renal podocytes (glomerular visceral epithelial cells), fenestrated endothelial cells, and an intervening glomerular basement membrane (7, 8) (Figure 2). Each kidney contains roughly 1 million individual glomeruli that together produce 180 liters of filtrate per day. The barrier restricts passage of large macromolecules such as albumin and cells while permitting free passage to water and small solutes (9). The most abundant and best-studied sialoglycoprotein in the glomerulus is podocalyxin (PC) (10). PC is found on glomerular endothelial cells and on the apical or free-floating surface of podocytes, where it connects to the actin cytoskeleton through Na+/H+ exchanger regulatory factor 2 (NHERF2) and phosphorylated ezrin (11, 12). Loss of the interaction between PC and the actin cytoskeleton is associated with the nephrotic syndrome, a disorder characterized by proteinuria and dramatic structural change of the podocytes due to collapse and flattening of the actin-based foot processes (13). Loss of sialylation of PC occurs in rodents injected with sialidase, puromycin aminonucleoside, or protamine sulfate, which neutralizes negative charges (14–16). All of these compounds cause an abrupt onset of proteinuria and foot process fusion. Simultaneous infusion of sialic acid prevents the proteinuria and podocyte effacement observed with puromycin injection, presumably due to resialylation of critical glomerular proteins (17). Taken together, these findings underscore the importance of protein sialylation in the control of glomerular structure and function.
The glomerular filtration barrier, with and without sialylated proteins. Blood enters the glomerular capillaries and is filtered across the endothelium and the basement membrane and through the filtration slits between podocyte foot processes to produce the primary urinary filtrate. In healthy glomeruli, this barrier restricts the passage of macromolecules but is highly permeable to water and small solutes. In this issue of the JCI, Galeano et al. (1) show that a mutation (M712T) in Gne/Mnk in mice results in a reduction in the number of sialic acid residues on critical glomerular proteins such as PC that are found on the apical surface of podocytes. Loss of sialylated proteins is associated with foot process fusion or collapse, splitting of the glomerular basement membrane (GBM), and loss of rbc and proteins such as albumin into the urine. Dietary supplementation with ManNAc prolongs life in mutant GneM712T/M712T mice and improves ultrastructure of the glomerular filtration barrier.
Mice homozygous for the GneM712T/M712T mutation die within days of birth with growth retardation and major structural abnormalities in the kidneys. The urinary space and renal tubules in these mice are packed with red blood cells and protein without any obvious inflammation, suggesting a major defect in formation and function of the glomerular filtration barrier. Accordingly, electron micrographs show segmental splitting of the glomerular basement membrane and fusion or absence of foot processes (Figure 2). Formation of the foot processes requires slit diaphragm (SD) proteins that are found in specialized intercellular junctions between foot processes. Phosphorylation of the SD protein nephrin (NPHS1) results in recruitment of Nck (non-catalytic region of tyrosine kinase) SH2-containing adaptor proteins and actin cytoskeletal reorganization (18–21). Although Galeano et al. (1) show that the SD proteins NPHS1 and podocin (NPHS2) are expressed in glomeruli from Gne mutants at wild-type levels, their subcellular localization and/or phosphorylation status were not examined.
Sialylation of PC is reduced in glomeruli from GneM712T/M712T mutant mice (1). Because PC is known to associate with actin cytoskeletal proteins in the podocytes, and loss of this interaction disrupts glomerular barrier function, hyposialylation might explain the renal defect in the GneM712T/M712T mutants. However, there are significant differences in phenotypes between PC KO mice and the GneM712T/M712T mutants. PC KO mice also die in the perinatal period with renal defects that include failure of SD formation, fusion of foot processes, and a complete lack of filtration with no urine in the bladder (21). However, no splitting of the glomerular basement membrane or leakage of red blood cells into the renal tubules was observed. In addition, PC KO mice exhibit extrarenal defects such as hernias and omphaloceles (protrusions of the intestine and omentum through a hernia in the abdominal wall near the navel) due to the broad expression of PC during fetal life. By contrast, no other defects were observed in the GneM712T/M712T mutants in any other organs including muscle, heart, or lungs. Furthermore, Gne KO mice rescued by a Gne transgene harboring another common mutation (V572L, which is found in patients of Japanese origin with HIBM) survived but develop muscle defects and fibrosis of the diaphragm and heart. Interestingly, some of these mice died suddenly, although the cause was unclear. Although the authors stated that other internal organs had no defects, analysis of renal function was not described in detail (22).
Why are the glomerular defects apparently more profound in GneM712T/M712T mutants than in PC KO mice or other animal models of sialylation defects? Several possible explanations exist. While PC is expressed by the podocyte, the endothelium and glomerular basement membranes also make significant contributions to the glomerular filtration barrier, and it is reasonable to assume that proteins in these compartments may also require sialic acid for normal functioning. In keeping with this hypothesis, PC is also expressed by endothelial cells, and splitting of the glomerular basement membrane (with hematuria) seen in the GneM712T/M712T mutants is reminiscent of the lesion seen in Alport syndrome, a disease caused by mutations in the collagen type IV α5 (COL4a5), COL4a4, and COL4a3 genes; the proteins encoded by these genes are major constituents of the glomerular basement membrane (23).
Strikingly, Galeano et al. (1) show that supplementation of the pregnant mouse’s diet with ManNAc, a precursor to sialic acid that readily crosses cell membranes, prolongs survival of the GneM712T/M712Tmice. This treatment was associated with an almost 10-fold enhancement of GNE enzyme activity in the kidney. Furthermore, sialylation of PC was also significantly increased in the kidneys of treated mutants, and there was marked improvement in ultrastructure of the filtration barrier.
Admittedly, there are striking differences between the human and mouse phenotypes caused by the identical GNE/Gne mutant alleles. Further understanding of the nature of this species difference could provide important insights into the regulation of protein sialylation in the kidney and its contribution to filtration barrier function. There is abundant evidence that human glomerular proteins are sialylated and that alterations in sialylation patterns can be associated with disease, although sialylation defects of individual proteins have not been reported. Thus, based on the dramatic effects of dietary ManNAc supplementation in this very aggressive model of glomerular pathology along with the simplicity and tolerability of this intervention, it seems plausible that this pathway might be useful as a more general therapeutic approach for glomerular diseases of many causes. This approach is especially attractive, as current therapies are nonspecific, associated with significant adverse side effects, and are often ineffective.
S.E. Quaggin thanks Dontscho Kerjaschki for critical review and helpful comments. S.E. Quaggin is the recipient of a Canada Research Chair Tier II, an Emerald Foundation award, and a Premier’s of Ontario Research of Excellence Award. This work was funded through Canadian Institutes of Health Research grant MOP 77756, National Cancer Institute of Canada grant 16002, and a Kidney Foundation of Canada Award.
Address correspondence to: Susan E. Quaggin, The Samuel Lunenfeld Research Institute, Room 855Q, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada. Phone: (416) 586-4800 ext. 2859; Fax: (416) 586-8588; E-mail: [email protected].
Nonstandard abbreviations used: Gne, uridine diphospho–N-acetylglucosamine 2-epimerase; HIBM, hereditary inclusion body myopathy; ManNAc, N-acetylmannosamine; Mnk, ManNAc kinase; PC, podocalyxin; SD, slit diaphragm.
Conflict of interest: The author is the recipient of a research grant from Genzyme and is a scientific consultant for Genentech.
Reference information: J. Clin. Invest.117:1480–1483 (2007). doi:10.1172/JCI32482.
See the related article at Mutation in the key enzyme of sialic acid biosynthesis causes severe glomerular proteinuria and is rescued by N-acetylmannosamine.