Ferroportin 1 is expressed basolaterally in rat kidney proximal tubule cells and iron excess increases its membrane trafficking

Ferroportin 1 (FPN1) is an iron export protein expressed in liver and duodenum, as well as in reticuloendothelial macrophages. Previously, we have shown that divalent metal transporter 1 (DMT1) is expressed in late endosomes and lysosomes of the kidney proximal tubule (PT), the nephron segment responsible for the majority of solute reabsorption. We suggested that following receptor mediated endocytosis of transferrin filtered by the glomerulus, DMT1 exports iron liberated from transferrin into the cytosol. FPN1 is also expressed in the kidney yet its role remains obscure. As a first step towards determining the role of renal FPN1, we localized FPN1 in the PT. FPN1 was found to be located in association with the basolateral PT membrane and within the cytosolic compartment. FPN1 was not expressed on the apical brush‐border membrane of PT cells. These data support a role for FPN1 in vectorial export of iron out of PT cells. Furthermore, under conditions of iron loading of cultured PT cells, FPN1 was trafficked to the plasma membrane suggesting a coordinated cellular response to export excess iron and limit cellular iron concentrations.     

Purinergic signaling in the lumen of a normal nephron and in remodeled PKD encapsulated cysts

The nephron is the functional unit of the kidney. Blood and plasma are continually filtered within the glomeruli that begin each nephron. Adenosine 5' triphosphate (ATP) and its metabolites are freely filtered by each glomerulus and enter the lumen of each nephron beginning at the proximal convoluted tubule (PCT). Flow rate, osmolality, and other mechanical or chemical stimuli for ATP secretion are present in each nephron segment. These ATP-release stimuli are also different in each nephron segment due to water or salt permeability or impermeability along different luminal membranes of the cells that line each nephron segment. Each of the above stimuli can trigger additional ATP release into the lumen of a nephron segment. Each nephron-lining epithelial cell is a potential source of secreted ATP. Together with filtered ATP and its metabolites derived from the glomerulus, secreted ATP and adenosine derived from cells along the nephron are likely the principal two of several nucleotide and nucleoside candidates for renal autocrine and paracrine ligands within the tubular fluid of the nephron. This minireview discusses the first principles of purinergic signaling as they relate to the nephron and the urinary bladder. The review discusses how the lumen of a renal tubule presents an ideal purinergic signaling microenvironment. The review also illustrates how remodeled and encapsulated cysts in autosomal dominant polycystic kidney disease (ADPKD) and remodeled pseudocysts in autosomal recessive PKD (ARPKD) of the renal collecting duct likely create an even more ideal microenvironment for purinergic signaling. Once trapped in these closed microenvironments, purinergic signaling becomes chronic and likely plays a significant epigenetic and detrimental role in the secondary progression of PKD, once the remodeling of the renal tissue has begun. In PKD cystic microenvironments, we argue that normal purinergic signaling within the lumen of the nephron provides detrimental acceleration of ADPKD once remodeling is complete.     

Renal expression and functions of aquaporin 1 and aquaporin 4 in cattle

Abstract

Aquaporin (AQP) 1 and AQP 4 are members of the aquaporin water channel family that play an important role in reabsorption of water from the renal tubular fluid to concentrate urine. Studies of renal AQPs have been performed in human, rodents, sheep, dogs and horses. We studied nephron segment-specific expression of AQP 1 and AQP 4 using immunohistochemical staining on paraffin sections of bovine kidneys. AQP 1 was moderately expressed in endothelium of the cortical capillary network, vasa recta, and glomerular capillaries. AQP 4 was moderately expressed only in cytoplasm of epithelial cells in proximal tubules. We concluded that AQP 1 and AQP 4 in the bovine kidney showed some differences from other species in renal trans-epithelial water transport.     

Induction of steady-state glomeruloid sphere by self-assembly from human embryonic kidney cells

The glomerulus is a network of capillaries known as a tuft, located at the beginning of a nephron in the kidney. Here we describe a novel method for the induction of a macroscopically visible three-dimensional glomerulus-like sphere (GLS). This procedure did not require any additional cytokines and completed the formation of spheres within 24?h. After the formation was complete, GLS maintained a steady state for at least five days without proliferation and without a decrease in viability. Therefore, this procedure assists various assays for a prolong period of time. Overall, our protocol allows for a very simple mixing of cells from different sources to obtain fine-grained and highly dispersed GLSs. The kidney filtration barrier is a unique structure characterized by a complex three-dimensional framework of podocytes and endothelial cells. GLS exhibited the induction of many podocyte-specific gene profiles similar to those in adult human kidneys, suggesting that the sphere formation process is important for the maturation of podocytes. Focal segmental glomerulosclerosis (FSGS) is one of the major causes of steroid-resistant nephrotic syndrome, and some circulating permeability factors in the patient's serum FSGS have been implicated in the pathogenesis of the disease. Serum from patients with FSGS induced the collapse of GLS, which imitates the appearance of glomerulosclerosis in patients. In conclusion, the investigation and use of GLS may provide a novel method to elucidate the molecular mechanisms underlying complicated and unexplained events in glomeruli in a similar condition in adult kidneys.     

Kidney regeneration through nephron neogenesis in medaka

Although renal regeneration is limited to repair of the proximal tubule in mammals, some bony fish are capable of renal regeneration through nephron neogenesis in the event of renal injury. We previously reported that nephron development in the medaka mesonephros is characterized by four histologically distinct stages, generally referred to as condensed mesenchyme, nephrogenic body, relatively small nephron, and the mature nephron. Developing nephrons are positive for wt1 expression during the first three of these stages. In the present study, we examined the regenerative response to renal injury, artificially induced by the administration of sublethal amounts of gentamicin in adult medaka. Similar to previous reports in other animals, the renal tubular epithelium and the glomerulus of the medaka kidney exhibited severe damage after exposure to this agent. However, kidneys showed substantial recovery after gentamicin administration, and a significant number of developing nephrons appeared 14 days after gentamicin administration (P < 0.01). Similarly, the expression of wt1 in developing nephrons also indicated the early stages of nephrogenesis. These findings show that medaka has the ability to regenerate kidney through nephron neogenesis during adulthood and that wt1 is a suitable marker for detecting nephrogenesis.     

Renal ontogeny in the rhesus monkey (Macaca mulatta) and directed differentiation of human embryonic stem cells towards kidney precursors

The development of the metanephric kidney was studied immunohistochemically across gestation in monkeys to identify markers of cell specification, and to aid in developing experimental paradigms for renal precursor differentiation from human embryonic stem cells (hESC). PAX2, an important kidney developmental marker, was expressed at the tips of the ureteric bud, in the surrounding condensing mesenchyme, and in the renal vesicle. Vimentin, a mesenchymal and renal marker, was strongly expressed in the metanephric blastema then found to be limited to the glomerulus and interstitial cells of the medulla and cortex. A model of gene expression based on human and nonhuman primate renal ontogeny was developed and incorporated into studies of hESC differentiation. Spontaneous hESC differentiation revealed markers of metanephric mesenchyme (OSR1, PAX2, SIX2, WT1) that increased over time, followed by upregulation of kidney precursor markers (EYA1, LIM1, CD24). Directed hESC differentiation was also evaluated with the addition of retinoic acid, Activin-A, and BMP-4 or BMP-7, and using different culture substrate conditions. Of the culture substrates studied, gelatin most closely recapitulated the anticipated directed developmental pattern of renal gene expression. No differences were found when BMP-4 and BMP-7 were compared with baseline conditions. PAX2 and Vimentin immunoreactivity in differentiating hESC was also similar to the renal precursor patterns reported for human fetal kidneys and findings described in rhesus monkeys. The results of these studies are as follows: (1) provide additional data to support that rhesus monkey kidney development parallels that of humans, and (2) provide a useful model for hESC directed differentiation towards renal precursors.     

金雕肾脏的组织学观察

利用生物显微技术观察了金雕Aquila chrysaetos肾脏的组织结构.结果表明,金雕肾实质由许多肾小叶构成,每个肾小叶可分为皮质和髓质两部分.肾单位由一个肾小体和一条与其相连的肾小管构成.肾小体由肾小囊和肾小球组成.肾小管分为近曲小管、髓袢、远曲小管和连接小管.集合管分为小叶周集合小管和髓质集合管两部分.具有发达的极周细胞.     

Nephrogenesis and the renal renin-angiotensin system in fetal sheep: effects of intrauterine growth restriction during late gestation

Previous studies have shown that intrauterine growth restriction (IUGR) can impair nephrogenesis, but uncertainties remain about the importance of the gestational timing of the insult and the effects on the renal renin-angiotensin system (RAS). We therefore hypothesized that induction of IUGR during late gestation alters the RAS, and this is associated with a decrease in nephron endowment. Our aims were to determine the effects of IUGR induced during the later stages of nephrogenesis on 1) nephron number; 2) mRNA expression of angiotensin AT(1) and AT(2) receptors, angiotensinogen, and renin genes in the kidney; and 3) the size of maculae densae. IUGR was induced in fetal sheep (n = 7) by umbilical-placental embolization from 110 to 130 days of the approximately 147-day gestation; saline-infused fetuses served as controls (n = 7). Samples of cortex from the left kidney were frozen, and the right kidney was perfusion fixed. Total kidney volume, nephron number, renal corpuscle volume, total maculae densae volume, and the volume of macula densa per glomerulus were stereologically estimated. mRNA expression of AT(1) and AT(2) receptors, angiotensinogen, and renin in the renal cortex was determined. In IUGR fetuses at 130 days, body and kidney weights were significantly reduced and nephron number was reduced by 24%. There was no difference in renin, angiotensinogen, or AT(1) and AT(2) receptor mRNA expression levels in the IUGR kidneys compared with controls. We conclude that fetal growth restriction late in nephrogenesis can lead to a marked reduction in nephron endowment but does not affect renal corpuscle or macula densa size, or renal RAS gene expression.     

OCRL1 Modulates Cilia Length in Renal Epithelial Cells

Lowe syndrome is an X-linked disorder characterized by cataracts at birth, mental retardation and progressive renal malfunction that results from loss of function of the OCRL1 (oculocerebrorenal syndrome of Lowe) protein. OCRL1 is a lipid phosphatase that converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 4-phosphate. The renal pathogenesis of Lowe syndrome patients has been suggested to result from alterations in membrane trafficking, but this cannot fully explain the disease progression. We found that knockdown of OCRL1 in zebrafish caused developmental defects consistent with disruption of ciliary function, including body axis curvature, pericardial edema, hydrocephaly and impaired renal clearance. In addition, cilia in the proximal tubule of the zebrafish pronephric kidney were longer in ocrl morphant embryos. We also found that knockdown of OCRL1 in polarized renal epithelial cells caused elongation of the primary cilium and disrupted formation of cysts in three-dimensional cultures. Calcium release in response to ATP was blunted in OCRL1 knockdown cells, suggesting changes in signaling that could lead to altered cell function. Our results suggest a new role for OCRL1 in renal epithelial cell function that could contribute to the pathogenesis of Lowe syndrome.     

Site-specific Expression of IQGAP1, a Key Mediator of Cytoskeleton, in Mouse Renal Tubules

IQGAP1 is a multifunctional junction molecule that is involved in cell migration, proliferation, differentiation, cell polarity, and cell–cell adhesion. It is highly expressed in the kidney and has recently been identified in the glomerular basement membrane as a nephrin-associated protein. However, the distribution of IQGAP1 in renal tubular epithelial cells is unknown. We performed confocal microscopic studies to localize IQGAP1 in each nephron segment using dual immunofluorescence staining with various antibodies against segment-specific markers. We found that IQGAP1 was strongly expressed in the distal convoluted tubule (DCT), collecting duct, and macula densa and moderately in the thick ascending limb and proximal tubule. In the DCT, the IQGAP1–F-actin complex forms a comb-like structure with multiple parallel spikes sitting on the basal membrane. In the macula densa cells, IQGAP1 is strongly expressed in the apical membrane, whereas in type A intercalated cells, IQGAP1 is expressed in the basolateral membrane, where it colocalizes with anion exchanger 1, and in principal cells, it is diffusely expressed. In conclusion, we showed the expression and subcellular localization of IQGAP1 in various nephron segments. The site-specific expression pattern of this potent modulator of multiple biological pathways in the renal tubules suggests that IQGAP1 may have multiple important roles in various renal functions. (J Histochem Cytochem 56:659–666, 2008)     

Ultrastructural characterization of the pronephric glomerulus development in zebrafish

The zebrafish pronephros is a valuable model for studying kidney development and diseases. Ultrastructural studies have revealed that zebrafish and mammals share similarities in nephron structures such as podocytes, slit diaphragms, glomerular basement membrane, and endothelium. However, the basic ultrastructural features of the pronephric glomerulus during glomerulogenesis have not been characterized. To understand these features, it is instructive to consider the developmental process of the pronephros glomerulus. Here, we describe the ultrastructural features of pronephric glomerulus in detail from 24 h hours post‐fertilization (hpf) to 144 hpf, the period during which the pronephric glomerulus develops from initiation to its mature morphology. The pronephric glomerulus underwent progressive morphogenesis from 24 to 72 hpf, and presumptive glomerular cells were observed ventral to the aorta region at 24 hpf. The nascent glomerular basement membrane and initial lumen were formed at 36 hpf. A lumen was clearly visible in the region of the pronephros at 48 hpf. At 60 hpf, the pronephric glomerulus contained more patches of capillaries. After these transformations, the complex capillary vessel networks had formed inside the glomerulus, which was surrounded by podocyte bodies with elaborate foot processes as well as well‐formed glomerular basement membrane by 72 hpf. The number of renal glomerular cells rapidly increased, and the glomerulus presented its delicate structural features by 96 hpf. Morphogenesis was completed at 120 hpf with the final formation of the pronephric glomerulus. J. Morphol. 277:1104–1112, 2016. © 2016 Wiley Periodicals, Inc.     

Membrane progestin receptors alpha and gamma in renal epithelium

Sex hormones have broader effects than regulating reproductive functions. Recent identification of membrane progestin receptors expressed in kidney prompted us to investigate their putative involvement in the renal effects of this hormone. We first focused our investigations on mPRalpha and gamma by analyzing three parameters 1/ their distribution along the mouse nephron and their subcellular location in native kidney, 2/ the ability of progesterone to stimulate ERK pathway and/or Ca(2+) release from internal stores in native kidney structures and 3/ the cellular localization of mPRalpha and its molecular determinants in heterologous expression system. We observed that 1/ mPRalpha expression is restricted to proximal tubules of both male and female mice whereas mPRgamma exhibits a much broader expression all along the nephron except the glomerulus, 2/ mPRalpha and gamma are not localized at the plasma membrane in native kidney, 3/ this expression does not permit either progesterone-induced ERK phosphorylation or Ca(2+) release and 4/ in HEK transfected cells, mPRalpha localizes in the endoplasmic reticulum (ER) due to a C-terminal ER retention motif (-KXX). Therefore, we have characterized mPRs in kidney but their role in renal physiology remains to be elucidated.     

Differential Expression of Specific Dermatan Sulfate Domains in Renal Pathology

Dermatan sulfate (DS), also known as chondroitin sulfate (CS)-B, is a member of the linear polysaccharides called glycosaminoglycans (GAGs). The expression of CS/DS and DS proteoglycans is increased in several fibrotic renal diseases, including interstitial fibrosis, diabetic nephropathy, mesangial sclerosis and nephrosclerosis. Little, however, is known about structural alterations in DS in renal diseases. The aim of this study was to evaluate the renal expression of two different DS domains in renal transplant rejection and glomerular pathologies. DS expression was evaluated in normal renal tissue and in kidney biopsies obtained from patients with acute interstitial or vascular renal allograft rejection, patients with interstitial fibrosis and tubular atrophy (IF/TA), and from patients with focal segmental glomerulosclerosis (FSGS), membranous glomerulopathy (MGP) or systemic lupus erythematosus (SLE), using our unique specific anti-DS antibodies LKN1 and GD3A12. Expression of the 4/2,4-di-O-sulfated DS domain recognized by antibody LKN1 was decreased in the interstitium of transplant kidneys with IF/TA, which was accompanied by an increased expression of type I collagen, decorin and transforming growth factor beta (TGF-β), while its expression was increased in the interstitium in FSGS, MGP and SLE. Importantly, all patients showed glomerular LKN1 staining in contrast to the controls. Expression of the IdoA-Gal-NAc4SDS domain recognized by GD3A12 was similar in controls and patients. Our data suggest a role for the DS domain recognized by antibody LKN1 in renal diseases with early fibrosis. Further research is required to delineate the exact role of different DS domains in renal fibrosis.     

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure¹. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury¹. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized^1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules^1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types³. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely^1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals^4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish⁶. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish^7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.     

Tight junction claudins and the kidney in sickness and in health

The epithelial cell tight junction has several functions including the control of paracellular transport between epithelial cells. Renal paracellular transport has been long recognized to exhibit unique characteristics within different segments of the nephron, functions as an important component of normal renal physiology and has been speculated to contribute to renal related pathology if functioning abnormally. The discovery of a large family of tight junction associated 4-transmembrane spanning domain proteins named claudins has advanced our understanding on how the paracellular permeability properties of tight junctions are determined. In the kidney, claudins are expressed in a nephron-specific pattern and are major determinants of the paracellular permeability of tight junctions in different nephron segments. The combination of nephron segment claudin expression patterns, inherited renal diseases, and renal epithelial cell culture models is providing important clues about how tight junction claudin molecules function in different segments of the nephron under normal and pathological conditions. This review discusses early observations of renal tubule paracellular transport and more recent information on the discovery of the claudin family of tight junction associated membrane proteins and how they relate to normal renal function as well as diseases of the human kidney.     

The role of SMARCAL1 in replication fork stability and telomere maintenance

SMARCAL1 (SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A-Like 1), also known as HARP, is an ATP-dependent annealing helicase that stabilizes replication forks during DNA damage. Mutations in this gene are the cause of Schimke immune-osseous dysplasia (SIOD), an autosomal recessive disorder characterized by T-cell immunodeficiency and growth dysfunctions. In this review, we summarize the main roles of SMARCAL1 in DNA repair, telomere maintenance and replication fork stability in response to DNA replication stress.     

Renal microvasculature of the black bear (Ursus americanus)

This study of the Black bear (Ursus americanus) was undertaken to provide basic information related to structural modifications in the renal microvasculature that might provide insight into the drastic alteration in renal urinary output that occurs during winter sleep. Vascular casts, as well as light microscopy and scanning electron microscopy, were used to study the vascular components of the juxtaglomerular complex and related vessels. Histologically, arterial cushions were readily identified at the origin of the afferent arterioles. In the area of the juxtaglomerular complex, the wall of the afferent arteriole appeared to be highly modified. The smooth muscle cells at this site demonstrated a change in morphology and orientation, and the diameter of the arteriole was altered. The pattern of the vascular casts at the origin of the afferent arteriole varied from that portion at the glomerulus, suggesting a modification of the vascular wall near the renal corpuscle. Although the morphology of the renal microvasculature of the Black bear is similar to that of other mammals in some aspects, it is dissimilar to that of other carnivores and of the human kidney in that there are structural modifications of the afferent arteriole that may contribute to a reduction of blood flow to the nephron during winter sleep.     

Functional genetic variation in aminopeptidase A (ENPEP): lack of clear association with focal and segmental glomerulosclerosis (FSGS)

The aminopeptidase A (APA) ectopeptidase is an integral membrane-bound zinc metalloprotease that cleaves aspartic and glutamic acidic residues from the N-terminus of a number of protein substrates that includes angiotensin II. Angiotensin II, the most vasoactive component of the renin-angiotensin-aldosterone (RAAS) pathway, can contribute to renal disease by causing an increase in arterial blood pressure leading to glomerular injury and fibrosis. APA is expressed in many organs, including the kidney where it localizes mainly to the podocyte cell membrane and brush borders of the proximal tubule cells. Antibodies directed to the APA peptide can induce an acute massive albuminuria in wild-type BALB/c mice after intravenous injection. We examined whether variants in the APA encoding gene (ENPEP) are more frequent in individuals with the proteinuric disease focal and segmental glomerulosclerosis (FSGS) compared to control individuals. The ENPEP coding sequence was re-sequenced in 188 FSGS patients and 48 controls. Genetic variants were further genotyped in 181 individuals without any known kidney disease. We then examined the effect of the non-synonymous coding variants identified on their cell surface APA activity after transfection in COS-1 cells. Several of these ENPEP variants lead to reproducibly altered APA activity. However, we did not see a clear correlation between the presence of a functional ENPEP variant and FSGS. However, the existence of these variants with marked effect on APA activity suggests that both rare and common variation in ENPEP may contribute to the development of renal and hypertensive disorders and warrants further study.