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Fabry disease is associated with progressive reduction in glomerular podocyte mass in young patients
S80
Abstracts
174 Fabry disease is associated with progressive reduction in glomerular podocyte mass in young patients Behzad Najafiana, Michael Mauerb, aUniversity of Washington, Seattle, WA, USA, bUniversity of Minnesota, Minneapolis, WA, USA Background: Chronic kidney disease is a major complication of Fabry nephropathy. There is growing evidence that podocytes play an important role in progression of Fabry nephropathy. We previously showed that podocyte GL-3 inclusion density [Vv(Inc/PC] and foot process width (FPW) progressively increase with age. We aimed to examine if these findings are associated with a change in glomerular podocyte mass in young Fabry patients. Renal biopsies from 12 (M/ F = 8/4) Fabry patients, age 12 [4-19] year, median [range], urine protein/creatinine (UPCR) 40 [0-223] μg/min, glomerular filtration rate (GFR) = 106 [90-183] ml/min/1.73 m2 were studied. Volume of glomeruli occupied by podocytes and FPW were estimated by electron microscopy stereology. The results were correlated with age and renal function. Results: Despite direct relationship between age and Vv(Inc/PC) (r = 0.67, p = 0.02), Vv(PC/glom) showed inverse relationship with age (r = -0.65, p = 0.02), while, no relationship was found between age and Vv(Cap/glom) or Vv(Mes/glom), confirming a loss in glomerular podocyte mass. 46 ± 5% of glomerular volume was composed of podocytes, 30 ± 5% glomerular capillaries and 14 ± 3% mesangium and about 10% urinary space between the capillaries. UPCR directly correlated with age (r = 0.61, p = 0.01), FPW(r = 0.66, p = 0.01) and Vv(Inc/PC), and inversely correlated with Vv(PC/glom). Vv(PC/Nglom) inversely correlated with FPW (r = -0.59, p = 0.046). Conclusions: Although we did not count podocyte in this study, since GL-3 accumulation leads to podocyte enlargement, our observation of reduced glomerular podocyte mass is strongly suggestive that Fabry disease is associated with progressive podocyte injury (FPW increase) and loss with age in young patients, both of which correlate with UPCR. These results are confirmatory to our other studies that show urinary podocyte loss increases with age in Fabry patients and correlates with UPCR. doi:10.1016/j.ymgme.2013.12.186
175 Heterogeneity of podocyte involvement in females with Fabry disease is associated with podocyte injury Behzad Najafiana, Michael Mauerb, aUniversity of Washington, Seattle, WA, USA, bUniversity of Minnesota, Minneapolis, MN, USA Fabry disease is caused by X-linked deficiency of α-galactosidase A coded by GLA gene which leads to intracellular accumulation of globotriaosulceramide (GL3) inclusions. Progressive renal failure is a major complication of this diseases, which although less severe in females, occurs in 40% of female patients. We hypothesized that the % podocytes that carry the active mutant GLA due to random X-inactivation in the glomeruli is related to podocyte injury and proteinuria. Kidney biopsies from 12 enzyme replacement naive females with Fabry disease, age 15 [8-63], median [range] were studied by electron microscopy and compared with 4 male patients. 51 [13-100]% of podocytes in glomeruli did not have GL3 inclusions (NFPC) in females. Interglomerular variation of %NFPC per glomerulus (%NFPC/glom) was less than 10% in biopsies. %NFPC/glom correlated with age in females (r = 0.65, p = 0.02), suggesting progressive loss of podocytes with Fabry phenotype with aging. There was an inverse relationship between %NFPC/glom and foot
process width (FPW) (r = -0.75, p = 0.007) after adjustment for age. However, no relationship was found between %NFPC/glom and urine protein creatinine ratio. In addition, GL3 volume density in podocytes with Fabry phenotype (FPC) directly correlated with FPW. GL3 volume density was virtually similar in podocyte with GL3 inclusions (FPC) in females and males, suggestive of absence of efficient cross-correction between FPC and NFPC. These findings are suggestive of a relationship between podocyte mosaicism and injury in female patients. Kidney biopsy by providing information about podocyte mosaicism may help to stratify females with Fabry disease for kidney failure risk. doi:10.1016/j.ymgme.2013.12.187
176 High-content screening using metabolic lysosomal enzyme probes John J. Nalewaya, Fiona K. Harlana, Nicola Longob, Marzia Pasqualib, Robert H. Batchelora, Jason S. Luska, aMarker Gene Technologies, Inc., Eugene, OR, USA, bThe University of Utah School of Medicine, Salt Lake City, UT, USA Lysosomes are acidic cytoplasmic organelles present in all nucleated mammalian cells and are involved in a variety of cellular processes. Defects in lysosomal enzyme activity have been associated with a variety of diseases including Parkinson, Tay-Sachs, Sandhoff, Krabbe and Gaucher syndromes. New lysosomal staining probes useful for labeling lysosomes in a live-cell format and capable of monitoring lysosomal metabolic enzyme activity have been developed. These new targeted probes are based upon fluorogenic substrates that have a low pKa value for optimum fluorescence at the lower physiological pH values found in lysosomes, contain enzymatically-cleavable functions for measurement of specific enzyme activities as well as targeting groups to direct their accumulation to the lysosomes using a live-cell staining format. Application to High-Content Screening (HCS) using cells derived from leukocytes and fibroblasts from patients with metachromatic leukodystrophy, Maroteaux-Lamy syndrome, and Gaucher I, II and III as well as healthy human fibroblast and leukocyte control cells are presented. The ability to monitor the effect of secondary therapeutic agents on lysosomal enzyme activity is also presented as a method of HCS screening for new therapeutic agents in these areas. Current leads include structural analogs of N-butyldeoxynojirimycin (miglustat) as well as an unrelated small molecule (M131941) that also appears to function as a chaperone. This work was supported in part by NIH Grant 5R44NS073225-04. doi:10.1016/j.ymgme.2013.12.188
177 Peculiarities of hemostasis in children with Hunter syndrome L.S. Namazova-Baranova, O.B. Gordeeva, N.D. Vashakmadze, M.A. Babaykina, E.G. Chernavina, A.K. Gevorkyan, L.M. Kuzenkova, T.V. Podkletnova, Scientific Center of Children’s Health, Moscow, Russian Federation Issues of platelet and plasmatic hemostasis levels in patients with Hunter syndrome have not been sufficiently studied, which is important to improve treatment of arterial hypertonia with disaggregants and anticoagulants. The aim is to study condition of the hemostatic system in children with Hunter disease treated with antihypertensive drugs.
Abstracts
174 Fabry disease is associated with progressive reduction in glomerular podocyte mass in young patients Behzad Najafiana, Michael Mauerb, aUniversity of Washington, Seattle, WA, USA, bUniversity of Minnesota, Minneapolis, WA, USA Background: Chronic kidney disease is a major complication of Fabry nephropathy. There is growing evidence that podocytes play an important role in progression of Fabry nephropathy. We previously showed that podocyte GL-3 inclusion density [Vv(Inc/PC] and foot process width (FPW) progressively increase with age. We aimed to examine if these findings are associated with a change in glomerular podocyte mass in young Fabry patients. Renal biopsies from 12 (M/ F = 8/4) Fabry patients, age 12 [4-19] year, median [range], urine protein/creatinine (UPCR) 40 [0-223] μg/min, glomerular filtration rate (GFR) = 106 [90-183] ml/min/1.73 m2 were studied. Volume of glomeruli occupied by podocytes and FPW were estimated by electron microscopy stereology. The results were correlated with age and renal function. Results: Despite direct relationship between age and Vv(Inc/PC) (r = 0.67, p = 0.02), Vv(PC/glom) showed inverse relationship with age (r = -0.65, p = 0.02), while, no relationship was found between age and Vv(Cap/glom) or Vv(Mes/glom), confirming a loss in glomerular podocyte mass. 46 ± 5% of glomerular volume was composed of podocytes, 30 ± 5% glomerular capillaries and 14 ± 3% mesangium and about 10% urinary space between the capillaries. UPCR directly correlated with age (r = 0.61, p = 0.01), FPW(r = 0.66, p = 0.01) and Vv(Inc/PC), and inversely correlated with Vv(PC/glom). Vv(PC/Nglom) inversely correlated with FPW (r = -0.59, p = 0.046). Conclusions: Although we did not count podocyte in this study, since GL-3 accumulation leads to podocyte enlargement, our observation of reduced glomerular podocyte mass is strongly suggestive that Fabry disease is associated with progressive podocyte injury (FPW increase) and loss with age in young patients, both of which correlate with UPCR. These results are confirmatory to our other studies that show urinary podocyte loss increases with age in Fabry patients and correlates with UPCR. doi:10.1016/j.ymgme.2013.12.186
175 Heterogeneity of podocyte involvement in females with Fabry disease is associated with podocyte injury Behzad Najafiana, Michael Mauerb, aUniversity of Washington, Seattle, WA, USA, bUniversity of Minnesota, Minneapolis, MN, USA Fabry disease is caused by X-linked deficiency of α-galactosidase A coded by GLA gene which leads to intracellular accumulation of globotriaosulceramide (GL3) inclusions. Progressive renal failure is a major complication of this diseases, which although less severe in females, occurs in 40% of female patients. We hypothesized that the % podocytes that carry the active mutant GLA due to random X-inactivation in the glomeruli is related to podocyte injury and proteinuria. Kidney biopsies from 12 enzyme replacement naive females with Fabry disease, age 15 [8-63], median [range] were studied by electron microscopy and compared with 4 male patients. 51 [13-100]% of podocytes in glomeruli did not have GL3 inclusions (NFPC) in females. Interglomerular variation of %NFPC per glomerulus (%NFPC/glom) was less than 10% in biopsies. %NFPC/glom correlated with age in females (r = 0.65, p = 0.02), suggesting progressive loss of podocytes with Fabry phenotype with aging. There was an inverse relationship between %NFPC/glom and foot
process width (FPW) (r = -0.75, p = 0.007) after adjustment for age. However, no relationship was found between %NFPC/glom and urine protein creatinine ratio. In addition, GL3 volume density in podocytes with Fabry phenotype (FPC) directly correlated with FPW. GL3 volume density was virtually similar in podocyte with GL3 inclusions (FPC) in females and males, suggestive of absence of efficient cross-correction between FPC and NFPC. These findings are suggestive of a relationship between podocyte mosaicism and injury in female patients. Kidney biopsy by providing information about podocyte mosaicism may help to stratify females with Fabry disease for kidney failure risk. doi:10.1016/j.ymgme.2013.12.187
176 High-content screening using metabolic lysosomal enzyme probes John J. Nalewaya, Fiona K. Harlana, Nicola Longob, Marzia Pasqualib, Robert H. Batchelora, Jason S. Luska, aMarker Gene Technologies, Inc., Eugene, OR, USA, bThe University of Utah School of Medicine, Salt Lake City, UT, USA Lysosomes are acidic cytoplasmic organelles present in all nucleated mammalian cells and are involved in a variety of cellular processes. Defects in lysosomal enzyme activity have been associated with a variety of diseases including Parkinson, Tay-Sachs, Sandhoff, Krabbe and Gaucher syndromes. New lysosomal staining probes useful for labeling lysosomes in a live-cell format and capable of monitoring lysosomal metabolic enzyme activity have been developed. These new targeted probes are based upon fluorogenic substrates that have a low pKa value for optimum fluorescence at the lower physiological pH values found in lysosomes, contain enzymatically-cleavable functions for measurement of specific enzyme activities as well as targeting groups to direct their accumulation to the lysosomes using a live-cell staining format. Application to High-Content Screening (HCS) using cells derived from leukocytes and fibroblasts from patients with metachromatic leukodystrophy, Maroteaux-Lamy syndrome, and Gaucher I, II and III as well as healthy human fibroblast and leukocyte control cells are presented. The ability to monitor the effect of secondary therapeutic agents on lysosomal enzyme activity is also presented as a method of HCS screening for new therapeutic agents in these areas. Current leads include structural analogs of N-butyldeoxynojirimycin (miglustat) as well as an unrelated small molecule (M131941) that also appears to function as a chaperone. This work was supported in part by NIH Grant 5R44NS073225-04. doi:10.1016/j.ymgme.2013.12.188
177 Peculiarities of hemostasis in children with Hunter syndrome L.S. Namazova-Baranova, O.B. Gordeeva, N.D. Vashakmadze, M.A. Babaykina, E.G. Chernavina, A.K. Gevorkyan, L.M. Kuzenkova, T.V. Podkletnova, Scientific Center of Children’s Health, Moscow, Russian Federation Issues of platelet and plasmatic hemostasis levels in patients with Hunter syndrome have not been sufficiently studied, which is important to improve treatment of arterial hypertonia with disaggregants and anticoagulants. The aim is to study condition of the hemostatic system in children with Hunter disease treated with antihypertensive drugs.