Piezo, Nephrocyte Function, and Slit Diaphragm Maintenance in Drosophila.

BACKGROUND: The Piezo gene encodes a highly conserved cell membrane protein responsible for sensing pressure. The glomerular kidney and the slit diaphragm filtration structure depend on pressure for filtration. However, how Piezo is involved in kidney function and in maintaining the slit diaphragm filtration structure is not clear. METHODS: We used Drosophila pericardial nephrocytes, filtration kidney cells with striking structural and functional similarities to human podocytes, in a loss-of-function model (mutant and knockdown) to study the roles of Piezo in nephrocyte filtration and function. RESULTS: Piezo is highly expressed at the invaginated membranes (lacuna channels) of nephrocytes. A Piezo loss-of-function mutant showed significant nephrocyte functional decline. Nephrocyte-specific silencing of Piezo showed disruption of the slit diaphragm filtration structure and significant functional defects. Electron microscopy showed that silencing Piezo in nephrocytes leads to reduced slit diaphragm density and abnormal shape of lacuna channels. Moreover, the Piezo-deficient nephrocytes showed internalized slit diaphragm component proteins, reduced autophagy, increased ER stress, and reduced calcium influx. CONCLUSIONS: Together, our findings suggest that Piezo plays an important role in the calcium homeostasis of nephrocytes and is required for maintaining nephrocyte function and the slit diaphragm filtration structure.

Pathogenesis of HIV associated nephropathy in children and adolescents: taking a hard look 40 years later in the era of gene-environmental interaction.

HIV-associated nephropathy (HIVAN) is a kidney disease that affects mainly people of African ancestry with a high HIV-1 viral load. New antiretroviral therapies (ART) have been highly efficient preventing and improving the outcome of HIVAN. However, providing chronic ART to children and adolescents living with HIV (CALWH) remains a significant challenge all over the world. More that 2.5 million CALWH, including those living in Sub-Saharan Africa, continue to be at high risk of developing HIVAN. Much of our understanding of the pathogenesis of HIVAN is based on studies conducted in transgenic mice and adults with HIVAN. However, CALWH may experience different health outcomes, risk factors, and susceptibilities to HIVAN in comparison to adults. This article reviews the progress made over the last 40 years in understanding the pathogenesis of HIVAN in CALWH, focusing on how the HIV virus, alongside genetic and environmental factors, contributes to the development of this disease. The landmark discovery that two risks alleles of the Apolipoprotein-1 (APOL1) gene play a critical role in HIVAN has significantly advanced our understanding of the disease's pathogenesis. However, we still need to understand why renal inflammation persists despite ART and determine whether the kidney may harbor HIV reservoirs that need to be eliminated to cure HIV permanently. For these reasons, we emphasize reviewing how HIV-1 infects renal cells, affects their growth and regeneration, and discussing how inflammatory cytokines and APOL1 affect the outcome of childhood HIVAN.

Association of circulating fibroblast growth factor-2 with progression of HIV-chronic kidney diseases in children.

BACKGROUND: Children living with HIV frequently show high plasma levels of fibroblast growth factor-2 (FGF-2/bFGF). FGF-2 accelerates the progression of several experimental kidney diseases; however, the role of circulating FGF-2 in childhood HIV-chronic kidney diseases (HIV-CKDs) is unknown. We carried out this study to determine whether high plasma FGF-2 levels were associated with the development of HIV-CKDs in children. METHODS: The plasma and urine FGF-2 levels were measured in 84 children (< 12 years of age) living with HIV during the pre-modern antiretroviral era, and followed for at least 3 years to determine the prevalence of proteinuria and HIV-CKDs. We also assessed the distribution of the kidney FGF-2 binding sites by autoradiography and Alcian blue staining, and explored potential mechanisms by which circulating FGF-2 may precipitate HIV-CKDs in cultured kidney epithelial and mononuclear cells derived from children with HIV-CKDs. RESULTS: High plasma FGF-2 levels were associated with a high viral load. Thirteen children (~ 15%) developed HIV-CKDs and showed a large reservoir of FGF-2 low-affinity binding sites in the kidney, which can facilitate the recruitment of circulating FGF-2. Children with high plasma and urine FGF-2 levels had 73-fold increased odds (95% CI 9-791) of having HIV-CKDs relative to those with normal FGF-2 values. FGF-2 induced the proliferation and decreased the expression of APOL-1 mRNA in podocytes, and increased the attachment and survival of infected mononuclear cells cultured from children with HIV-CKDs. CONCLUSIONS: High plasma FGF-2 levels appear to be an additional risk factor for developing progressive childhood HIV-CKDs.

Childhood HIV-associated nephropathy: 36 years later.

HIV-associated nephropathy (HIVAN) predominantly affects people of African ancestry living with HIV who do not receive appropriate antiretroviral therapy (ART). Childhood HIVAN is characterized by heavy proteinuria and decreased kidney function. Kidney histology shows mesangial expansion, classic or collapsing glomerulosclerosis, and microcystic renal tubular dilatation leading to kidney enlargement. The pathogenesis of HIVAN involves the kidney recruitment of inflammatory cells and the infection of kidney epithelial cells. In addition, both viral and genetic factors play key roles in this disease. Modern ART has improved the outcome and decreased the prevalence of childhood HIVAN. However, physicians have had modest success providing chronic ART to children and adolescents, and we continue to see children with HIVAN all over the world. This article discusses the progress made during the last decade in our understanding of the pathogenesis and treatment of childhood HIVAN, placing particular emphasis on the mechanisms that mediate the infection of kidney epithelial cells, and the roles of cytokines, the HIV-Tat gene, and the Apolipoprotein-1 (APOL1) gene risk variants in this disease. In view of the large number of children living with HIV at risk of developing HIVAN, better prevention and treatment programs are needed to eradicate this disease.

A Drosophila model system to assess the function of human monogenic podocyte mutations that cause nephrotic syndrome.

Many genetic mutations have been identified as monogenic causes of nephrotic syndrome (NS), but important knowledge gaps exist in the roles of these genes in kidney cell biology and renal diseases. More animal models are needed to assess the functions of these genes in vivo, and to determine how they cause NS in a timely manner. Drosophila nephrocytes and human podocytes share striking similarities, but to what degree these known NS genes play conserved roles in nephrocytes remains unknown. Here we systematically studied 40 genes associated with NS, including 7 that have not previously been analysed for renal function in an animal model. We found that 85% of these genes are required for nephrocyte functions, suggesting that a majority of human genes known to be associated with NS play conserved roles in renal function from flies to humans. To investigate functional conservation in more detail, we focused on Cindr, the fly homolog of the human NS gene CD2AP. Silencing Cindr in nephrocytes led to dramatic nephrocyte functional impairment and shortened life span, as well as collapse of nephrocyte lacunar channels and effacement of nephrocyte slit diaphragms. These phenotypes could be rescued by expression of a wild-type human CD2AP gene, but not a mutant allele derived from a patient with CD2AP-associated NS. We conclude that the Drosophila nephrocyte can be used to elucidate clinically relevant molecular mechanisms underlying the pathogenesis of most monogenic forms of NS, and to efficiently generate personalized in vivo models of genetic renal diseases bearing patient-specific mutations.

Optimizing the AKI definition during first postnatal week using Assessment of Worldwide Acute Kidney Injury Epidemiology in Neonates (AWAKEN) cohort.

BACKGROUND: Neonates with serum creatinine (SCr) rise ≥0.3 mg/dL and/or ≥50% SCr rise are more likely to die, even when controlling for confounders. These thresholds have not been tested in newborns. We hypothesized that different gestational age (GA) groups require different SCr thresholds. METHODS: Neonates in Assessment of Worldwide Acute Kidney Epidemiology in Neonates (AWAKEN) with ≥1 SCr on postnatal days 1-2 and ≥1 SCr on postnatal days 3-8 were assessed. We compared the mortality predictability of SCr absolute (≥0.3 mg/dL) vs percent (≥50%) rise. Next, we determine usefulness of combining absolute with percent rise. Finally, we determined the optimal absolute, percent, and maximum SCr thresholds that provide the highest mortality area under curve (AUC) and specificity for different GA groups. RESULTS: The ≥0.3 mg/dL rise outperformed ≥50% SCr rise. Addition of percent rise did not improve mortality predictability. The optimal SCr thresholds to predict AUC and specificity were ≥0.3 and ≥0.6 mg/dL for ≤29 weeks GA, and ≥0.1 and ≥0.3 mg/dL for >29 week GA. The maximum SCr value provides great specificity. CONCLUSION: Unique SCr rise cutoffs for different GA improves outcome prediction. Percent SCr rise does not add value to the neonatal AKI definition.

A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele or Dietary Coenzyme Q10 Supplementation.

Clinical studies have identified patients with nephrotic syndrome caused by mutations in genes involved in the biosynthesis of coenzyme Q10 (CoQ10), a lipid component of the mitochondrial electron transport chain and an important antioxidant. However, the cellular mechanisms through which these mutations induce podocyte injury remain obscure. Here, we exploited the striking similarities between Drosophila nephrocytes and human podocytes to develop a Drosophila model of these renal diseases, and performed a systematic in vivo analysis assessing the role of CoQ10 pathway genes in renal function. Nephrocyte-specific silencing of Coq2, Coq6, and Coq8, which are genes involved in the CoQ10 pathway that have been associated with genetic nephrotic syndrome in humans, induced dramatic adverse changes in these cells. In particular, silencing of Coq2 led to an abnormal localization of slit diaphragms, collapse of lacunar channels, and more dysmorphic mitochondria. In addition, Coq2-deficient nephrocytes showed elevated levels of autophagy and mitophagy, increased levels of reactive oxygen species, and increased sensitivity to oxidative stress. Dietary supplementation with CoQ10 at least partially rescued these defects. Furthermore, expressing the wild-type human COQ2 gene specifically in nephrocytes rescued the defective protein uptake, but expressing the mutant allele derived from a patient with COQ2 nephropathy did not. We conclude that transgenic Drosophila lines carrying mutations in the CoQ10 pathway genes are clinically relevant models with which to explore the pathogenesis of podocyte injury and could serve as a new platform to test novel therapeutic approaches.

Transmembrane TNF-α Facilitates HIV-1 Infection of Podocytes Cultured from Children with HIV-Associated Nephropathy.

Studies have shown that podocytes and renal tubular epithelial cells from patients with HIV-associated nephropathy (HIVAN) express HIV-1 transcripts, suggesting that productive infection of renal epithelial cells precipitates development of HIVAN. However, podocytes and renal tubular epithelial cells do not express CD4 receptors, and it is unclear how these cells become productively infected in vivo We investigated the mechanisms underlying the infection by HIV-1 of podocytes cultured from the urine of children with HIVAN. We observed low-level productive infection on exposure of these cells to primary cell-free HIV-1 supernatants. However, envelope-defective recombinant HIV-1 did not infect the renal epithelial cell lines. Moreover, treatment of podocytes to inhibit endocytic transport or dynamin activity or remove cell surface heparan sulfate proteoglycans reduced infection efficiency. Transfection of CD4- 293T cells with a cDNA expression library developed from a podocyte cell line derived from a child with HIVAN led to the identification of TNF-α as a possible mediator of HIV-1 infection. Overexpression of transmembrane TNF-α in cultured CD4- renal tubular epithelial cells, 293T cells, and HeLa cells enabled the infection of these cells; exposure to soluble TNF-α did not. Immunohistochemistry showed TNF-α expression in podocytes of renal sections from children with HIVAN. Furthermore, we found that TNF-α enhanced NF-κB activation and integration of HIV-1 into the podocyte DNA. Finally, inhibition of dynamin activity blocked TNF-α-mediated infection. These data establish a role for transmembrane TNF-α in facilitating the viral entry and integration of HIV-1 into the DNA of renal epithelial cells.

APOL1-G1 in Nephrocytes Induces Hypertrophy and Accelerates Cell Death.

People of African ancestry carrying certain APOL1 mutant alleles are at elevated risk of developing renal diseases. However, the mechanisms underlying APOL1-associated renal diseases are unknown. Because the APOL1 gene is unique to humans and some primates, new animal models are needed to understand the function of APOL1 in vivo We generated transgenic Drosophila fly lines expressing the human APOL1 wild type allele (G0) or the predominant APOL1 risk allele (G1) in different tissues. Ubiquitous expression of APOL1 G0 or G1 in Drosophila induced lethal phenotypes, and G1 was more toxic than was G0. Selective expression of the APOL1 G0 or G1 transgene in nephrocytes, fly cells homologous to mammalian podocytes, induced increased endocytic activity and accumulation of hemolymph proteins, dextran particles, and silver nitrate. As transgenic flies with either allele aged, nephrocyte function declined, cell size increased, and nephrocytes died prematurely. Compared with G0-expressing cells, however, G1-expressing cells showed more dramatic phenotypes, resembling those observed in cultured mammalian podocytes overexpressing APOL1-G1. Expressing the G0 or G1 APOL1 transgene in nephrocytes also impaired the acidification of organelles. We conclude that expression of an APOL1 transgene initially enhances nephrocyte function, causing hypertrophy and subsequent cell death. This new Drosophila model uncovers a novel mechanism by which upregulated expression of APOL1-G1 could precipitate renal disease in humans. Furthermore, this model may facilitate the identification of APOL1-interacting molecules that could serve as new drug targets to treat APOL1-associated renal diseases.

A new approach to define acute kidney injury in term newborns with hypoxic ischemic encephalopathy.

BACKGROUND: Current definitions of acute kidney injury (AKI) are not sufficiently sensitive to identify all newborns with AKI during the first week of life. METHODS: To determine whether the rate of decline of serum creatinine (SCr) during the first week of life can be used to identify newborns with AKI, we reviewed the medical records of 106 term neonates at risk of AKI who were treated with hypothermia for hypoxic ischemic encephalopathy (HIE). RESULTS: Of the newborns enrolled in the study, 69 % showed a normal rate of decline of SCr to ≥50 % and/or reached SCr levels of ≤0.6 mg/dl before the 7th day of life, and therefore had an excellent clinical outcome (control group). Thirteen newborns with HIE (12 %) developed AKI according to an established neonatal definition (AKI-KIDGO group), and an additional 20 newborns (19 %) showed a rate of decline of SCr of <33, <40, and <46 % from birth to days 3, 5, or 7 of life, respectively (delayed rise in estimated SCr clearance group). Compared to the control group, newborns in the other two groups required more days of mechanical ventilation and vasopressor drugs and had higher gentamicin levels, more fluid overload, lower urinary epidermal growth factor levels, and a prolonged length of stay. CONCLUSIONS: The rate of decline of SCr provides a sensitive approach to identify term newborns with AKI during the first week of life.

Angiopoietin-1 prevents severe bleeding complications induced by heparin-like drugs and fibroblast growth factor-2 in mice.

Critically ill children can develop bleeding complications when treated with heparin-like drugs. These events are usually attributed to the anticoagulant activity of these drugs. However, previous studies showed that fibroblast growth factor-2 (FGF-2), a heparin-binding growth factor released in the circulation of these patients, could precipitate intestinal hemorrhages in mice treated with the heparin-like drug pentosan polysulfate (PPS). Yet very little is known about how FGF-2 induces bleeding complications in combination with heparin-like drugs. Here, we examined the mechanisms by which circulating FGF-2 induces intestinal hemorrhages in mice treated with PPS. We used a well-characterized mouse model of intestinal hemorrhages induced by FGF-2 plus PPS. Adult FVB/N mice were infected with adenovirus carrying Lac-Z or a secreted form of recombinant human FGF-2, and injected with PPS, at doses that do not induce bleeding complications per se. Mice treated with FGF-2 in combination with PPS developed an intestinal inflammatory reaction that increased the permeability and disrupted the integrity of submucosal intestinal vessels. These changes, together with the anticoagulant activity of PPS, induced lethal hemorrhages. Moreover, a genetically modified form of the endothelial ligand angiopoietin-1 (Ang-1*), which has powerful antipermeability and anti-inflammatory activity, prevented the lethal bleeding complications without correcting the anticoagulant status of these mice. These findings define new mechanisms through which FGF-2 and Ang-1* modulate the outcome of intestinal bleeding complications induced by PPS in mice and may have wider clinical implications for critically ill children treated with heparin-like drugs.

The basic domain of HIV-tat transactivating protein is essential for its targeting to lipid rafts and regulating fibroblast growth factor-2 signaling in podocytes isolated from children with HIV-1-associated nephropathy.

Podocyte injury has a critical role in the pathogenesis of HIV-associated nephropathy (HIVAN). The HIV-1 transactivator of transcription (Tat), combined with fibroblast growth factor-2 (FGF-2), can induce the dedifferentiation and proliferation of cultured human podocytes. Cellular internalization of Tat requires interactions with heparan sulfate proteoglycans and cholesterol-enriched lipid rafts (LRs). However, the specific distribution of Tat in human podocytes and its ability to associate with LRs have not been documented. Here, we found that Tat is preferentially recruited to LRs in podocytes isolated from children with HIVAN. Furthermore, we identified arginines in the basic domain (RKKRRQRRR) of Tat as essential for (1) targeting Tat to LRs, (2) Tat-mediated increases in the expression of Rho-A and matrix metalloproteinase-9 in LRs, and (3) Tat-mediated enhancement of FGF-2 signaling in human podocytes and HIV-transgenic mouse kidneys and the exacerbation of renal lesions in these mice. Tat carrying alanine substitutions in the basic domain (AKKAAQAAA) remained localized in the cytosol and did not associate with LRs or enhance FGF-2 signaling in cultured podocytes. These results show the specific association of Tat with LRs in podocytes isolated from children with HIVAN, confirm Tat as a regulator of FGF-2 signaling in LRs, and identify the key domain of Tat responsible for promoting these effects and aggravating renal injury in HIV-transgenic mice. Moreover, these results provide a molecular framework for developing novel therapies to improve the clinical outcome of children with HIVAN.

Clinical practice guideline for the management of chronic kidney disease in patients infected with HIV: 2014 update by the HIV Medicine Association of the Infectious Diseases Society of America.

It is important to realize that guidelines cannot always account for individual variation among patients. They are not intended to supplant physician judgment with respect to particular patients or special clinical situations. IDSA considers adherence to these guidelines to be voluntary, with the ultimate determination regarding their application to be made by the physician in the light of each patient's individual circumstances.

HIV-1 Nef acts in synergy with APOL1-G1 to induce nephrocyte cell death in a new Drosophila model of HIV-related kidney diseases.

Background: People carrying two APOL1 risk alleles (RA) G1 or G2 are at greater risk of developing HIV-associated nephropathy (HIVAN). Studies in transgenic mice showed that the expression of HIV-1 genes in podocytes, and nef in particular, led to HIVAN. However, it remains unclear whether APOL1-RA and HIV-1 Nef interact to induce podocyte cell death. Method: We generated transgenic (Tg) flies that express APOL1-G1 (derived from a child with HIVAN) and HIV-1 nef specifically in the nephrocytes, the fly equivalent of mammalian podocytes, and assessed their individual and combined effects on the nephrocyte filtration structure and function. Results: We found that HIV-1 Nef acts in synergy with APOL1-G1 resulting in nephrocyte structural and functional defects. Specifically, HIV-1 Nef itself can induce endoplasmic reticulum (ER) stress without affecting autophagy. Furthermore, Nef exacerbates the organelle acidification defects and autophagy reduction induced by APOL1-G1. The synergy between HIV-1 Nef and APOL1-G1 is built on their joint effects on elevating ER stress, triggering nephrocyte dysfunction and ultimately cell death. Conclusions: Using a new Drosophila model of HIV-1-related kidney diseases, we identified ER stress as the converging point for the synergy between HIV-1 Nef and APOL1-G1 in inducing nephrocyte cell death. Given the high relevance between Drosophila nephrocytes and human podocytes, this finding suggests ER stress as a new therapeutic target for HIV-1 and APOL1-associated nephropathies.

Development of an animal model of nephrocalcinosis via selective dietary sodium and chloride depletion.

BACKGROUND: Nephrocalcinosis (NC) is an important clinical problem seen in critically ill preterm neonates treated with loop diuretics. No reliable animal models are available to study the pathogenesis of NC in preterm infants. The purpose of this study was to develop a reproducible and clinically relevant animal model of NC for these patients and to explore the impact of extracellular fluid (ECF) volume contraction induced by sodium and chloride depletion in this process. METHODS: Three-week-old weanling Sprague-Dawley rats were fed diets deficient in either chloride or sodium or both. A subgroup of rats from each dietary group was injected daily with furosemide (40 mg/kg i.p.). RESULTS: Rats fed a control diet, with or without furosemide, or a chloride-depleted diet alone, did not develop NC. By contrast, 50% of the rats injected with furosemide and fed the chloride-depleted diet developed NC. Moreover, 94% of the rats fed the combined sodium- and chloride-depleted diet developed NC, independently of furosemide use. NC was associated with the development of severe ECF volume contraction; hypochloremic, hypokalemic, metabolic alkalosis; increased phosphaturia; and growth retardation. CONCLUSION: Severe ECF volume contraction induced by chronic sodium and chloride depletion appears to play an important role in the pathogenesis of NC.

A pilot study of urinary fibroblast growth factor-2 and epithelial growth factor as potential biomarkers of acute kidney injury in critically ill children.

BACKGROUND: Acute kidney injury (AKI) increases the morbidity of critically ill children. Thus, it is necessary to identify better renal biomarkers to follow the outcome of these patients. This prospective case-control study explored the clinical value of a urinary biomarker profile comprised of neutrophil gelatinase lipocalin (uNGAL), fibroblast growth factor-2 (uFGF-2), and epidermal growth factor (uEGF) to follow these patients. METHODS: Urine samples were collected from 21 healthy children, and 39 critically ill children (mean age 7.5 years ± 6.97 SD) admitted to a pediatric intensive care unit with sepsis or requiring extra corporeal membrane oxygenation (ECMO). uNGAL, uFGF-2, and uEGF levels were measured using ELISA kits during the first 24 h of admission to PICU, at peak of illness, and upon resolution of the critical illness. RESULTS: On admission, the uNGAL and uFGF-2 levels were increased, and the uEGF levels were decreased, in critically ill children with AKI (n = 19) compared to those without AKI (n = 20), and healthy controls. A biomarker score using the combined cut-off values of uNGAL, uFGF-2, and uEGF (AUC = 0.90) showed the highest specificity to identify children with AKI, relative to each biomarker alone. uNGAL and uFGF-2 on admission showed high sensitivity and specificity to predict mortality (AUC = 0.82). CONCLUSIONS: The biomarker profile comprised of uNGAL, uFGF-2, and uEGF increased the specificity to detect AKI in critically ill children, when compared to each biomarker used alone. uNGAL and uFGF-2 may also predict the risk of death. Further validation of these findings in a large sample size is warranted.

A novel urinary biomarker profile to identify acute kidney injury (AKI) in critically ill neonates: a pilot study.

BACKGROUND: The goal of this study was to assess the value of a urinary biomarker profile comprised of neutrophil gelatinase-associated lipocalin (NGAL), fibroblast growth factor-2 (FGF-2), and epidermal growth factor (EGF), to detect acute kidney injury (AKI) in critically ill neonates. METHODS: We conducted a prospective cohort pilot study of at-risk neonates treated in a level IIIC neonatal intensive care unit (NICU) with therapeutic hypothermia (HT) (n = 25) or extracorporeal membrane oxygenation (ECMO) (n = 10). Urine was collected at baseline, 48 h of illness, and > 24 h post-recovery of their corresponding treatments. Control samples were collected from 27 healthy newborns. The data were expressed as urinary concentrations and values normalized for urinary creatinine. AKI was defined as the presence of oliguria >24 h and/or elevated serum creatinine (SCr), or the failure to improve the estimated creatinine clearance (eCCL) by >50% post-recovery. Non-parametric statistical tests and ROC analyses were used to interpret the data. RESULTS: Fifteen at-risk newborns had AKI. In the first 48 h of illness, the urinary levels of NGAL and FGF-2 had high sensitivity but poor specificity to identify neonates with AKI. At recovery, low urinary EGF levels identified neonates with AKI with a sensitivity of 74% and specificity of 84%. Overall, in the early stages of a critical illness, the urinary levels of NGAL and FGF-2 were sensitive, but not specific, to identify neonates at risk of AKI. Low EGF levels post-recovery identified critically ill neonates with AKI. CONCLUSIONS: These findings require validation in larger prospective studies.