Research into HIBM lacks an understanding of basic pathogenic mechanisms, an animal model, and an effective therapy. We took a first step toward addressing these issues by creating a Gne gene–targeted knockin mouse mimicking the M712T mutation of Persian-Jewish HIBM patients. With the exception of 1 male, homozygous mutated (GneM712T/M712T
) mice did not survive past age P3 (Figure D) and showed no muscle pathology at age P2 (Supplemental Figure 1). The lack of early myopathic features recapitulates the human HIBM phenotype. In both cases, the muscle pathology may occur late or be attenuated because a modicum of sialic acid is provided through the actions of residual Gne/Mnk enzymatic activities (11
) (Figure , A and B, and Figure F) supplemented by ancillary enzymes such as GlcNAc kinase (NAGK) (19
) and generic sugar kinases. Eventually, the GneM712T/M712T
mice may develop myopathic features, if they can be maintained well past weaning. Even very young mutant mice exhibited hyposialylation of PSA-NCAM (i.e., in brain) (Figure D), previously reported in skeletal muscle of HIBM patients (15
) and in embryonic stem cells of Gne
knockout mice (16
Remarkably, the GneM712T/M712T
mice clearly differed from HIBM patients in their manifestation of an apparently lethal renal phenotype. The mice exhibited early glomerular disease, with red blood cell infiltrates (Figure , A–D), effacement of podocyte foot processes, and segmental splitting of the lamina densa of the GBM (Figure B), resulting in death within 72 hours after birth. In our group of human HIBM patients, we have no indication of renal abnormalities. Laboratory findings were all within normal ranges, including levels of blood urea nitrogen, creatinine, and urine 24-hour protein and glucose (32
). We know of no reports of abnormal urinary laboratory values in patients with HIBM.
We showed that Gne/Mnk localized to mouse kidney glomeruli (Figure E), a site with high sialic acid concentrations (33
). Humans and mice differ in the relative importance of sialic acid to the kidney, and they certainly differ in the type of sialic acid present. Most mammalian species utilize the sialic acid Neu5Gc (N
-glycolylneuraminic acid), but humans have lost the ability to synthesize Neu5Gc (35
) and rely on Neu5Ac as their main sialic acid. Protein glycosylation patterns also vary; the glomerular sialoprotein podocalyxin itself differs widely among species in the contingent of O
- and N
-linked glycosylation sites in its extracellular aminoterminal region (36
). Future studies, such as employing the Cre-Lox system to create conditional Gne knockouts (22
), might shed light on these issues.
The morphologic abnormalities of podocytes and GBM in the GneM712T/M712T
mouse represent surprising and important findings. Podocytes are glomerular epithelial cells that provide the architecture of the glomerular filtration apparatus, including interdigitating foot processes, slit diaphragms, and the intercellular urinary spaces (23
). The negatively charged sialic acid residues on glycoproteins such as α-dystroglycan, α3β1-integrin, and podocalyxin are essential for the function of podocyte foot processes (24
) and they act as antiadhesion molecules, assisting in maintaining an open urinary space, filtration slits, and Bowman space. Disruption of podocalyxin or neutralization of its negative charge resulted in dissociation of podocalyxin from the actin cytoskeleton and led to foot process effacement (39
). Indeed, we demonstrated the presence of hyposialylated podocalyxin in GneM712T/M712T
mouse kidney extracts (Figure E) along with effacement of foot processes and malformed filtration slits (Figure B). Some forms of glomerular disease (such as minimal change nephrosis; ref. 40
) exhibit hyposialylation of foot process glycoproteins, with subsequent deformation of podocyte membranes and proteinuria (33
). Effacement of podocyte foot processes has also been reported for mice lacking podocalyxin (44
), nephrin (45
), or the giant protocadherin mFAT1 (46
), all leading to proteinuria and death within 72 hours of birth. Other laboratory models have been created to study the phenomenon of effacement of foot processes due to general hyposialylation; these include sialidase inoculation in mice, resulting in hyposialylation of podocalyxin (41
), supplementation studies with purine aminonucleoside (47
), and perfusion of a polycation protamine sulfate (48
). To our knowledge, our GneM712T/M712T
knockin mouse is the first genetic
model demonstrating podocyte flattening and fusion events due to hyposialylation, resulting in proteinuria.
Interestingly, our mutant mice also displayed hematuria, a feature that can be attributed to structural GBM defects (Figure B), which allow red blood cells to gain access to Bowman space, as occurs in thin GBM disease (49
). The segmental GBM splitting in our knockin mice might have resulted from defects in expression levels or charge of specific proteins. In contrast to the findings on podocalyxin (Figure E), testing of other compartment-specific markers, some of which are affected in renal GBM disorders, did not reveal any defects in expression levels in GneM712T/M712T
kidneys. These included GBM markers laminin-1, laminin β1, and Col4A3
(dysregulated in Alport disease) (25
) (Figure C and Supplemental Figure 2) and the podocyte marker podocin (data not shown) (38
). Advanced investigations are required to further elucidate the glomerular phenotype in our GneM712T/M712T
mice. Nevertheless, the severe renal findings of podocytopathy and GBM splitting are likely responsible for the proteinuria, hematuria, and early death in our mice.
Although the exact cause of neonatal lethality in the GneM712T/M712T
mice remains elusive, their rescue by ManNAc suggests that sialic acid deficiency was involved. Outcome parameters included survival (Figure A), improved renal histology (Figure , B–D), including a markedly improved integrity of the GBM (Figure , C and D), less flattened and fused podocyte foot processes (Figure , C and D), increased sialylation of renal podocalyxin (Figure E), and increased sialylation of brain PSA-NCAM (Figure D), all likely related to augmented production of sialic acid. Since ManNAc supplementation increased skeletal muscle Gne epimerase activity (Figure F) and Gne/Mnk protein expression (Figure , A and B), we suggest that ManNAc improved catalytic activity by stabilizing the normal and the mutant enzymes. Similar stabilization effects on other proteins have been demonstrated using natural or artificial ligands or chaperones (51
). The effects of ManNAc on Gne
mutations other than M712T deserve further investigation.
mice can be used to determine the optimal sialic acid repletion therapy for HIBM. Our knockin mice may eventually develop myopathic features similar to the adult-onset myopathy of HIBM patients; treatment could then be geared toward preventing the specific muscle disease. We recently observed a transient but significant improvement in the muscle strength of HIBM patients who received intravenous immune globulin G, and it is hypothesized that this effect is mediated through the large sialic acid content provided by the immune globulin (32
). Other sources of sialic acid may also be reasonable candidates for treating HIBM, especially if they show efficacy in the appropriate mouse model.
In conclusion, we consider the uncharged, physiological monosaccharide ManNAc a promising therapeutic option for HIBM, particularly for patients harboring the M712T GNE
mutation. In addition, ManNAc may have therapeutic potential for some podocytopathies. Candidate disorders include minimal change nephrosis (40
), focal and segmental glomerulosclerosis (53
), membranous glomerulonephritis (54
), and other forms of unexplained idiopathic nephrotic syndrome (55
). The GneM712T/M712T
mice unexpectedly provide a unique opportunity to study basic mechanisms and targeted therapies of podocyte injury and/or GBM splitting, for which appropriate model systems are sparse (23
). This is especially true for disorders involving proteinuria and/or hematuria for which the etiology is unknown and may be related to changes in charge (sialylation) of GBM components. Increased survival of the M712T Gne
knockin mice could serve as an absolute outcome parameter for potential therapeutic interventions, and resolution of renal disease could provide a graded measure of response.