- Email: [email protected]
Lose the lipid: renoprotection conferred by Gb3 synthase knockout
commentary
www.kidney-international.org
Lose the lipid: renoprotection conferred by Gb3 synthase knockout Anata´lia Labilloy1 and Ora A. Weisz2 The ability of proximal tubule cells to internalize filtered proteins over a broad concentration range is essential for maintaining a protein-free urine but also renders these cells uniquely susceptible to cytotoxic damage. Morace et al. find that knockout of globotriaosylceramide synthase, an enzyme required for production of Gb3 and other members of the globo series of glycosphingolipids, impairs endocytic uptake of filtered proteins and preserves kidney function in mouse models of acute kidney injury. Kidney International (2019) 96, 270–272; https://doi.org/10.1016/j.kint.2019.03.034 Copyright ª 2019, International Society of Nephrology. Published by Elsevier Inc. All rights reserved.
see basic research on page 327
A
cute kidney injury (AKI) is a major cause of morbidity in children and adults, especially those who are critically ill, and when present it independently increases their overall risk of poor clinical outcomes and early death. Cells of the proximal tubule (PT) are particularly vulnerable to injury by ischemic, toxic (i.e., nephrotoxic drugs, radiocontrast agents, reactive oxygen species, heavy metals), or obstructive insults. Feedback mechanisms exist whereby diseased PT cells cause glomerular pathology, further exacerbating kidney disease.1 Currently, few interventions exist to prevent or limit AKI. A major function of the PT is to recapture proteins that escape the glomerular filtration barrier. Albumin is the most abundant protein in the plasma, and albuminuria or loss of albumin in the urine is a well-defined clinical marker of kidney injury, whether it originates from 1
Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA; and 2Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA Correspondence: Ora A. Weisz, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, 3550 Terrace St., Pittsburgh, Pennsylvania 15261, USA. E-mail: [email protected] 270
glomerular or PT dysfunction or a combination. Albumin is mainly internalized via clathrin-mediated endocytosis upon binding to the multiligand receptors megalin and cubilin, which are abundantly expressed in PT cells.2 Although only small amounts of protein enter the tubule lumen under normal conditions, PT cells are able to efficiently recover filtered proteins over a remarkable concentration range. The capability to internalize large amounts of protein upon exposure to an overload of abnormally filtered proteins is important for maintaining serum protein composition, but also predisposes PT cells to injury. Damage to the glomerular filtration barrier or release of myoglobin and hemoglobin into the circulation during rhabdomyolytic and hemolytic injuries results in dramatic increases in PT internalization of albumin, myoglobin, and hemoglobin, respectively. Under these conditions, exposure to saturated fatty acids and heme carried by filtered proteins can lead to PT damage, cell death, and interstitial inflammation and fibrosis. Similarly, the PT is prone to nephrotoxic damage from gentamicin and other aminoglycosides that once internalized upon binding to megalin and cubilin receptors, and can interfere with
phospholipid metabolism, protein synthesis and sorting, and mitochondrial function. Limiting the uptake of these proteins and drugs by impairing the PT endocytic pathway is predicted to protect PT cells and reduce kidney injury. Glycosphingolipids (GSLs) are ubiquitous membrane components composed of a long-chain sphingoid amine, a fatty acid, and a carbohydrate. These lipids can act as primary receptors for carbohydrate-binding proteins and can cluster in cholesterol-enriched membrane microdomains that play a primary role in protein sorting and intracellular trafficking. A variety of complex cellular processes, including ion transport, cellular proliferation and differentiation, and signal transduction, are modulated by GSLs. GSLs are highly abundant in the kidney, and extensive effort has been directed toward characterizing the GSL composition and distribution in different kidney cell types. Dysregulated GSL metabolism has been demonstrated in a variety of kidney diseases including diabetic nephropathy, polycystic kidney disease, and glomerulonephritis. Accumulation of the neutral GSL globotriaosylceramide Gb3 and its deacylated counterpart, globotriaosylsphingosine, in Fabry disease (due to mutations in the Gb3 hydrolyzing enzyme a-galactosidase A) causes significant kidney dysfunction, with proteinuria, tubulointerstitial disease, reduced glomerular filtration rate, and ultimately renal failure. Gb3 is also known to be a receptor for Shiga toxin, a potent bacterial toxin associated with hemolytic uremic syndrome, a leading cause of acute kidney injury in children. However, the normal function of Gb3 in the kidney, and particularly in the PT, remains unknown. Data from Morace and colleagues3 in this issue of Kidney International now suggest that Gb3 plays a role in the efficient reabsorption of filtered proteins and that reduction of this lipid in the PT can be renoprotective. Previous work by others has demonstrated that pharmacologic inhibition of GlcCer synthesis, an enzyme critical for synthesis of nearly all glycosphingolipids, confers resistance to renal damage by Shiga toxin in rats.4 The authors extend this finding by Kidney International (2019) 96, 270–286
commentary
a
b
c
d
Ceramide Control GlcCer
Ganglio Series (Gm3…)
Globo Series (Gb3 and Gb4)
Gb3S KO
Albumin uptake
Neolacto Series (Lc3…)
Gb3 Synthase
LacCer
Disease models
Control
Gb3S-/-
Gb3S-/-
Gb3 Megalin Cubilin Albumin
In normal PT cells, Gb3 colocalizes with megalin, cubilin, and internalized albumin
Slight decrease in PT albumin uptake/ Slight increase in albumin excretion
Significant renoprotection from myoglobin- and glycerol-induced kidney injury
Figure 1 | Gb3 synthase knockout is renoprotective against kidney injury. (a) Schematic of the glycosphingolipid synthesis pathway. Ceramide (Cer) is converted sequentially to Glucosylceramide (GlcCer) and then Lactosylceramide (LacCer), the precursor lipid for formation of neolacto-, globo- and ganglio-series lipids. The enzyme Gb3 synthase (Gb3S) is required for conversion of LacCer to globotriaosylceramide (Gb3) which can be further glycosylated to generate Gb4. (b) In proximal tubule (PT) cells, Gb3 localizes largely to intracellular albumin-positive compartments as well as with megalin and cubilin at the apical plasma membrane. (c) Knockdown of Gb3S in the absence of disease induces a doubling of (the normally very low) urinary albumin excretion in mice that likely reflects a slight reduction in PT albumin uptake. (d) In disease conditions, Gb3S knockdown provides significant renoprotection from myoglobin- and gentamicin-induced kidney injury. The renoprotective mechanism is unclear: loss of Gb3 could affect membrane fluidity or signaling pathway to impair endocytosis; alternatively, Gb3 may normally function as a coreceptor or as an alternative receptor for megalin-cubilin–mediated uptake of filtered ligands. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
showing that global knockout of Gb3 synthase (Gb3S) protects C57BL/6 mice from renal damage in AKI models of rhabdomyolysis and gentamicin toxicity (Figure 1). Similar results were obtained with a tubular knockout of uridine diphosphate-glucose:ceramide glucosyltransferase (the enzyme that synthesizes GlcCer). Additionally, they demonstrate that administration of the GlcCer inhibitor Genz123346 for 3 weeks also prevents damage from glycerol-induced rhabdomyolysis. Because (unlike in humans) Gb3S is not expressed in murine podocytes, the authors argue that changes in glomerular function or permeability do not contribute to the observed effects. However, changes in PT megalin or cubilin expression were not examined in the context of disease. The authors do include important controls to ensure that inflammatory responses and the extent of muscle injury are similar in wild-type and Gb3S–/– mice. These observations, together with the finding that myoglobin uptake by PT cells is qualitatively reduced in Gb3S–/– mice, lead the authors to legitimately conclude that Gb3 plays a role in maximizing the reabsorption of filtered megalin/cubilin ligands. Kidney International (2019) 96, 270–286
Although the protective effect of Gb3 synthase knockout in these disease models is clear, the effect of Gb3 in the PT uptake of filtered proteins under normal conditions is less obvious. For example, urinary albumin excretion was only incrementally higher (< 2-fold) in Gb3S–/– mice compared with wild-type mice, and this difference was not magnified when mice were fed high-fat diets. By contrast, albumin excretion is about 20-fold higher when cubilin and megalin receptors are knocked out.5 The authors use an enviable array of technologies to connect Gb3 with the endocytic machinery and examine the mechanism of Gb3 function in both mouse and human cells. Quantitative analysis using electron microscopy revealed a 2-fold increase in apical membrane invaginations and a slight but significant reduction in subapical vesicles in the PT cells of Gb3S–/– mice, suggestive of a global impairment in endocytic pathway function. Similarly, intravital studies showed a reduction in the rate of albumin spreading from the luminal aspect of PT cells toward the basal surface, interpreted by the authors as evidence of impaired endocytic traffic in Gb3S–/– mice. To address the question
of how Gb3 normally participates in endocytic uptake, the authors carefully examined the distribution of the lipid with superresolution microscopy. Partial colocalization was observed between Gb3 (visualized with STxB-Cy3) and megalin/cubilin at the apical plasma membrane and with FITC-albumin in intracellular compartments. To extend these studies to human cells, they created a CRISPR/Cas9 knockout model of Gb3S in HK-2 cells. Using a proximity ligation assay designed to detect interaction between 2 target proteins, they demonstrate close apposition of anti-Gb3 with anti-cubilin antibodies in parental HK2 cells and also document reduced albumin uptake in the Gb3S knockout clone. Based on these findings, the authors conclude that Gb3 either functions as an “attachment or capacity receptor” for megalin/cubilin or as a separate receptor system at the PT apical surface. Considering that both megalin and cubilin are very large proteins with multiple ligandbinding domains that extend far above the plane of the plasma membrane, it is difficult to envision how Gb3 might serve as an endocytic coreceptor, and other mechanisms by which Gb3 might 271
commentary
influence endocytic traffic are perhaps more likely. In addition to their multiple cell-signaling functions, GSLs have wellestablished roles in modulating membrane curvature and fluidity that could readily alter endocytic traffic in PT cells. Indeed, Shiga toxin binding to Gb3 at the plasma membrane is known to cause lipid reorganization that induces the formation of membrane invaginations.6 Although the role of membrane traffic versus other signaling functions of Gb3 in protection of the PT needs to be sorted out, either way this lipid plays an important role in PT function that deserves additional study. As with all multifaceted studies, this work has some limitations. Despite the careful controls, one cannot exclude the possibility that extrarenal effects contribute to results observed with the Gb3S global knockout mouse model. Additionally, the authors conclude that effects they observe are due to alterations in Gb3 levels, but Gb3S knockout results in multiple changes to the cellular GSL profile, including loss of Gb4 (synthesized from Gb3) and increased levels of lactosylceramide that in turn can be converted to neolacto-GSLs and gangliosides. Both Gb4 and the ganglioside Gm3 are abundantly expressed in mouse PTs.7,8 Moreover, STxB binding to Gb3 is known to induce changes in intracellular trafficking that might not be seen in the native state, and it is unclear whether some of the colocalization findings might have been artificially induced by the use of this reagent in living cells to detect Gb3. Finally, because the role of Gb3S in human podocytes could not be explored in this study, it is unclear how altering Gb3 levels in these cells might impact kidney function and how translatable these findings are to human kidney disease. Reducing exposures and susceptibility risk factors is the most effective way to prevent development or progression of AKI. However, this is not always feasible. Several agents that target specific pathways have been proposed as renoprotective with varied response in AKI animal models.9 This study shows a significant renoprotection achieved by GSL modulation in 2 common clinical scenarios in animal models, with potential management 272
applications. Although critical issues remain to be sorted out before concluding that this approach will work in humans, individuals who might benefit from such treatment would be those with genetic conditions that cause recurrent episodes of severe rhabdomyolysis such as glycogen storage disorders, fatty acid oxidation disorders, and mitochondrial myopathies, or when a long course of aminoglycoside treatment is anticipated in individuals with known susceptibility risk factors. The role of Gb3 modulation in preventing AKI due to other insults also needs to be further explored. DISCLOSURE All the authors declared no competing interests. REFERENCES 1. Lameire N, Vanbiesen W, Vanholder R. Acute renal failure. Lancet. 2005;365:417–430. 2. Eshbach ML, Weisz OA. Receptor-mediated endocytosis in the proximal tubule. Annu Rev Physiol. 2017;79:425–448.
3. Morace I, Pilz R, Federico G, et al. Renal globotriaosylceramide facilitates tubular albumin absorption and its inhibition protects against acute kidney injury. Kidney Int. 2019;96:327–341. 4. Silberstein C, Lucero MS, Zotta E, et al. A glucosylceramide synthase inhibitor protects rats against the cytotoxic effects of shiga toxin 2. Pediatr Res. 2011;69:390–394. 5. Weyer K, Andersen PK, Schmidt K, et al. Abolishment of proximal tubule albumin endocytosis does not affect plasma albumin during nephrotic syndrome in mice. Kidney Int. 2018;93:335–342. 6. Römer W, Berland L, Chambon V, et al. Shiga toxin induces tubular membrane invaginations for its uptake into cells. Nature. 2007;450:670–675. 7. Kaneko T, Tsubakihara Y, Fushimi H, et al. Histochemical and immunoelectron microscopic analysis of ganglioside GM3 in human kidney. Clin Exp Nephrol. 2015;19:403– 410. 8. Fujii Y, Numata S, Nakamura Y, et al. Murine glycosyltransferases responsible for the expression of globo-series glycolipids: cDNA structures, mRNA expression, and distribution of their products. Glycobiology. 2005;15:1257– 1267. 9. Yang Y, Song M, Liu Y, et al. Renoprotective approaches and strategies in acute kidney injury. Pharmacol Ther. 2016;163:58–73.
A zebrafish tale of parabiosis, podocytes, and proteinuria Brooke E. Chambers1 and Rebecca A. Wingert1 Glomerular damage is a harbinger of kidney dysfunction. Circulating permeability factors are implicated in causing primary and secondary damage to podocytes, leading to proteinuria and eventual progression to the nephrotic syndrome, but the mechanisms are not well understood. Müller-Deile et al. employed parabiosis with zebrafish embryos and found that a damaged glomerulus can impact a healthy one in a shared circulatory system. This methodology shows promise for elucidating kidney injury pathways in response to systemic disease. Kidney International (2019) 96, 272–275; https://doi.org/10.1016/j.kint.2019.04.041 Copyright ª 2019, International Society of Nephrology. Published by Elsevier Inc. All rights reserved.
see basic research on page 342
1
Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, Indiana, USA Correspondence: Rebecca A. Wingert, Department of Biological Sciences, University of Notre Dame, 100 Galvin Life Science Research Building, Notre Dame, Indiana 46556, USA. E-mail: [email protected]
T
he glomerulus is a spherical blood filter and first element of the nephron that is followed by the proximal tubule. The glomerulus consists of a dense mesh of capillaries composed of fenestrated endothelial cells that are surrounded by a basement membrane and encased with specialized epithelial cells called podocytes. The podocytes Kidney International (2019) 96, 270–286
www.kidney-international.org
Lose the lipid: renoprotection conferred by Gb3 synthase knockout Anata´lia Labilloy1 and Ora A. Weisz2 The ability of proximal tubule cells to internalize filtered proteins over a broad concentration range is essential for maintaining a protein-free urine but also renders these cells uniquely susceptible to cytotoxic damage. Morace et al. find that knockout of globotriaosylceramide synthase, an enzyme required for production of Gb3 and other members of the globo series of glycosphingolipids, impairs endocytic uptake of filtered proteins and preserves kidney function in mouse models of acute kidney injury. Kidney International (2019) 96, 270–272; https://doi.org/10.1016/j.kint.2019.03.034 Copyright ª 2019, International Society of Nephrology. Published by Elsevier Inc. All rights reserved.
see basic research on page 327
A
cute kidney injury (AKI) is a major cause of morbidity in children and adults, especially those who are critically ill, and when present it independently increases their overall risk of poor clinical outcomes and early death. Cells of the proximal tubule (PT) are particularly vulnerable to injury by ischemic, toxic (i.e., nephrotoxic drugs, radiocontrast agents, reactive oxygen species, heavy metals), or obstructive insults. Feedback mechanisms exist whereby diseased PT cells cause glomerular pathology, further exacerbating kidney disease.1 Currently, few interventions exist to prevent or limit AKI. A major function of the PT is to recapture proteins that escape the glomerular filtration barrier. Albumin is the most abundant protein in the plasma, and albuminuria or loss of albumin in the urine is a well-defined clinical marker of kidney injury, whether it originates from 1
Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA; and 2Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA Correspondence: Ora A. Weisz, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, 3550 Terrace St., Pittsburgh, Pennsylvania 15261, USA. E-mail: [email protected] 270
glomerular or PT dysfunction or a combination. Albumin is mainly internalized via clathrin-mediated endocytosis upon binding to the multiligand receptors megalin and cubilin, which are abundantly expressed in PT cells.2 Although only small amounts of protein enter the tubule lumen under normal conditions, PT cells are able to efficiently recover filtered proteins over a remarkable concentration range. The capability to internalize large amounts of protein upon exposure to an overload of abnormally filtered proteins is important for maintaining serum protein composition, but also predisposes PT cells to injury. Damage to the glomerular filtration barrier or release of myoglobin and hemoglobin into the circulation during rhabdomyolytic and hemolytic injuries results in dramatic increases in PT internalization of albumin, myoglobin, and hemoglobin, respectively. Under these conditions, exposure to saturated fatty acids and heme carried by filtered proteins can lead to PT damage, cell death, and interstitial inflammation and fibrosis. Similarly, the PT is prone to nephrotoxic damage from gentamicin and other aminoglycosides that once internalized upon binding to megalin and cubilin receptors, and can interfere with
phospholipid metabolism, protein synthesis and sorting, and mitochondrial function. Limiting the uptake of these proteins and drugs by impairing the PT endocytic pathway is predicted to protect PT cells and reduce kidney injury. Glycosphingolipids (GSLs) are ubiquitous membrane components composed of a long-chain sphingoid amine, a fatty acid, and a carbohydrate. These lipids can act as primary receptors for carbohydrate-binding proteins and can cluster in cholesterol-enriched membrane microdomains that play a primary role in protein sorting and intracellular trafficking. A variety of complex cellular processes, including ion transport, cellular proliferation and differentiation, and signal transduction, are modulated by GSLs. GSLs are highly abundant in the kidney, and extensive effort has been directed toward characterizing the GSL composition and distribution in different kidney cell types. Dysregulated GSL metabolism has been demonstrated in a variety of kidney diseases including diabetic nephropathy, polycystic kidney disease, and glomerulonephritis. Accumulation of the neutral GSL globotriaosylceramide Gb3 and its deacylated counterpart, globotriaosylsphingosine, in Fabry disease (due to mutations in the Gb3 hydrolyzing enzyme a-galactosidase A) causes significant kidney dysfunction, with proteinuria, tubulointerstitial disease, reduced glomerular filtration rate, and ultimately renal failure. Gb3 is also known to be a receptor for Shiga toxin, a potent bacterial toxin associated with hemolytic uremic syndrome, a leading cause of acute kidney injury in children. However, the normal function of Gb3 in the kidney, and particularly in the PT, remains unknown. Data from Morace and colleagues3 in this issue of Kidney International now suggest that Gb3 plays a role in the efficient reabsorption of filtered proteins and that reduction of this lipid in the PT can be renoprotective. Previous work by others has demonstrated that pharmacologic inhibition of GlcCer synthesis, an enzyme critical for synthesis of nearly all glycosphingolipids, confers resistance to renal damage by Shiga toxin in rats.4 The authors extend this finding by Kidney International (2019) 96, 270–286
commentary
a
b
c
d
Ceramide Control GlcCer
Ganglio Series (Gm3…)
Globo Series (Gb3 and Gb4)
Gb3S KO
Albumin uptake
Neolacto Series (Lc3…)
Gb3 Synthase
LacCer
Disease models
Control
Gb3S-/-
Gb3S-/-
Gb3 Megalin Cubilin Albumin
In normal PT cells, Gb3 colocalizes with megalin, cubilin, and internalized albumin
Slight decrease in PT albumin uptake/ Slight increase in albumin excretion
Significant renoprotection from myoglobin- and glycerol-induced kidney injury
Figure 1 | Gb3 synthase knockout is renoprotective against kidney injury. (a) Schematic of the glycosphingolipid synthesis pathway. Ceramide (Cer) is converted sequentially to Glucosylceramide (GlcCer) and then Lactosylceramide (LacCer), the precursor lipid for formation of neolacto-, globo- and ganglio-series lipids. The enzyme Gb3 synthase (Gb3S) is required for conversion of LacCer to globotriaosylceramide (Gb3) which can be further glycosylated to generate Gb4. (b) In proximal tubule (PT) cells, Gb3 localizes largely to intracellular albumin-positive compartments as well as with megalin and cubilin at the apical plasma membrane. (c) Knockdown of Gb3S in the absence of disease induces a doubling of (the normally very low) urinary albumin excretion in mice that likely reflects a slight reduction in PT albumin uptake. (d) In disease conditions, Gb3S knockdown provides significant renoprotection from myoglobin- and gentamicin-induced kidney injury. The renoprotective mechanism is unclear: loss of Gb3 could affect membrane fluidity or signaling pathway to impair endocytosis; alternatively, Gb3 may normally function as a coreceptor or as an alternative receptor for megalin-cubilin–mediated uptake of filtered ligands. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
showing that global knockout of Gb3 synthase (Gb3S) protects C57BL/6 mice from renal damage in AKI models of rhabdomyolysis and gentamicin toxicity (Figure 1). Similar results were obtained with a tubular knockout of uridine diphosphate-glucose:ceramide glucosyltransferase (the enzyme that synthesizes GlcCer). Additionally, they demonstrate that administration of the GlcCer inhibitor Genz123346 for 3 weeks also prevents damage from glycerol-induced rhabdomyolysis. Because (unlike in humans) Gb3S is not expressed in murine podocytes, the authors argue that changes in glomerular function or permeability do not contribute to the observed effects. However, changes in PT megalin or cubilin expression were not examined in the context of disease. The authors do include important controls to ensure that inflammatory responses and the extent of muscle injury are similar in wild-type and Gb3S–/– mice. These observations, together with the finding that myoglobin uptake by PT cells is qualitatively reduced in Gb3S–/– mice, lead the authors to legitimately conclude that Gb3 plays a role in maximizing the reabsorption of filtered megalin/cubilin ligands. Kidney International (2019) 96, 270–286
Although the protective effect of Gb3 synthase knockout in these disease models is clear, the effect of Gb3 in the PT uptake of filtered proteins under normal conditions is less obvious. For example, urinary albumin excretion was only incrementally higher (< 2-fold) in Gb3S–/– mice compared with wild-type mice, and this difference was not magnified when mice were fed high-fat diets. By contrast, albumin excretion is about 20-fold higher when cubilin and megalin receptors are knocked out.5 The authors use an enviable array of technologies to connect Gb3 with the endocytic machinery and examine the mechanism of Gb3 function in both mouse and human cells. Quantitative analysis using electron microscopy revealed a 2-fold increase in apical membrane invaginations and a slight but significant reduction in subapical vesicles in the PT cells of Gb3S–/– mice, suggestive of a global impairment in endocytic pathway function. Similarly, intravital studies showed a reduction in the rate of albumin spreading from the luminal aspect of PT cells toward the basal surface, interpreted by the authors as evidence of impaired endocytic traffic in Gb3S–/– mice. To address the question
of how Gb3 normally participates in endocytic uptake, the authors carefully examined the distribution of the lipid with superresolution microscopy. Partial colocalization was observed between Gb3 (visualized with STxB-Cy3) and megalin/cubilin at the apical plasma membrane and with FITC-albumin in intracellular compartments. To extend these studies to human cells, they created a CRISPR/Cas9 knockout model of Gb3S in HK-2 cells. Using a proximity ligation assay designed to detect interaction between 2 target proteins, they demonstrate close apposition of anti-Gb3 with anti-cubilin antibodies in parental HK2 cells and also document reduced albumin uptake in the Gb3S knockout clone. Based on these findings, the authors conclude that Gb3 either functions as an “attachment or capacity receptor” for megalin/cubilin or as a separate receptor system at the PT apical surface. Considering that both megalin and cubilin are very large proteins with multiple ligandbinding domains that extend far above the plane of the plasma membrane, it is difficult to envision how Gb3 might serve as an endocytic coreceptor, and other mechanisms by which Gb3 might 271
commentary
influence endocytic traffic are perhaps more likely. In addition to their multiple cell-signaling functions, GSLs have wellestablished roles in modulating membrane curvature and fluidity that could readily alter endocytic traffic in PT cells. Indeed, Shiga toxin binding to Gb3 at the plasma membrane is known to cause lipid reorganization that induces the formation of membrane invaginations.6 Although the role of membrane traffic versus other signaling functions of Gb3 in protection of the PT needs to be sorted out, either way this lipid plays an important role in PT function that deserves additional study. As with all multifaceted studies, this work has some limitations. Despite the careful controls, one cannot exclude the possibility that extrarenal effects contribute to results observed with the Gb3S global knockout mouse model. Additionally, the authors conclude that effects they observe are due to alterations in Gb3 levels, but Gb3S knockout results in multiple changes to the cellular GSL profile, including loss of Gb4 (synthesized from Gb3) and increased levels of lactosylceramide that in turn can be converted to neolacto-GSLs and gangliosides. Both Gb4 and the ganglioside Gm3 are abundantly expressed in mouse PTs.7,8 Moreover, STxB binding to Gb3 is known to induce changes in intracellular trafficking that might not be seen in the native state, and it is unclear whether some of the colocalization findings might have been artificially induced by the use of this reagent in living cells to detect Gb3. Finally, because the role of Gb3S in human podocytes could not be explored in this study, it is unclear how altering Gb3 levels in these cells might impact kidney function and how translatable these findings are to human kidney disease. Reducing exposures and susceptibility risk factors is the most effective way to prevent development or progression of AKI. However, this is not always feasible. Several agents that target specific pathways have been proposed as renoprotective with varied response in AKI animal models.9 This study shows a significant renoprotection achieved by GSL modulation in 2 common clinical scenarios in animal models, with potential management 272
applications. Although critical issues remain to be sorted out before concluding that this approach will work in humans, individuals who might benefit from such treatment would be those with genetic conditions that cause recurrent episodes of severe rhabdomyolysis such as glycogen storage disorders, fatty acid oxidation disorders, and mitochondrial myopathies, or when a long course of aminoglycoside treatment is anticipated in individuals with known susceptibility risk factors. The role of Gb3 modulation in preventing AKI due to other insults also needs to be further explored. DISCLOSURE All the authors declared no competing interests. REFERENCES 1. Lameire N, Vanbiesen W, Vanholder R. Acute renal failure. Lancet. 2005;365:417–430. 2. Eshbach ML, Weisz OA. Receptor-mediated endocytosis in the proximal tubule. Annu Rev Physiol. 2017;79:425–448.
3. Morace I, Pilz R, Federico G, et al. Renal globotriaosylceramide facilitates tubular albumin absorption and its inhibition protects against acute kidney injury. Kidney Int. 2019;96:327–341. 4. Silberstein C, Lucero MS, Zotta E, et al. A glucosylceramide synthase inhibitor protects rats against the cytotoxic effects of shiga toxin 2. Pediatr Res. 2011;69:390–394. 5. Weyer K, Andersen PK, Schmidt K, et al. Abolishment of proximal tubule albumin endocytosis does not affect plasma albumin during nephrotic syndrome in mice. Kidney Int. 2018;93:335–342. 6. Römer W, Berland L, Chambon V, et al. Shiga toxin induces tubular membrane invaginations for its uptake into cells. Nature. 2007;450:670–675. 7. Kaneko T, Tsubakihara Y, Fushimi H, et al. Histochemical and immunoelectron microscopic analysis of ganglioside GM3 in human kidney. Clin Exp Nephrol. 2015;19:403– 410. 8. Fujii Y, Numata S, Nakamura Y, et al. Murine glycosyltransferases responsible for the expression of globo-series glycolipids: cDNA structures, mRNA expression, and distribution of their products. Glycobiology. 2005;15:1257– 1267. 9. Yang Y, Song M, Liu Y, et al. Renoprotective approaches and strategies in acute kidney injury. Pharmacol Ther. 2016;163:58–73.
A zebrafish tale of parabiosis, podocytes, and proteinuria Brooke E. Chambers1 and Rebecca A. Wingert1 Glomerular damage is a harbinger of kidney dysfunction. Circulating permeability factors are implicated in causing primary and secondary damage to podocytes, leading to proteinuria and eventual progression to the nephrotic syndrome, but the mechanisms are not well understood. Müller-Deile et al. employed parabiosis with zebrafish embryos and found that a damaged glomerulus can impact a healthy one in a shared circulatory system. This methodology shows promise for elucidating kidney injury pathways in response to systemic disease. Kidney International (2019) 96, 272–275; https://doi.org/10.1016/j.kint.2019.04.041 Copyright ª 2019, International Society of Nephrology. Published by Elsevier Inc. All rights reserved.
see basic research on page 342
1
Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, Indiana, USA Correspondence: Rebecca A. Wingert, Department of Biological Sciences, University of Notre Dame, 100 Galvin Life Science Research Building, Notre Dame, Indiana 46556, USA. E-mail: [email protected]
T
he glomerulus is a spherical blood filter and first element of the nephron that is followed by the proximal tubule. The glomerulus consists of a dense mesh of capillaries composed of fenestrated endothelial cells that are surrounded by a basement membrane and encased with specialized epithelial cells called podocytes. The podocytes Kidney International (2019) 96, 270–286