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1.
Toxicol Pathol ; 51(4): 160-175, 2023 06.
Article in English | MEDLINE | ID: mdl-37632371

ABSTRACT

Assessment of hypertensive tubulopathy for more than fifty animal models of hypertension in experimental pathology employs criteria that do not correspond to lesional descriptors for tubular lesions in clinical pathology. We provide a critical appraisal of experimental hypertension with the same approach used to estimate hypertensive renal tubulopathy in humans. Four models with different pathogenesis of hypertension were analyzed-chronic angiotensin (Ang) II-infused and renin-overexpressing (TTRhRen) mice, spontaneously hypertensive (SHR), and Goldblatt two-kidney one-clip (2K1C) rats. Mouse models, SHR, and the nonclipped kidney in 2K1C rats had no regular signs of hypertensive tubulopathy. Histopathology in animals was mild and limited to variations in the volume density of tubular lumen and epithelium, interstitial space, and interstitial collagen. Affected kidneys in animals demonstrated lesion values that are significantly different compared with healthy controls but correspond to mild damage if compared with hypertensive humans. The most substantial human-like hypertensive tubulopathy was detected in the clipped kidney of 2K1C rats. For the first time, our study demonstrated the regular presence of chronic progressive nephropathy (CPN) in relatively young mice and rats with induced hypertension. Because CPN may confound the assessment of rodent models of hypertension, proliferative markers should be used to verify nonhypertensive tubulopathy.


Subject(s)
Hypertension , Pathology, Clinical , Humans , Rats , Mice , Animals , Rats, Inbred SHR , Kidney , Disease Models, Animal
2.
Diabetes ; 71(6): 1282-1298, 2022 06 01.
Article in English | MEDLINE | ID: mdl-35275988

ABSTRACT

Excessive production of renal reactive oxygen species (ROS) plays a major role in diabetic kidney disease (DKD). Here, we provide key findings demonstrating the predominant pathological role of the pro-oxidant enzyme NADPH oxidase 5 (NOX5) in DKD, independent of the previously characterized NOX4 pathway. In patients with diabetes, we found increased expression of renal NOX5 in association with enhanced ROS formation and upregulation of ROS-sensitive factors early growth response 1 (EGR-1), protein kinase C-α (PKC-α), and a key metabolic gene involved in redox balance, thioredoxin-interacting protein (TXNIP). In preclinical models of DKD, overexpression of NOX5 in Nox4-deficient mice enhances kidney damage by increasing albuminuria and augmenting renal fibrosis and inflammation via enhanced ROS formation and the modulation of EGR1, TXNIP, ERK1/2, PKC-α, and PKC-ε. In addition, the only first-in-class NOX inhibitor, GKT137831, appears to be ineffective in the presence of NOX5 expression in diabetes. In vitro, silencing of NOX5 in human mesangial cells attenuated upregulation of EGR1, PKC-α, and TXNIP induced by high glucose levels, as well as markers of inflammation (TLR4 and MCP-1) and fibrosis (CTGF and collagens I and III) via reduction in ROS formation. Collectively, these findings identify NOX5 as a superior target in human DKD compared with other NOX isoforms such as NOX4, which may have been overinterpreted in previous rodent studies.


Subject(s)
Diabetes Mellitus , Diabetic Nephropathies , Animals , Diabetic Nephropathies/genetics , Diabetic Nephropathies/metabolism , Fibrosis , Humans , Inflammation/metabolism , Mice , NADPH Oxidase 4/genetics , NADPH Oxidase 5/genetics , NADPH Oxidase 5/metabolism , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Reactive Oxygen Species/metabolism
3.
PLoS One ; 17(2): e0264136, 2022.
Article in English | MEDLINE | ID: mdl-35176122

ABSTRACT

Current research on hypertension utilizes more than fifty animal models that rely mainly on stable increases in systolic blood pressure. In experimental hypertension, grading or scoring of glomerulopathy in the majority of studies is based on a wide range of opinion-based histological changes that do not necessarily comply with lesional descriptors for glomerular injury that are well-established in clinical pathology. Here, we provide a critical appraisal of experimental hypertensive glomerulopathy with the same approach used to assess hypertensive glomerulopathy in humans. Four hypertensive models with varying pathogenesis were analyzed-chronic angiotensin II infused mice, mice expressing active human renin in the liver (TTRhRen), spontaneously hypertensive rats (SHR), and Goldblatt two-kidney one-clip rats (2K1C). Analysis of glomerulopathy utilized the same criteria applied in humans-hyalinosis, focal segmental glomerulosclerosis (FSGS), ischemic, hypertrophic and solidified glomeruli, or global glomerulosclerosis (GGS). Data from animal models were compared to human reference values. Kidneys in TTRhRen mice, SHR and the nonclipped kidneys in 2K1C rats had no sign of hyalinosis, FSGS or GGS. Glomerulopathy in these groups was limited to variations in mesangial and capillary compartment volumes, with mild increases in collagen deposition. Histopathology in angiotensin II infused mice corresponded to mesangioproliferative glomerulonephritis, but not hypertensive glomerulosclerosis. The number of nephrons was significantly reduced in TTRhRen mice and SHR, but did not correlate with severity of glomerulopathy. The most substantial human-like glomerulosclerotic lesions, including FSGS, ischemic obsolescent glomeruli and GGS, were found in the clipped kidneys of 2K1C rats. The comparison of affected kidneys to healthy control in animals produces lesion values that are numerically impressive but correspond to mild damage if compared to humans. Animal studies should be standardized by employing the criteria and classifications established in human pathology to make experimental and human data fully comparable for comprehensive analysis and model improvements.


Subject(s)
Angiotensin II/toxicity , Disease Models, Animal , Glomerulosclerosis, Focal Segmental/pathology , Hypertension, Renal/pathology , Hypertension/complications , Nephritis/pathology , Nephrosclerosis/pathology , Animals , Glomerulosclerosis, Focal Segmental/etiology , Glomerulosclerosis, Focal Segmental/metabolism , Humans , Hypertension/chemically induced , Hypertension, Renal/etiology , Hypertension, Renal/metabolism , Male , Nephritis/etiology , Nephritis/metabolism , Nephrosclerosis/etiology , Nephrosclerosis/metabolism , Rats , Rats, Inbred SHR , Vasoconstrictor Agents/toxicity
4.
Am J Vet Res ; 83(4): 349-355, 2022 Jan 28.
Article in English | MEDLINE | ID: mdl-35092667

ABSTRACT

OBJECTIVE: To compare single and triplicate applanation tonometry values across previous intraocular pressure (IOP) studies in dogs. ANIMALS: 116 ophthalmologically normal dogs. PROCEDURES: Triplicate IOP readings (n = 1432) from studies evaluating effect of anesthetic protocols were analyzed to estimate a range of probable differences between averaged triplicate and first, averaged and lowest, and first and lowest IOPs. The decrease in variability with triplicate measurements and the magnitude of effects on statistical power were quantified. RESULTS: The 2.5th to 97.5th interpercentile range for differences of averaged triplicate values minus first IOP readings was -3 to 2.7 mm Hg; for averaged minus lowest: 0 to 3.7 mm Hg; for first minus lowest: 0 to 5 mm Hg. The 95% prediction interval for differences in study group means (n = 160 groups, n = 5 to 11 eyes per group) based on averaged minus first measurements was -1.0 to 0.9 mm Hg with associated SDs reduced by 4% on average. Analysis of previous studies using averaged instead of first IOP values resulted in minimal decreases in SEs of 3-9% (0.03 to 0.09 mm Hg). Of 11 comparisons found significant with averaged data, 2 (18%) were found nonsignificant with first measurements. Of 96 comparisons found nonsignificant with averaged data, 3 (3%) were found significant with first measurements. CLINICAL RELEVANCE: With applanation tonometry in ophthalmologically normal dogs, no clinically meaningful difference was found between the first, lowest, or averaged triplicate IOP measurements, but the first reading has a larger variance and hence will result in lower statistical power.


Subject(s)
Intraocular Pressure , Tonometry, Ocular , Animals , Dogs , Eye , Manometry/veterinary , Tonometry, Ocular/methods , Tonometry, Ocular/veterinary
5.
Diabetologia ; 62(9): 1712-1726, 2019 09.
Article in English | MEDLINE | ID: mdl-31222503

ABSTRACT

AIMS/HYPOTHESIS: Excessive production of reactive oxygen species (ROS) plays a detrimental role in the progression of diabetic kidney disease (DKD). Renal oxidative stress activates proinflammatory cytokines, chemokines and profibrotic factors in DKD. Increased expression of the prooxidant enzyme NADPH oxidase (NOX) 5 in kidneys of diabetic individuals has been hypothesised to correlate with renal injury and progression of DKD. Since the gene encoding NOX5 is not expressed in the mouse genome, we examined the effect of inducible human NOX5 expression in renal cells, selectively in either endothelial cells or vascular smooth muscle cells (VSMCs)/mesangial cells in a model of insulin-deficient diabetes, the Akita mouse. METHODS: Renal structural injury, including glomerulosclerosis, mesangial expansion and extracellular matrix protein accumulation, as well as renal inflammation, ROS formation and albuminuria, were examined in the NOX5 transgenic Akita mouse model of DKD. RESULTS: Expression of NOX5 in either endothelial cells or VSMCs/mesangial cells in diabetic Akita mice was associated with increased renal inflammation (monocyte chemoattractant protein-1, NF-κB and toll-like receptor-4) and glomerulosclerosis, as well as upregulation of protein kinase C-α and increased expression of extracellular matrix genes (encoding collagen III, fibronectin and α-smooth muscle actin) and proteins (collagen IV), most likely mediated via enhanced renal ROS production. The effect of VSMC/mesangial cell-specific NOX5 expression resulted in more pronounced renal fibrosis in comparison with endothelial cell-specific NOX5 expression in diabetic mice. In addition, albuminuria was significantly increased in diabetic VEcad+NOX5+ mice (1192 ± 194 µg/24 h) when compared with diabetic VEcad+NOX5- mice (770 ± 98 µg/24 h). Furthermore, the regulatory components of NOX5 activation, including heat shock protein 90 and transient receptor potential cation channel subfamily C member 6, were upregulated only in the presence of both NOX5 and diabetes. CONCLUSIONS/INTERPRETATION: The findings from this study highlight the importance of NOX5 in promoting diabetes-related renal injury and provide the rationale for the development of a selective NOX5 inhibitor for the prevention and/or treatment of DKD.


Subject(s)
Albuminuria/metabolism , Fibrosis/metabolism , Inflammation/metabolism , Kidney/metabolism , Albuminuria/pathology , Animals , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Diabetic Nephropathies/metabolism , Disease Models, Animal , Fibrosis/pathology , Humans , Inflammation/pathology , Kidney/pathology , Mice , Mice, Transgenic , Muscle, Smooth, Vascular/metabolism , NADPH Oxidase 5/metabolism , Oxidative Stress/physiology , Reactive Oxygen Species/metabolism
6.
PLoS One ; 14(5): e0216734, 2019.
Article in English | MEDLINE | ID: mdl-31112562

ABSTRACT

Remodeling of spatially heterogeneous arterial trees is routinely quantified on tissue sections by averaging linear dimensions, with lack of comparison between different organs and models. The impact of experimental models or hypertension treatment modalities on organ-specific vascular remodeling remains undefined. A wide variety of arterial remodeling types has been demonstrated for hypertensive models, which include differences across organs. The purpose of this study was to reassess methods for measurement of arterial remodeling and to establish a morphometric algorithm for standard and comparable quantification of vascular remodeling in hypertension in different vascular beds. We performed a novel and comprehensive morphometric analysis of terminal arteries in the brain, heart, lung, liver, kidney, spleen, stomach, intestine, skin, skeletal muscle, and adrenal glands of control and Goldblatt hypertensive rats on routinely processed tissue sections. Mean dimensions were highly variable but grouping them into sequential 5 µm intervals permitted creation of reliable linear regression equations and complex profiles. Averaged arterial dimensions demonstrated seven remodeling patterns that were distinct from conventional inward-outward and hypertrophic-eutrophic definitions. Numerical modeling predicted at least nineteen variants of arterial spatial conformations. Recognition of remodeling variants was not possible using averaged dimensions, their ratios, or the remodeling and growth indices. To distinguish remodeling patterns, a three-dimensional modeling was established and tested. The proposed algorithm permits quantitative analysis of arterial remodeling in different organs and may be applicable for comparative studies between animal hypertensive models and human hypertension. Arterial wall tapering is the most important factor to consider in arterial morphometry, while perfusion fixation with vessel relaxation is not necessary. Terminal arteries in organs undergo the same remodeling pattern in Goldblatt rats, except for organs with hemodynamics affected by the arterial clip. The existing remodeling nomenclature should be replaced by a numerical classification applicable to any type of arterial remodeling.


Subject(s)
Hypertension, Renovascular/pathology , Vascular Remodeling , Algorithms , Animals , Arteries/diagnostic imaging , Arteries/pathology , Computer Simulation , Coronary Vessels/diagnostic imaging , Coronary Vessels/pathology , Disease Models, Animal , Hemodynamics , Humans , Hypertension, Renovascular/diagnostic imaging , Hypertension, Renovascular/physiopathology , Imaging, Three-Dimensional , Male , Models, Anatomic , Organ Specificity , Pulmonary Artery/diagnostic imaging , Pulmonary Artery/pathology , Rats , Rats, Wistar , Renal Artery/diagnostic imaging , Renal Artery/pathology , Vascular Remodeling/physiology
7.
Am J Physiol Renal Physiol ; 315(4): F954-F966, 2018 10 01.
Article in English | MEDLINE | ID: mdl-29873512

ABSTRACT

Mutations in α-actinin-4 (actinin-4) result in hereditary focal segmental glomerulosclerosis (FSGS) in humans. Actinin-4 mutants induce podocyte injury because of dysregulation of the cytoskeleton and proteotoxicity. Injury may be associated with endoplasmic reticulum (ER) stress and polyubiquitination of proteins. We assessed if the chemical chaperone 4-phenylbutyrate (4-PBA) can ameliorate the proteotoxicity of an actinin-4 mutant. Actinin-4 K255E, which causes FSGS in humans (K256E in the mouse), showed enhanced ubiquitination, accelerated degradation, aggregate formation, and enhanced association with filamentous (F)-actin in glomerular epithelial cells (GECs). The mutant disrupted ER function and stimulated autophagy. 4-PBA reduced actinin-4 K256E aggregation and its tight association with F-actin. Transgenic mice that express actinin-4 K256E in podocytes develop podocyte injury, proteinuria, and FSGS in association with glomerular ER stress. Treatment of these mice with 4-PBA in the drinking water over a 10-wk period significantly reduced albuminuria and ER stress. Another drug, celastrol, which enhanced expression of ER and cytosolic chaperones in GECs, tended to reduce actinin-4 aggregation but did not decrease the tight association of actinin-4 K256E with F-actin and did not reduce albuminuria in actinin-4 K256E transgenic mice. Thus, chemical chaperones, such as 4-PBA, may represent a novel therapeutic approach to certain hereditary glomerular diseases.


Subject(s)
Actinin/genetics , Kidney Glomerulus/injuries , Mutation/genetics , Proteostasis/genetics , Actin Cytoskeleton/metabolism , Animals , Disease Models, Animal , Endoplasmic Reticulum/metabolism , Glomerulosclerosis, Focal Segmental/metabolism , Kidney Glomerulus/metabolism , Mice, Transgenic , Podocytes/metabolism , Proteinuria/metabolism
8.
Clin Sci (Lond) ; 132(13): 1453-1470, 2018 07 18.
Article in English | MEDLINE | ID: mdl-29739827

ABSTRACT

Neuronal ubiquitin C-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme that maintains intracellular ubiquitin pools and promotes axonal transport. Uchl1 deletion in mice leads to progressive axonal degeneration, affecting the dorsal root ganglion that harbors axons emanating to the kidney. Innervation is a crucial regulator of renal hemodynamics, though the contribution of neuronal UCHL1 to this is unclear. Immunofluorescence revealed significant neuronal UCHL1 expression in mouse kidney, including periglomerular axons. Glomerular filtration rate trended higher in 6-week-old Uchl1-/- mice, and by 12 weeks of age, these displayed significant glomerular hyperfiltration, coincident with the onset of neurodegeneration. Angiotensin converting enzyme inhibition had no effect on glomerular filtration rate of Uchl1-/- mice indicating that the renin-angiotensin system does not contribute to the observed hyperfiltration. DCE-MRI revealed increased cortical renal blood flow in Uchl1-/- mice, suggesting that hyperfiltration results from afferent arteriole dilation. Nonetheless, hyperglycemia, cyclooxygenase-2, and nitric oxide synthases were ruled out as sources of hyperfiltration in Uchl1-/- mice as glomerular filtration rate remained unchanged following insulin treatment, and cyclooxygenase-2 and nitric oxide synthase inhibition. Finally, renal nerve dysfunction in Uchl1-/- mice is suggested given increased renal nerve arborization, decreased urinary norepinephrine, and impaired vascular reactivity. Uchl1-deleted mice demonstrate glomerular hyperfiltration associated with renal neuronal dysfunction, suggesting that neuronal UCHL1 plays a crucial role in regulating renal hemodynamics.


Subject(s)
Glomerular Filtration Rate/physiology , Neurodegenerative Diseases/physiopathology , Ubiquitin Thiolesterase/physiology , Animals , Arterioles/physiopathology , Cyclooxygenase 2/metabolism , Glucose Intolerance/physiopathology , Kidney/innervation , Kidney/metabolism , Mice, Knockout , Neurodegenerative Diseases/metabolism , Neurons/metabolism , Nitric Oxide Synthase/metabolism , Renal Artery/physiopathology , Renal Circulation/physiology , Renin-Angiotensin System/physiology , Ubiquitin Thiolesterase/deficiency , Ubiquitin Thiolesterase/metabolism , Vascular Resistance/physiology
9.
Am J Physiol Renal Physiol ; 315(2): F353-F363, 2018 08 01.
Article in English | MEDLINE | ID: mdl-29667913

ABSTRACT

Loss of ubiquitin COOH-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme required for neuronal function, led to hyperphosphatemia accompanied by phosphaturia in mice, while calcium homeostasis remained intact. We therefore investigated the mechanisms underlying the phosphate imbalance in Uchl1-/- mice. Interestingly, phosphaturia was not a result of lower renal brush border membrane sodium-phosphate cotransporter expression as sodium-phosphate cotransporter 2a and 2c expression levels was similar to wild-type levels. Plasma parathyroid hormone and fibroblast growth factor 23 levels were not different; however, fibroblast growth factor 23 mRNA levels were significantly increased in femur homogenates from Uchl1-/- mice. Full-length and soluble α-klotho levels were comparable in kidneys from wild-type and Uchl1-/- mice; however, soluble α-klotho was reduced in Uchl1-/- mice urine. Consistent with unchanged components of 1,25(OH)2D3 metabolism (i.e., CYP27B1 and CYP24A1), sodium-phosphate cotransporter 2b protein levels were not different in ileum brush borders from Uchl1-/- mice, suggesting that the intestine is not the source of hyperphosphatemia. Nonetheless, when Uchl1-/- mice were fed a low-phosphate diet, plasma phosphate, urinary phosphate, and fractional excretion of phosphate were significantly attenuated and comparable to levels of low-phosphate diet-fed wild-type mice. Our findings demonstrate that Uchl1-deleted mice exhibit perturbed phosphate homeostasis, likely consequent to decreased urinary soluble α-klotho, which can be rescued with a low-phosphate diet. Uchl1-/- mice may provide a useful mouse model to study mild perturbations in phosphate homeostasis.


Subject(s)
Diet , Glucuronidase/deficiency , Hyperphosphatemia/enzymology , Hypophosphatemia, Familial/enzymology , Kidney/enzymology , Phosphates/metabolism , Ubiquitin Thiolesterase/deficiency , Animals , Calcitriol/blood , Disease Models, Animal , Femur/metabolism , Fibroblast Growth Factor-23 , Fibroblast Growth Factors/genetics , Fibroblast Growth Factors/metabolism , Gene Deletion , Genetic Predisposition to Disease , Glucuronidase/urine , Homeostasis , Hyperphosphatemia/blood , Hyperphosphatemia/genetics , Hyperphosphatemia/urine , Hypophosphatemia, Familial/blood , Hypophosphatemia, Familial/genetics , Hypophosphatemia, Familial/urine , Intestinal Absorption , Klotho Proteins , Mice, Knockout , Parathyroid Hormone/blood , Phenotype , Phosphates/blood , Phosphates/urine , Ubiquitin Thiolesterase/genetics
10.
Lab Invest ; 98(3): 360-370, 2018 03.
Article in English | MEDLINE | ID: mdl-29251736

ABSTRACT

PGE2 regulates glomerular hemodynamics, renin secretion, and tubular transport. This study examined the contribution of PGE2 EP1 receptors to sodium and water homeostasis. Male EP1-/- mice were bred with hypertensive TTRhRen mice (Htn) to evaluate blood pressure and kidney function at 8 weeks of age in four groups: wildtype (WT), EP1-/-, Htn, HtnEP1-/-. Blood pressure and water balance were unaffected by EP1 deletion. COX1 and mPGE2 synthase were increased and COX2 was decreased in mice lacking EP1, with increases in EP3 and reductions in EP2 and EP4 mRNA throughout the nephron. Microdissected proximal tubule sglt1, NHE3, and AQP1 were increased in HtnEP1-/-, but sglt2 was increased in EP1-/- mice. Thick ascending limb NKCC2 was reduced in the cortex but increased in the medulla. Inner medullary collecting duct (IMCD) AQP1 and ENaC were increased, but AVP V2 receptors and urea transporter-1 were reduced in all mice compared to WT. In WT and Htn mice, PGE2 inhibited AVP-water transport and increased calcium in the IMCD, and inhibited sodium transport in cortical collecting ducts, but not in EP1-/- or HtnEP1-/- mice. Amiloride (ENaC) and hydrochlorothiazide (pendrin inhibitor) equally attenuated the effect of PGE2 on sodium transport. Taken together, the data suggest that EP1 regulates renal aquaporins and sodium transporters, attenuates AVP-water transport and inhibits sodium transport in the mouse collecting duct, which is mediated by both ENaC and pendrin-dependent pathways.


Subject(s)
Dinoprostone/metabolism , Hypertension/metabolism , Kidney Tubules, Collecting/metabolism , Receptors, Prostaglandin E, EP1 Subtype/metabolism , Sodium/metabolism , Animals , Aquaporins/metabolism , Blood Pressure , Calcium/metabolism , Glomerular Filtration Rate , Male , Mice , Prostaglandin-E Synthases/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Sodium-Hydrogen Exchanger 3/metabolism , Solute Carrier Family 12, Member 1/metabolism
11.
Diabetes ; 66(10): 2691-2703, 2017 10.
Article in English | MEDLINE | ID: mdl-28747378

ABSTRACT

NADPH oxidase-derived excessive production of reactive oxygen species (ROS) in the kidney plays a key role in mediating renal injury in diabetes. Pathological changes in diabetes include mesangial expansion and accumulation of extracellular matrix (ECM) leading to glomerulosclerosis. There is a paucity of data about the role of the Nox5 isoform of NADPH oxidase in animal models of diabetic nephropathy since Nox5 is absent in the mouse genome. Thus, we examined the role of Nox5 in human diabetic nephropathy in human mesangial cells and in an inducible human Nox5 transgenic mouse exposed to streptozotocin-induced diabetes. In human kidney biopsies, Nox5 was identified to be expressed in glomeruli, which appeared to be increased in diabetes. Colocalization demonstrated Nox5 expression in mesangial cells. In vitro, silencing of Nox5 in human mesangial cells was associated with attenuation of the hyperglycemia and TGF-ß1-induced enhanced ROS production, increased expression of profibrotic and proinflammatory mediators, and increased TRPC6, PKC-α, and PKC-ß expression. In vivo, vascular smooth muscle cell/mesangial cell-specific overexpression of Nox5 in a mouse model of diabetic nephropathy showed enhanced glomerular ROS production, accelerated glomerulosclerosis, mesangial expansion, and ECM protein (collagen IV and fibronectin) accumulation as well as increased macrophage infiltration and expression of the proinflammatory chemokine MCP-1. Collectively, this study provides evidence of a role for Nox5 and its derived ROS in promoting progression of diabetic nephropathy.


Subject(s)
Diabetic Nephropathies/metabolism , NADPH Oxidases/metabolism , Animals , Blotting, Western , Cell Line , Diabetic Nephropathies/genetics , Enzyme-Linked Immunosorbent Assay , Humans , Inflammation/metabolism , Kidney/metabolism , Kidney Glomerulus/metabolism , Mesangial Cells/metabolism , Mice , Mice, Transgenic , NADPH Oxidases/genetics , Protein Kinase C beta/metabolism , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain Reaction
12.
Clin Sci (Lond) ; 128(8): 465-81, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25630236

ABSTRACT

Since the first demonstration of Nox enzyme expression in the kidney in the early 1990s and the subsequent identification of Nox4, or RENOX, a decade later, it has become apparent that the Nox family of reactive oxygen species (ROS) generating enzymes plays an integral role in the normal physiological function of the kidney. As our knowledge of Nox expression patterns and functions in various structures and specialized cell types within the kidney grows, so does the realization that Nox-derived oxidative stress contributes significantly to a wide variety of renal pathologies through their ability to modify lipids and proteins, damage DNA and activate transcriptional programmes. Diverse studies demonstrate key roles for Nox-derived ROS in kidney fibrosis, particularly in settings of chronic renal disease such as diabetic nephropathy. As the most abundant Nox family member in the kidney, much emphasis has been placed on the role of Nox4 in this setting. However, an ever growing body of work continues to uncover key roles for other Nox family members, not only in diabetic kidney disease, but in a diverse array of renal pathological conditions. The objective of the present review is to highlight the latest novel developments in renal Nox biology with an emphasis not only on diabetic nephropathy but many of the other renal disease contexts where oxidative stress is implicated.


Subject(s)
Kidney Diseases/enzymology , NADPH Oxidases/metabolism , Animals , Humans , Isoenzymes/metabolism , Kidney Diseases/pathology , NADPH Oxidases/chemistry
14.
Biochim Biophys Acta ; 1842(7): 1028-40, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24662305

ABSTRACT

Renal ubiquitin C-terminal hydrolase L1 (UCHL1) is upregulated in a subset of human glomerulopathies, including focal segmental glomerulosclerosis (FSGS), where it may serve to promote ubiquitin pools for degradation of cytotoxic proteins. In the present study, we tested whether UCHL1 is expressed in podocytes of a mouse model of ACTN4-associated FSGS. Podocyte UCHL1 protein was detected in glomeruli of K256E-ACTN4(pod+)/UCHL1+/+ mice. UCHL1+/- mice were intercrossed with K256E-ACTN4(pod+) mice and monitored for features of glomerular disease. 10-week-old K256E-ACTN4(pod+)/UCHL1-/- mice exhibited significantly ameliorated albuminuria, glomerulosclerosis, tubular pathology and blood pressure. Interestingly, while UCHL1 deletion diminished both tubular and glomerular apoptosis, WT1-positive nuclei were unchanged. Finally, UCHL1 levels correlated positively with poly-ubiquitinated proteins but negatively with K256E-α-actinin-4 levels, implying reduced K256E-α-actinin-4 proteolysis in the absence of UCHL1. Our data suggest that UCHL1 upregulation in ACTN4-associated FSGS fuels the proteasome and that UCHL1 deletion may impair proteolysis and thereby preserve K256E/wt-α-actinin-4 heterodimers, maintaining podocyte cytoskeletal integrity and protecting the glomerular filtration barrier.


Subject(s)
Actinin/genetics , Glomerulosclerosis, Focal Segmental/genetics , Sequence Deletion , Ubiquitin Thiolesterase/genetics , Actinin/metabolism , Animals , Cytoskeleton/genetics , Cytoskeleton/metabolism , Disease Models, Animal , Genetic Predisposition to Disease , Glomerulosclerosis, Focal Segmental/enzymology , Glomerulosclerosis, Focal Segmental/metabolism , Kidney Glomerulus/enzymology , Kidney Glomerulus/metabolism , Mice , Mice, Knockout , Podocytes/metabolism , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Up-Regulation
15.
Am J Physiol Endocrinol Metab ; 304(11): E1188-98, 2013 Jun 01.
Article in English | MEDLINE | ID: mdl-23531619

ABSTRACT

Renal podocyte apoptosis is an early event of diabetic nephropathy progression. Insulin action is critical for podocyte survival. Previous studies demonstrated that Src homology-2 domain-containing phosphatase-1 (SHP-1) is elevated in renal cortex of type 1 diabetic mice; we hypothesized that hyperglycemia-induced SHP-1 expression may affect insulin actions in podocytes. Type 1 diabetic Akita mice (Ins2(+/C96Y)) developed elevated foot process effacement and podocyte apoptosis compared with control littermate mice (Ins2(+/+)). In contrast to Ins2(+/+) mice, insulin-stimulated protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) phosphorylation were remarkably reduced in renal podocytes of Akita mice. This renal insulin resistance was associated with elevated SHP-1 expression in the glomeruli. Cultured podocytes exposed to high glucose concentration (HG; 25 mM) for 96 h exhibited high levels of apoptotic markers and caspase-3/7 enzymatic activity. HG exposure raised mRNA and protein levels of SHP-1 and reduced the insulin-signaling pathway in podocytes. Overexpression of dominant-negative SHP-1 in podocytes prevented HG effects and restored insulin actions. Elevated SHP-1 expression induced by high glucose levels was directly associated with insulin receptor-ß in vitro and in vivo to prevent insulin-stimulated Akt and ERK phosphorylation. In conclusion, our results showed that high levels of SHP-1 expression in glomeruli cause insulin resistance and podocyte loss, thereby contributing to diabetic nephropathy.


Subject(s)
Hyperglycemia/metabolism , Insulin/metabolism , Podocytes/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Receptor, Insulin/metabolism , Animals , Apoptosis/drug effects , Apoptosis/physiology , Caspase 3/metabolism , Cell Line , Cells, Cultured , Diabetic Nephropathies/genetics , Diabetic Nephropathies/metabolism , Glucose/pharmacology , Hyperglycemia/genetics , Insulin Resistance/physiology , Male , Mice , Mice, Inbred C57BL , Phosphorylation/drug effects , Phosphorylation/physiology , Podocytes/drug effects , Protein Tyrosine Phosphatase, Non-Receptor Type 6/genetics , Receptor, Insulin/genetics , Signal Transduction/drug effects , Signal Transduction/physiology
16.
Clin Sci (Lond) ; 124(3): 191-202, 2013 Feb.
Article in English | MEDLINE | ID: mdl-22920224

ABSTRACT

Nox (NADPH oxidase)-derived ROS (reactive oxygen species) have been implicated in the development of diabetic nephropathy. Of the Nox isoforms in the kidney, Nox4 is important because of its renal abundance. In the present study, we tested the hypothesis that GKT136901, a Nox1/4 inhibitor, prevents the development of nephropathy in db/db (diabetic) mice. Six groups of male mice (8-week-old) were studied: (i) untreated control db/m, (ii) low-dose GKT136901-treated db/m (30 mg/kg of body weight per day), (iii) high-dose GKT136901-treated db/m (90 mg/kg of body weight per day), (iv) untreated db/db; (v) low dose GKT136901-treated db/db; and (vi) high-dose GKT136901-treated db/db. GKT136901, in chow, was administered for 16 weeks. db/db mice developed diabetes and nephropathy as evidenced by hyperglycaemia, albuminuria and renal injury (mesangial expansion, tubular dystrophy and glomerulosclerosis). GKT136901 treatment had no effect on plasma glucose or BP (blood pressure) in any of the groups. Plasma and urine TBARSs (thiobarbituric acid-reacting substances) levels, markers of systemic and renal oxidative stress, respectively, were increased in diabetic mice. Renal mRNA expression of Nox4, but not of Nox2, increased, Nox1 was barely detectable in db/db. Expression of the antioxidant enzyme SOD-1 (superoxide dismutase 1) decreased in db/db mice. Renal content of fibronectin, pro-collagen, TGFß (transforming growth factor ß) and VCAM-1 (vascular cell adhesion molecule 1) and phosphorylation of ERK1/2 (extracellular-signal-regulated kinase 1/2) were augmented in db/db kidneys, with no change in p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase). Treatment reduced albuminuria, TBARS and renal ERK1/2 phosphorylation and preserved renal structure in diabetic mice. Our findings suggest a renoprotective effect of the Nox1/4 inhibitor, possibly through reduced oxidative damage and decreased ERK1/2 activation. These phenomena occur independently of improved glucose control, suggesting GKT136901-sensitive targets are involved in complications of diabetes rather than in the disease process.


Subject(s)
Diabetes Mellitus, Type 2/complications , Diabetic Nephropathies/prevention & control , NADPH Oxidases/antagonists & inhibitors , Pyrazoles/pharmacology , Pyridones/pharmacology , Albuminuria/prevention & control , Albuminuria/urine , Animals , Blood Glucose/analysis , Blood Pressure/drug effects , Blotting, Western , Body Weight/drug effects , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/urine , Diabetic Nephropathies/etiology , Diabetic Nephropathies/physiopathology , Disease Models, Animal , Gene Expression Regulation, Enzymologic/drug effects , Kidney/drug effects , Kidney/metabolism , Kidney/pathology , Male , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinases/metabolism , NADH, NADPH Oxidoreductases/antagonists & inhibitors , NADH, NADPH Oxidoreductases/genetics , NADH, NADPH Oxidoreductases/metabolism , NADPH Oxidase 1 , NADPH Oxidase 4 , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Superoxide Dismutase-1 , Thiobarbituric Acid Reactive Substances/analysis , Transforming Growth Factor beta/metabolism , Vascular Cell Adhesion Molecule-1/metabolism
17.
PLoS One ; 7(5): e37649, 2012.
Article in English | MEDLINE | ID: mdl-22629438

ABSTRACT

Angiotensin-converting enzyme 2 (ACE2) is expressed in the kidney and may be a renoprotective enzyme, since it converts angiotensin (Ang) II to Ang-(1-7). ACE2 has been detected in urine from patients with chronic kidney disease. We measured urinary ACE2 activity and protein levels in renal transplant patients (age 54 yrs, 65% male, 38% diabetes, n = 100) and healthy controls (age 45 yrs, 26% male, n = 50), and determined factors associated with elevated urinary ACE2 in the patients. Urine from transplant subjects was also assayed for ACE mRNA and protein. No subjects were taking inhibitors of the renin-angiotensin system. Urinary ACE2 levels were significantly higher in transplant patients compared to controls (p = 0.003 for ACE2 activity, and p≤0.001 for ACE2 protein by ELISA or western analysis). Transplant patients with diabetes mellitus had significantly increased urinary ACE2 activity and protein levels compared to non-diabetics (p<0.001), while ACE2 mRNA levels did not differ. Urinary ACE activity and protein were significantly increased in diabetic transplant subjects, while ACE mRNA levels did not differ from non-diabetic subjects. After adjusting for confounding variables, diabetes was significantly associated with urinary ACE2 activity (p = 0.003) and protein levels (p<0.001), while female gender was associated with urinary mRNA levels for both ACE2 and ACE. These data indicate that urinary ACE2 is increased in renal transplant recipients with diabetes, possibly due to increased shedding from tubular cells. Urinary ACE2 could be a marker of renal renin-angiotensin system activation in these patients.


Subject(s)
Diabetes Mellitus/urine , Kidney Transplantation , Peptidyl-Dipeptidase A/urine , Adult , Aged , Angiotensin II/urine , Angiotensin-Converting Enzyme 2 , Female , Humans , Kidney/metabolism , Male , Middle Aged , RNA, Messenger/urine , Renin-Angiotensin System/physiology , Sex Factors
18.
J Signal Transduct ; 2011: 563128, 2011.
Article in English | MEDLINE | ID: mdl-21808733

ABSTRACT

Focal segmental glomerulosclerosis (FSGS) is an important cause of proteinuria and nephrotic syndrome in humans. The pathogenesis of FSGS may be associated with glomerular visceral epithelial cell (GEC; podocyte) injury, leading to apoptosis, detachment, and "podocytopenia", followed by glomerulosclerosis. Mutations in α-actinin-4 are associated with FSGS in humans. In cultured GECs, α-actinin-4 mediates adhesion and cytoskeletal dynamics. FSGS-associated α-actinin-4 mutants show increased binding to actin filaments, compared with the wild-type protein. Expression of an α-actinin-4 mutant in mouse podocytes in vivo resulted in proteinuric FSGS. GECs that express mutant α-actinin-4 show defective spreading and motility, and such abnormalities could alter the mechanical properties of the podocyte, contribute to cytoskeletal disruption, and lead to injury. The potential for mutant α-actinin-4 to injure podocytes is also suggested by the characteristics of this mutant protein to form microaggregates, undergo ubiquitination, impair the ubiquitin-proteasome system, enhance endoplasmic reticulum stress, and exacerbate apoptosis.

19.
Am J Pathol ; 177(5): 2290-9, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20889563

ABSTRACT

SLK expression and activity are increased during kidney development and recovery from renal ischemia-reperfusion injury. In cultured cells, SLK promotes F-actin destabilization as well as apoptosis, partially via the p38 kinase pathway. To better understand the effects of SLK in vivo, a transgenic mouse model was developed where SLK was expressed in a podocyte-specific manner using the mouse nephrin promoter. Offspring of four founder mice carried the SLK transgene. Among male transgenic mice, 66% developed albuminuria at approximately 3 months of age, and the albuminuric mice originated from three of four founders. Overall, the male transgenic mice demonstrated about fivefold greater urinary albumin/creatinine compared with male non-transgenic mice. Transgenic and non-transgenic female mice did not develop albuminuria, suggesting that females were less susceptible to glomerular filtration barrier damage than their male counterparts. In transgenic mice, electron microscopy revealed striking podocyte injury, including poorly formed or effaced foot processes, and edematous and vacuolated cell bodies. By immunoblotting, nephrin expression was decreased in glomeruli of the albuminuric transgenic mice. Activation-specific phosphorylation of p38 was increased in transgenic mice compared with non-transgenic animals. Glomeruli of SLK transgenic mice showed around 30% fewer podocytes, and a reduction in F-actin compared with control glomeruli. Thus, podocyte SLK overexpression in vivo results in injury and podocyte loss, consistent with the effects of SLK in cultured cells.


Subject(s)
Albuminuria/metabolism , Podocytes/enzymology , Podocytes/pathology , Protein Serine-Threonine Kinases/metabolism , Actinin/genetics , Actinin/metabolism , Animals , Cytoskeleton/metabolism , Female , Humans , Kidney Glomerulus/cytology , Kidney Glomerulus/metabolism , Kidney Glomerulus/pathology , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Transgenic , Podocytes/ultrastructure , Protein Serine-Threonine Kinases/genetics , Transgenes , p38 Mitogen-Activated Protein Kinases/metabolism
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