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1.
FASEB Bioadv ; 1(8): 498-510, 2019 Aug.
Article in English | MEDLINE | ID: mdl-31825015

ABSTRACT

Podocytes are key cells in maintaining the integrity of the glomerular filtration barrier and preventing albuminuria. Glycogen synthase kinase 3 (GSK3) is a multi-functional serine/threonine kinase existing as two distinct but related isoforms (α and ß). In the podocyte it has previously been reported that inhibition of the ß isoform is beneficial in attenuating a variety of glomerular disease models but loss of both isoforms is catastrophic. However, it is not known what the role of GSK3α is in these cells. We now show that GSK3α is present and dynamically modulated in podocytes. When GSK3α is transgenically knocked down specifically in the podocytes of mice it causes mild but significant albuminuria by 6-weeks of life. Its loss also does not protect in models of diabetic or Adriamycin-induced nephropathy. In vitro deletion of podocyte GSK3α causes cell death and impaired autophagic flux suggesting it is important for this key cellular process. Collectively this work shows that GSK3α is important for podocyte health and that augmenting its function may be beneficial in treating glomerular disease.

2.
Nat Commun ; 10(1): 403, 2019 01 24.
Article in English | MEDLINE | ID: mdl-30679422

ABSTRACT

Albuminuria affects millions of people, and is an independent risk factor for kidney failure, cardiovascular morbidity and death. The key cell that prevents albuminuria is the terminally differentiated glomerular podocyte. Here we report the evolutionary importance of the enzyme Glycogen Synthase Kinase 3 (GSK3) for maintaining podocyte function in mice and the equivalent nephrocyte cell in Drosophila. Developmental deletion of both GSK3 isoforms (α and ß) in murine podocytes causes late neonatal death associated with massive albuminuria and renal failure. Similarly, silencing GSK3 in nephrocytes is developmentally lethal for this cell. Mature genetic or pharmacological podocyte/nephrocyte GSK3 inhibition is also detrimental; producing albuminuric kidney disease in mice and nephrocyte depletion in Drosophila. Mechanistically, GSK3 loss causes differentiated podocytes to re-enter the cell cycle and undergo mitotic catastrophe, modulated via the Hippo pathway but independent of Wnt-ß-catenin. This work clearly identifies GSK3 as a critical regulator of podocyte and hence kidney function.


Subject(s)
Albuminuria/metabolism , Glycogen Synthase Kinase 3/metabolism , Kidney Diseases/metabolism , Kidney/physiology , Podocytes/metabolism , Albuminuria/blood , Albuminuria/pathology , Albuminuria/urine , Animals , Cell Cycle , Cell Line , Disease Models, Animal , Drosophila , Gene Deletion , Gene Silencing , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3 beta/drug effects , Glycogen Synthase Kinase 3 beta/genetics , Glycogen Synthase Kinase 3 beta/metabolism , Hippo Signaling Pathway , Kaplan-Meier Estimate , Kidney/pathology , Kidney Diseases/blood , Kidney Diseases/pathology , Kidney Diseases/urine , Male , Mice , Podocytes/enzymology , Podocytes/pathology , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proteomics , Rats, Wistar , Renal Insufficiency , Verteporfin/pharmacology , beta Catenin/metabolism
3.
Am J Physiol Renal Physiol ; 314(3): F454-F461, 2018 03 01.
Article in English | MEDLINE | ID: mdl-29167171

ABSTRACT

Hemolytic uremic syndrome (HUS) is major global health care issue as it is the leading cause of acute kidney injury in children. It is a triad of acute kidney injury, microangiopathic hemolytic anemia, and thrombocytopenia. In recent years, major advances in our understanding of complement-driven inherited rare forms of HUS have been achieved. However, in children 90% of cases of HUS are associated with a Shiga toxin-producing enteric pathogen. The precise pathological mechanisms in this setting are yet to be elucidated. The purpose of this review is to discuss advances in our understanding of the pathophysiology underlying HUS and identify the key questions yet to be answered by the scientific community.


Subject(s)
Acute Kidney Injury/etiology , Atypical Hemolytic Uremic Syndrome/etiology , Complement Activation , Complement System Proteins/immunology , Escherichia coli Infections/microbiology , Shiga-Toxigenic Escherichia coli/pathogenicity , Thrombotic Microangiopathies/etiology , Acute Kidney Injury/genetics , Acute Kidney Injury/immunology , Acute Kidney Injury/microbiology , Animals , Atypical Hemolytic Uremic Syndrome/genetics , Atypical Hemolytic Uremic Syndrome/immunology , Atypical Hemolytic Uremic Syndrome/microbiology , Complement Activation/genetics , Complement System Proteins/genetics , Genetic Predisposition to Disease , Humans , Phenotype , Prognosis , Risk Factors , Thrombotic Microangiopathies/genetics , Thrombotic Microangiopathies/immunology , Thrombotic Microangiopathies/microbiology
4.
Arch Dis Child Educ Pract Ed ; 99(2): 73-9, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24163356

ABSTRACT

There have been phenomenal advances in our understanding of renal biology over the last 20 years through our ability to define the genetic mutations causing kidney disease in children. This review will take you through a trip down the nephron and highlight how these conditions may present to the paediatrician and the molecular basis for their biological effects.


Subject(s)
DNA/genetics , Genetic Markers/genetics , Genetic Predisposition to Disease , Kidney Diseases/genetics , Nephrons , Child , Humans
5.
Am J Physiol Renal Physiol ; 305(2): F182-8, 2013 Jul 15.
Article in English | MEDLINE | ID: mdl-23698113

ABSTRACT

Podocytes are critically important for maintaining the integrity of the glomerular filtration barrier and preventing albuminuria. Recently, it has become clear that to achieve this, they need to be insulin sensitive and produce an optimal amount of VEGF-A. In other tissues, insulin has been shown to regulate VEGF-A release, but this has not been previously examined in the podocyte. Using in vitro and in vivo approaches, in the present study, we now show that insulin regulates VEGF-A in the podocyte in both mice and humans via the insulin receptor (IR). Insulin directly increased VEGF-A mRNA levels and protein production in conditionally immortalized wild-type human and murine podocytes. Furthermore, when podocytes were rendered insulin resistant in vitro (using stable short hairpin RNA knockdown of the IR) or in vivo (using transgenic podocyte-specific IR knockout mice), podocyte VEGF-A production was impaired. Importantly, in vivo, this occurs before the development of any podocyte damage due to podocyte insulin resistance. Modulation of VEGF-A by insulin in the podocyte may be another important factor in the development of glomerular disease associated with conditions in which insulin signaling to the podocyte is deranged.


Subject(s)
Insulin/metabolism , Podocytes/metabolism , Vascular Endothelial Growth Factor A/metabolism , Animals , Humans , Insulin Resistance , Mice , Mice, Knockout , RNA, Messenger/metabolism
6.
J Pathol ; 230(1): 95-106, 2013 May.
Article in English | MEDLINE | ID: mdl-23299523

ABSTRACT

Podocytes are crucial for preventing the passage of albumin into the urine and, when lost, are associated with the development of albuminuria, renal failure and cardiovascular disease. Podocytes have limited capacity to regenerate, therefore pro-survival mechanisms are critically important. Insulin-like growth factor-II (IGF-II) is a potent survival and growth factor; however, its major function is thought to be in prenatal development, when circulating levels are high. IGF-II has only previously been reported to continue to be expressed in discrete regions of the brain into adulthood in rodents, with systemic levels being undetectable. Using conditionally immortalized human and ex vivo adult mouse cells of the glomerulus, we demonstrated the podocyte to be the major glomerular source and target of IGF-II; it signals to this cell via the IGF-I receptor via the PI3 kinase and MAPK pathways. Functionally, a reduction in IGF signalling causes podocyte cell death in vitro and glomerular disease in vivo in an aged IGF-II transgenic mouse that produces approximately 60% of IGF-II due to a lack of the P2 promoter of this gene. Collectively, this work reveals the fundamental importance of IGF-II in the mature podocyte for glomerular health across mammalian species.


Subject(s)
Insulin-Like Growth Factor II/metabolism , Podocytes/cytology , Podocytes/metabolism , Signal Transduction/physiology , Aging/physiology , Animals , Cell Line, Transformed , Cell Survival/physiology , Humans , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/metabolism , Insulin-Like Growth Factor II/genetics , Kidney Glomerulus/cytology , Kidney Glomerulus/physiology , Mesangial Cells/cytology , Mesangial Cells/metabolism , Mice , Mice, Transgenic , RNA, Small Interfering/genetics
7.
Diabetologia ; 52(9): 1944-52, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19533082

ABSTRACT

AIMS/HYPOTHESIS: Peroxisome proliferator-activated receptor (PPAR) gamma agonists are used increasingly in the treatment of type 2 diabetes. In the context of renal disease, PPARgamma agonists reduce microalbuminuria in diabetic nephropathy; however, the mechanisms underlying this effect are unknown. Glomerular podocytes are newly characterised insulin-sensitive cells and there is good evidence that they are targeted in diabetic nephropathy. In this study we investigated the functional and molecular effects of the PPARgamma agonist rosiglitazone on human podocytes. METHODS: Conditionally immortalised human podocytes were cultured with rosiglitazone and functional effects were measured with glucose-uptake assays. The effect of rosiglitazone on glucose uptake was also measured in 3T3-L1 adipocytes, nephrin-deficient podocytes, human glomerular endothelial cells, proximal tubular cells and podocytes treated with the NEFA palmitate. The role of the glucose transporter GLUT1 was investigated with immunofluorescence and small interfering RNA knockdown and the plasma membrane expression of GLUT1 was determined with bis-mannose photolabelling. RESULTS: Rosiglitazone significantly increased glucose uptake in wild-type podocytes and this was associated with translocation of GLUT1 to the plasma membrane. This effect was blocked with GLUT1 small interfering RNA. Nephrin-deficient podocytes, glomerular endothelial cells and proximal tubular cells did not increase glucose uptake in response to either insulin or rosiglitazone. Furthermore, rosiglitazone significantly increased basal and insulin-stimulated glucose uptake when podocytes were treated with the NEFA palmitate. CONCLUSIONS/INTERPRETATION: In conclusion, rosiglitazone has a direct and protective effect on glucose uptake in wild-type human podocytes. This represents a novel mechanism by which PPARgamma agonists may improve podocyte function in diabetic nephropathy.


Subject(s)
Glucose Transporter Type 1/metabolism , Glucose/metabolism , Hypoglycemic Agents/pharmacology , Kidney Glomerulus/metabolism , Podocytes/metabolism , Thiazolidinediones/pharmacology , Biological Transport/drug effects , Cell Culture Techniques , Cell Line , Cell Membrane/drug effects , Cell Membrane/metabolism , DNA Primers , Glucose Transporter Type 1/drug effects , Glucose Transporter Type 1/genetics , Humans , Kidney Glomerulus/drug effects , Kinetics , Podocytes/drug effects , RNA/genetics , Rosiglitazone , Transfection
9.
Kidney Int ; 71(6): 569-73, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17228362

ABSTRACT

Acute renal disease is common in sub-Saharan Africa, with high mortality. Its etiology is poorly understood; quartan malaria owing to Plasmodium malariae was implicated in previous series. Few previous studies have included histological data; furthermore, much of the literature pre-dates the human immunodeficiency virus (HIV) epidemic. We report prospective analysis of acute proteinuric renal disease in children in rural Uganda. Clinical and laboratory data are presented on 65 patients (aged 2-14 years, mean 8.4; 35 male, 30 female) in 41 of whom histological diagnosis was obtained by renal biopsy. The most frequent histological finding was endocapillary proliferative glomerulonephritis (GN) in 27/41 cases, in 20 of which eosinophils were very prominent. No cases showed features of HIV nephropathy. Malarial films were positive in 11 cases: all owing to Plasmodium falciparum. Patients were treated with diuretics, antihypertensives, and supportive measures. Corticosteroids were rarely used, being reserved for patients with minimal changes on renal biopsy. Clinical outcomes were fair: 91% of patients survived to discharge. We conclude that acute GN is common in children in Uganda, that an unusual eosinophilic proliferative GN is the most frequent histological finding, that HIV is not implicated as an important factor in this age group, and that good outcomes can be achieved using simple clinical and laboratory diagnostic methods. Renal biopsy in selected cases is feasible and helpful, especially in allowing rational use of corticosteroids and other potentially toxic treatments. Symptomatic treatments and careful supportive care will allow the majority of children to recover.


Subject(s)
Eosinophils/pathology , Glomerulonephritis/epidemiology , Glomerulonephritis/pathology , Adolescent , Biopsy , Child , Child, Preschool , Eosinophilia/diagnosis , Eosinophilia/epidemiology , Eosinophilia/pathology , Female , Glomerulonephritis/diagnosis , Humans , Kidney Glomerulus/pathology , Male , Prevalence , Prospective Studies , Uganda/epidemiology
10.
Kidney Int ; 70(6): 1038-45, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16837924

ABSTRACT

Glucocorticoids are widely used in the treatment of human glomerular diseases, but their mode of action is poorly understood particularly in steroid-sensitive nephrotic syndrome, which is most common in childhood and is characterized by a lack of inflammation in the kidney. The podocyte is a key cell in the glomerulus in health and disease: until recently, human podocytes have been difficult to study in vitro. We have developed a conditionally immortalized human podocyte cell line transfected with a temperature-sensitive simian virus 40 transgene: when the transgene is inactivated in vitro, these cells adopt the phenotype of differentiated podocytes. We have used these cells to evaluate, using immunocytochemistry, reverse transcriptase-polymerase chain reaction, and Western blotting, direct effects of the glucocorticoid dexamethasone at concentrations designed to mimic in vivo therapeutic corticosteroid levels. Dexamethasone upregulated expression of nephrin and tubulin-alpha, and downregulated vascular endothelial growth factor. Effects on cell cycle were complex with downregulation of cyclin kinase inhibitor p21 and augmentation of podocyte survival, without any effect on apoptosis. We report cytokine production by human podocytes, especially interleukin (IL)-6 and -8; IL-6 expression was suppressed by dexamethasone. These potent direct effects on podocytes illustrate a novel mode of action of glucocorticoids and suggest potential new therapeutic strategies for glomerular disease.


Subject(s)
Antigens, Polyomavirus Transforming , Dexamethasone/pharmacology , Glucocorticoids/pharmacology , Podocytes/drug effects , Simian virus 40/immunology , Cell Line, Transformed , Cell Survival/drug effects , Cell Transformation, Viral , Child, Preschool , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Dose-Response Relationship, Drug , Gene Expression Regulation/drug effects , Humans , Immunohistochemistry , Interleukin-6/metabolism , Membrane Proteins/metabolism , Tubulin/metabolism , Vascular Endothelial Growth Factor A/metabolism
11.
Arch Dis Child ; 88(11): 962-5, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14612355

ABSTRACT

BACKGROUND: The previous epidemiological study of paediatric nephrolithiasis in Britain was conducted more than 30 years ago. AIMS: To examine the presenting features, predisposing factors, and treatment strategies used in paediatric stones presenting to a British centre over the past five years. METHODS: A total of 121 children presented with a urinary tract renal stone, to one adult and one paediatric centre, over a five year period (1997-2001). All children were reviewed in a dedicated stone clinic and had a full infective and metabolic stone investigative work up. Treatment was assessed by retrospective hospital note review. RESULTS: A metabolic abnormality was found in 44% of children, 30% were classified as infective, and 26% idiopathic. Bilateral stones on presentation occurred in 26% of the metabolic group compared to 12% in the infective/idiopathic group (odds ratio 2.7, 95% CI 1.03 to 7.02). Coexisting urinary tract infection was common (49%) in the metabolic group. Surgically, minimally invasive techniques (lithotripsy, percutaneous nephrolithotomy, and endoscopy) were used in 68% of patients. CONCLUSIONS: There has been a shift in the epidemiology of paediatric renal stone disease in the UK over the past 30 years. Underlying metabolic causes are now the most common but can be masked by coexisting urinary tract infection. Treatment has progressed, especially surgically, with sophisticated minimally invasive techniques now employed. All children with renal stones should have a metabolic screen.


Subject(s)
Kidney Calculi/epidemiology , Adolescent , Age Distribution , Child , Child, Preschool , Female , Humans , Incidence , Infant , Kidney Calculi/etiology , Kidney Calculi/surgery , Male , Metabolic Diseases/complications , Minimally Invasive Surgical Procedures/methods , Retrospective Studies , Risk Factors , Sex Distribution , Urinary Tract Infections/complications
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