Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common inherited kidney disorder, caused mainly by mutations in genes encoding for Polycystin 1 or (PC-1) Polycystin 2 (PC-2). Previously published studies provide evidence that a defective glucose metabolism is present in renal cells lacking PC-1, in the kidneys from a PKD mouse model, and in kidneys from ADPKD patients. Metabolomics analysis revealed that in the absence of a functional PC-1 cells preferentially rely on aerobic glycolysis, rather than oxidative phosphorylation. The switch to glycolytic metabolism can be the consequence of mitochondrial respiratory defects, and thus we aim to determine the contribution of mitochondrial dysfunction in the process of cytogenesis. To address this question we generated a novel mouse model bearing mitochondrial alteration exclusively in the tubular epithelial cells of distal tubules of the kidney. Among the candidate mitochondrial proteins, which conditional KO will result in mitochondrial impairment, we chose to inactivate the dynamin-related GTPase Optic Atrophy 1 (OPA1), thus generating a kidney-specific Opa1 KO mice (Opa1 KO, for short). OPA1 plays a key role in in mediating inner mitochondrial membrane fusion and membrane tethering that supports cristae structure. Consequently, OPA1 has been shown to participate to the regulation of important mitochondrial functions including apoptosis, respiratory capacity, mtDNA maintenance, and calcium homeostasis. Opa1 KO mice are born at the expected Mendelian ratios, are viable at birth, and indistinguishable from control littermates. However, Opa1 KO mice display growth retardation, and die precociously at around P70. Analysis of Opa1 KO kidneys indicates a progressive kidney enlargement from P30 up to P60, and a regression in kidney weight at P90. Histochemical examination reveals that Opa1 KO kidneys present progressive dilatations of some tubular lumens, but no cysts similar to those found in the mouse model of PKD were detected. We conclude that an impairment of mitochondrial fitness is not sufficient per se to drive cyst formation in mouse kidney. Kidney-cross sections stained with Dolichos biflorus agglutinin (DBA), a marker specific for collecting duct cells, demonstrates a remarkable increase in the amount of DBA-positive tubules, revealing that kidney expansion was due to Opa1 KO cells proliferation, a data confirmed by the analysis of Ki67 proliferation staining. The phenotype of Opa1 KO kidneys was totally unexpected, since an increase in the proliferation rate in Opa1 deficient cells has never been reported in the literature, in both in vitro and in vivo systems. The predicted decrease in mitochondrial respiration in Opa1 KO tissue was confirmed by the analysis of cytochrome c oxidase and succinate dehydrogenase enzyme activity. The physiology of the mutated kidney was investigated by collecting urine form Opa1 KO mice and controls. Opa1 KO mice display a progressive increase in the diuresis, a reduction in urine osmolarity, and urine acidification. In line with the polyuria, Opa1 KO mice also exhibit progressive polydipsia. The evaluation of the urinary concentration of several electrolytes indicates a general dilution of urine without a specific impairment of renal electrolyte transporters, while their total excretion is not influenced by the enhanced urine output. We conclude that in Opa1 KO kidneys the electrolyte handling is still conserved, while the urine concentration mechanism is impaired. Consistent with the enhanced water output, levels of Aquaporin 2 water channel are decreased in Opa1 KO kidneys. Our data indicate that an impairment of mitochondrial function caused by Opa1 depletion leads to an alteration in the physiology of the kidney. Further investigations will clarifying the molecular mechanisms underlying the relationship among mitochondria functionality, kidney epithelial cell proliferation and kidney failure.
Studio degli effetti della delezione nel rene del principale regolatore della fusione mitocondriale Opa1
Unravelling the outcome of renal depletion of the mitochondrial fusion master regulator Opa1
MASERATI, MARTINA
2017/2018
Abstract
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common inherited kidney disorder, caused mainly by mutations in genes encoding for Polycystin 1 or (PC-1) Polycystin 2 (PC-2). Previously published studies provide evidence that a defective glucose metabolism is present in renal cells lacking PC-1, in the kidneys from a PKD mouse model, and in kidneys from ADPKD patients. Metabolomics analysis revealed that in the absence of a functional PC-1 cells preferentially rely on aerobic glycolysis, rather than oxidative phosphorylation. The switch to glycolytic metabolism can be the consequence of mitochondrial respiratory defects, and thus we aim to determine the contribution of mitochondrial dysfunction in the process of cytogenesis. To address this question we generated a novel mouse model bearing mitochondrial alteration exclusively in the tubular epithelial cells of distal tubules of the kidney. Among the candidate mitochondrial proteins, which conditional KO will result in mitochondrial impairment, we chose to inactivate the dynamin-related GTPase Optic Atrophy 1 (OPA1), thus generating a kidney-specific Opa1 KO mice (Opa1 KO, for short). OPA1 plays a key role in in mediating inner mitochondrial membrane fusion and membrane tethering that supports cristae structure. Consequently, OPA1 has been shown to participate to the regulation of important mitochondrial functions including apoptosis, respiratory capacity, mtDNA maintenance, and calcium homeostasis. Opa1 KO mice are born at the expected Mendelian ratios, are viable at birth, and indistinguishable from control littermates. However, Opa1 KO mice display growth retardation, and die precociously at around P70. Analysis of Opa1 KO kidneys indicates a progressive kidney enlargement from P30 up to P60, and a regression in kidney weight at P90. Histochemical examination reveals that Opa1 KO kidneys present progressive dilatations of some tubular lumens, but no cysts similar to those found in the mouse model of PKD were detected. We conclude that an impairment of mitochondrial fitness is not sufficient per se to drive cyst formation in mouse kidney. Kidney-cross sections stained with Dolichos biflorus agglutinin (DBA), a marker specific for collecting duct cells, demonstrates a remarkable increase in the amount of DBA-positive tubules, revealing that kidney expansion was due to Opa1 KO cells proliferation, a data confirmed by the analysis of Ki67 proliferation staining. The phenotype of Opa1 KO kidneys was totally unexpected, since an increase in the proliferation rate in Opa1 deficient cells has never been reported in the literature, in both in vitro and in vivo systems. The predicted decrease in mitochondrial respiration in Opa1 KO tissue was confirmed by the analysis of cytochrome c oxidase and succinate dehydrogenase enzyme activity. The physiology of the mutated kidney was investigated by collecting urine form Opa1 KO mice and controls. Opa1 KO mice display a progressive increase in the diuresis, a reduction in urine osmolarity, and urine acidification. In line with the polyuria, Opa1 KO mice also exhibit progressive polydipsia. The evaluation of the urinary concentration of several electrolytes indicates a general dilution of urine without a specific impairment of renal electrolyte transporters, while their total excretion is not influenced by the enhanced urine output. We conclude that in Opa1 KO kidneys the electrolyte handling is still conserved, while the urine concentration mechanism is impaired. Consistent with the enhanced water output, levels of Aquaporin 2 water channel are decreased in Opa1 KO kidneys. Our data indicate that an impairment of mitochondrial function caused by Opa1 depletion leads to an alteration in the physiology of the kidney. Further investigations will clarifying the molecular mechanisms underlying the relationship among mitochondria functionality, kidney epithelial cell proliferation and kidney failure.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/22897