Haptoglobin Genotype-dependent Differences in Macrophage Lysosomal Oxidative Injury

The major function of the Haptoglobin (Hp) protein is to control trafficking of extracorpuscular hemoglobin (Hb) thru the macrophage CD163 receptor with degradation of the Hb in the lysosome. There is a common copy number polymorphism in the Hp gene (Hp 2 allele) that has been associated with a severalfold increased incidence of atherothrombosis in multiple longitudinal studies. Increased plaque oxidation and apoptotic markers have been observed in Hp 2-2 atherosclerotic plaques, but the mechanism responsible for this finding has not been determined. We proposed that the increased oxidative injury in Hp 2-2 plaques is due to an impaired processing of Hp 2-2-Hb complexes within macrophage lysosomes, thereby resulting in redox active iron accumulation, lysosomal membrane oxidative injury, and macrophage apoptosis. We sought to test this hypothesis in vitro using purified Hp-Hb complex and cells genetically manipulated to express CD163. CD163-mediated endocytosis and lysosomal degradation of Hp-Hb were decreased for Hp 2-2-Hb complexes. Confocal microscopy using lysotropic pH indicator dyes demonstrated that uptake of Hp 2-2-Hb complexes disrupted the lysosomal pH gradient. Cellular fractionation studies of lysosomes isolated from macrophages incubated with Hp 2-2-Hb complexes demonstrated increased lysosomal membrane oxidation and a loss of lysosomal membrane integrity leading to lysosomal enzyme leakage into the cytoplasm. Additionally, markers of apoptosis, DNA fragmentation, and active caspase 3 were increased in macrophages that had endocytosed Hp 2-2-Hb complexes. These data provide novel mechanistic insights into how the Hp genotype regulates lysosomal oxidative stress within macrophages after receptor-mediated endocytosis of Hb.     

Altered expression of iron transport proteins in streptozotocin-induced diabetic rat kidney

Diabetes mellitus is associated with altered iron homeostasis in both human and animal diabetic models. Iron is a metal oxidant capable of generating reactive oxygen species (ROS) and has been postulated to contribute to diabetic nephropathy. Two proteins involved in iron metabolism that are expressed in the kidney are the divalent metal transporter, DMT1 (Slc11a2), and the Transferrin Receptor (TfR). Thus, we investigated whether renal DMT1 or TfR expression is altered in diabetes, as this could potentially affect ROS generation and contribute to diabetic nephropathy. Rats were rendered diabetic with streptozotocin (STZ-diabetes) and renal DMT1 and TfR expression studied using semi-quantitative immunoblotting and immunofluorescence. In STZ-diabetic Sprague-Dawley rats, renal DMT1 expression was significantly reduced and TfR expression increased after 2 weeks. DMT1 downregulation was observed in both proximal tubules and collecting ducts. Renal DMT1 expression was also decreased in Wistar rats following 12 weeks of STZ-diabetes, an effect that was fully corrected by insulin-replacement but not by cotreatment with the aldose reductase inhibitor, sorbinil. Increased renal TfR expression was also observed in STZ-diabetic Wistar rats together with elevated cellular iron accumulation. Together these data demonstrate renal DMT1 downregulation and TfR upregulation in STZ-diabetes. Whilst the consequence of altered DMT1 expression on renal iron handling and oxidant damage remains to be determined, the attenuation of the putative lysosomal iron exit pathway in proximal tubules could potentially explain lysosomal iron accumulation reported in human diabetes and STZ-diabetic animals.     

Proximal tubule haptoglobin gene activation is an integral component of the acute kidney injury "stress response"

Haptoglobin (Hp) synthesis occurs almost exclusively in liver, and it is rapidly upregulated in response to stress. Because many of the pathways that initiate hepatic Hp synthesis are also operative during acute kidney injury (AKI), we tested whether AKI activates the renal cortical Hp gene. CD-1 mice were subjected to six diverse AKI models: ischemia-reperfusion, glycerol injection, cisplatin nephrotoxicity, myoglobinuria, endotoxemia, and bilateral ureteral obstruction. Renal cortical Hp gene induction was determined either 4-72 h or 1-3 wk later by measuring Hp mRNA and protein levels. Relative renal vs. hepatic Hp gene induction during endotoxemia was also assessed. Each form of AKI induced striking and sustained Hp mRNA increases, leading to ～10- to 100-fold renal Hp protein elevations (ELISA; Western blot). Immunohistochemistry, and isolated proximal tubule assessments, indicated that the proximal tubule was the dominant (if not only) site of the renal Hp increases. Corresponding urinary and plasma Hp elevations were surrogate markers of this response. Endotoxemia evoked 25-fold greater Hp mRNA increases in kidney vs. liver, indicating marked renal Hp gene reactivity. Clinical relevance of these findings was suggested by observations that urine samples from 16 patients with established AKI had statistically higher (～12×) urinary Hp levels than urine samples from either normal subjects or from 15 patients with chronic kidney disease. These AKI-associated urinary Hp increases mirrored those seen for urinary neutrophil gelatinase-associated lipoprotein, a well accepted AKI biomarker gene. In summary, these studies provide the first evidence that AKI evokes rapid, marked, and sustained induction of the proximal tubule Hp gene. Hp's known antioxidant, as well as its protean pro- and anti-inflammatory, actions imply potentially diverse effects on the evolution of acute tubular injury.     

ER stress contributes to renal proximal tubule injury by increasing SREBP-2-mediated lipid accumulation and apoptotic cell death

Renal proximal tubule injury is induced by agents/conditions known to cause endoplasmic reticulum (ER) stress, including cyclosporine A (CsA), an immunosuppressant drug with nephrotoxic effects. However, the underlying mechanism by which ER stress contributes to proximal tubule cell injury is not well understood. In this study, we report lipid accumulation, sterol regulatory element-binding protein-2 (SREBP-2) expression, and ER stress in proximal tubules of kidneys from mice treated with the classic ER stressor tunicamycin (Tm) or in human renal biopsy specimens showing CsA-induced nephrotoxicity. Colocalization of ER stress markers [78-kDa glucose regulated protein (GRP78), CHOP] with SREBP-2 expression and lipid accumulation was prominent within the proximal tubule cells exposed to Tm or CsA. Prolonged ER stress resulted in increased apoptotic cell death of lipid-enriched proximal tubule cells with colocalization of GRP78, SREBP-2, and Ca(2+)-independent phospholipase A(2) (iPLA(2)β), an SREBP-2 inducible gene with proapoptotic characteristics. In cultured HK-2 human proximal tubule cells, CsA- and Tm-induced ER stress caused lipid accumulation and SREBP-2 activation. Furthermore, overexpression of SREBP-2 or activation of endogenous SREBP-2 in HK-2 cells stimulated apoptosis. Inhibition of SREBP-2 activation with the site-1-serine protease inhibitor AEBSF prevented ER stress-induced lipid accumulation and apoptosis. Overexpression of the ER-resident chaperone GRP78 attenuated ER stress and inhibited CsA-induced SREBP-2 expression and lipid accumulation. In summary, our findings suggest that ER stress-induced SREBP-2 activation contributes to renal proximal tubule cell injury by dysregulating lipid homeostasis.     

Akt Links Insulin Signaling to Albumin Endocytosis in Proximal Tubule Epithelial Cells

Diabetes mellitus (DM) has become an epidemic, causing a significant decline in quality of life of individuals due to its multisystem involvement. Kidney is an important target organ in DM accounting for the majority of patients requiring renal replacement therapy at dialysis units. Microalbuminuria (MA) has been a valuable tool to predict end-organ damage in DM but its low sensitivity has driven research efforts to seek other alternatives. Albumin is taken up by albumin receptors, megalin and cubilin in the proximal tubule epithelial cells. We demonstrated that insulin at physiological concentrations induce albumin endocytosis through activation of protein kinase B (Akt) in proximal tubule epithelial cells. Inhibition of Akt by a phosphorylation deficient construct abrogated insulin induced albumin endocytosis suggesting a role for Akt in insulin-induced albumin endocytosis. Furthermore we demonstrated a novel interaction between Akt substrate 160kDa (AS160) and cytoplasmic tail of megalin. Mice with type 1 DM (T1D) displayed decreased Akt, megalin, cubilin and AS160 expression in their kidneys in association with urinary cubilin shedding preceding significant MA. Patients with T1D who have developed MA in the EDC (The Pittsburgh Epidemiology of Diabetes Complications) study demonstrated urinary cubilin shedding prior to development of MA. We hypothesize that perturbed insulin-Akt cascade in DM leads to alterations in trafficking of megalin and cubilin, which results in urinary cubilin shedding as a prelude to MA in early diabetic nephropathy. We propose that utilization of urinary cubilin shedding, as a urinary biomarker, will allow us to detect and intervene in diabetic nephropathy (DN) at an earlier stage.     

Expression of Toll-Like Receptor 2 in Glomerular Endothelial Cells and Promotion of Diabetic Nephropathy by Porphyromonas gingivalis Lipopolysaccharide

The toll-like receptor (TLR) has been suggested as a candidate cause for diabetic nephropathy. Recently, we have reported the TLR4 expression in diabetic mouse glomerular endothelium. The study here investigates the effects of the periodontal pathogen Porphyromonas gingivalis lipopolysaccharide (LPS) which is a ligand for TLR2 and TLR4 in diabetic nephropathy. In laser-scanning microscopy of glomeruli of streptozotocin- and a high fat diet feed-induced type I and type II diabetic mice, TLR2 localized on the glomerular endothelium and proximal tubule epithelium. The TLR2 mRNA was detected in diabetic mouse glomeruli by in situ hybridization and in real-time PCR of the renal cortex, the TLR2 mRNA amounts were larger in diabetic mice than in non-diabetic mice. All diabetic mice subjected to repeated LPS administrations died within the survival period of all of the diabetic mice not administered LPS and of all of the non-diabetic LPS-administered mice. The LPS administration promoted the production of urinary protein, the accumulation of type I collagen in the glomeruli, and the increases in IL-6, TNF-α, and TGF-β in the renal cortex of the glomeruli of the diabetic mice. It is thought that blood TLR ligands like Porphyromonas gingivalis LPS induce the glomerular endothelium to produce cytokines which aid glomerulosclerosis. Periodontitis may promote diabetic nephropathy.     

FGF1ΔHBS delays the progression of diabetic nephropathy in late-stage type 2 diabetes mouse model by alleviating renal inflammation,fibrosis, and apoptosis

Elderly adults are at higher risk for developing diabetic complications including diabetic nephropathy (DN), contributing to excess morbidity and mortality in elderly individuals. A non-mitogenic variant of fibroblast growth factor 1 (FGF1^ΔHBS) was demonstrated to prevent DN in an early-stage (2-month-old) type 2 diabetes (T2D) mouse model. The present study aimed to investigate the potential therapeutic effects of FGF1^ΔHBS against the progression of renal dysfunction in a late-stage T2D mouse model with established DN. Nine-month-old db/db mice were administered FGF1^ΔHBS every other day for 3 months. db/db mice at 12-month-old without FGF1^ΔHBS treatment exhibited high blood glucose level and elevated urine albumin-to-creatinine ratio. FGF1^ΔHBS treatment effectively reversed hyperglycemia, delayed the development of renal dysfunction, and reduced kidney size and weight. Furthermore, FGF1^ΔHBS treatment significantly prevented the progression of renal morphologic impairment. FGF1^ΔHBS treatment demonstrated anti-inflammatory and anti-fibrotic effects, with significantly decreased protein levels of key pro-inflammatory cytokines and pro-fibrotic factors in kidney. Moreover, FGF1^ΔHBS treatment greatly decreased apoptosis of renal tubular cells, accompanied by significant downregulation of the proapoptotic protein and upregulation of the antiapoptotic protein and peroxisome proliferator-activated receptor α (PPARα) expression in kidney. Mechanistically, FGF1^ΔHBS treatment directly protected mouse proximal tubule cells against palmitate-induced apoptosis, which was abolished by PPARα inhibition. In conclusion, this study demonstrated that FGF1^ΔHBS delays the progression of renal dysfunction likely through activating PPARα to prevent renal tubule cell death in late-stage T2D, exhibiting a promising translational potential in treating DN in elderly T2D individuals by ameliorating renal inflammation, fibrosis and apoptosis.     

DMT1 expression is increased in the lungs of hypotransferrinemic mice

Despite a lack of transferrin, hypotransferrinemic (Hp) mice demonstrate an accumulation of iron in peripheral organs including the lungs. One potential candidate for such transferrin-independent uptake of iron is divalent metal transporter-1 (DMT1), an established iron transporter. We tested the hypothesis that increased concentrations of iron in the lungs of Hp mice are associated with elevations in DMT1 expression. With the use of inductively coupled plasma emission spectroscopy, measurements of nonheme iron confirmed significantly elevated concentrations in the lung tissue of Hp mice relative to the wild-type mice. Western blot analyses for the expression of two isoforms of DMT1 in the Hp mice relative to the wild-type animals demonstrated an elevation for the isoform that lacks an iron-responsive element (IRE) with significant decrements in the expression of +IRE DMT1. With the use of immunohistochemistry, -IRE DMT1 was localized to both airway epithelial cells and alveolar macrophages in wild-type mice. Staining appeared increased in both types of cells in the Hp mice. Elevated concentrations of both tissue nonheme iron and expression of -IRE DMT1 in the Hp mice were associated with increased quantities of -IRE mRNA. There was no difference between wild-type and homozygotic Hp mice in the amount of mRNA for DMT1 +IRE. We conclude that differences between Hp and wild-type mice in nonheme iron concentrations were accompanied by increases in the expression of -IRE DMT1. Increased expression of -IRE DMT1 in the lungs of the Hp mice could be responsible for elevated concentrations of the metal in these tissues.     

Dextran is resistant to lysosomal digestion in kidney tubules

Low molecular weight dextran (Rheomacrodex) was infused into dextran resistant rats in a dose of 5 g/kg body weight. The kidneys were studied by electron microscopy at different time intervals after infusion using a special fixative for the demonstration of dextran. The lysosomes of proximal tubule cells gradually accumulated dextran which remained in small amounts even after 10 days. In separate kidney slice experiments the ability of dextran-loaded proximal tubule lysosomes to digest absorbed proteins was determined using 125I-labelled lysozyme. There were no changes in lysosomal protein digestion. Labelled dextran was resistant to digestion in vitro by homogenates of rat or rabbit kidney cortex or isolated rat lysosomal enzymes. It is concluded that the protein absorption pathway and lysosomal protein catabolism is unchanged after tubular uptake of dextran despite pronounced ultrastructural alterations to the lysosomal system and that dextran is resistant to lysosomal digestion in renal proximal tubules.     

Tubular proteinuria in mice and humans lacking the intrinsic lysosomal protein SCARB2/Limp-2

Deficiency of the intrinsic lysosomal protein human scavenger receptor class B, member 2 (SCARB2; Limp-2 in mice) causes collapsing focal and segmental glomerular sclerosis (FSGS) and myoclonic epilepsy in humans, but patients with no apparent kidney damage have recently been described. We now demonstrate that these patients can develop tubular proteinuria. To determine the mechanism, mice deficient in Limp-2, the murine homolog of SCARB2, were studied. Most low-molecular-weight proteins filtered by the glomerulus are removed in the proximal convoluted tubule (PCT) by megalin/cubilin-dependent receptor-mediated endocytosis. Expression of megalin and cubilin was unchanged in Limp-2(-/-) mice, however, and the initial uptake of injected Alexa Fluor 555-conjugated bovine serum albumin (Alexa-BSA) was similar to wild-type mice, indicating that megalin/cubilin-dependent, receptor-mediated endocytosis was unaffected. There was a defect in proteolysis of reabsorbed proteins in the Limp-2(-/-) mice, demonstrated by the persistence of Alexa-BSA in the PCT compared with controls. This was associated with the failure of the lysosomal protease cathepsin B to colocalize with Alexa-BSA and endogenous retinol-binding protein in kidneys from Limp-2(-/-) mice. The data suggest that tubular proteinuria in Limp-2(-/-) mice is due to failure of endosomes containing reabsorbed proteins to fuse with lysosomes in the proximal tubule of the kidney. Failure of proteolysis is a novel mechanism for tubular proteinuria.     

Immunocytochemical localization of cathepsin D in lysosomes of cortical collecting tubule cells of the rat kidney

Immunocytochemical localization of cathepsin D in rat renal tubules was investigated by means of indirect immunoenzyme and protein A--gold techniques. By light microscopy, fine granular staining was seen in the mesangial cells of glomeruli. Heavy reaction deposits were present in the cortical tubular segments and some of the medullary collecting tubules. The proximal tubules contained a few positive granules. Other segments were negative for cathepsin D. By electron microscopy, gold particles representing the antigenic sites for cathepsin D were present in cytoplasmic granules and multivesicular bodies of the segment of the cortical collecting tubule. These cytoplasmic granules were presumed to be digestive vacuoles (secondary lysosomes) from their morphological profile. The proximal tubule cells contained the very weakly labeled secondary lysosomes. No specific labeling was noted in other segments of the nephron. Control experiments confirmed the specificity of the immunostaining. Quantitative analysis of the labeling density in each subcellular compartment also confirmed that the main subcellular sites for cathepsin D are the secondary lysosomes and multivesicular bodies. The labeling density in these granules of the lysosomal system varied widely with the individual granules, suggesting that there is a considerable heterogeneity of enzyme content among the granules of the lysosomal system. The prominent presence of cathepsin D in the cortical collecting tubule suggests a certain segment-specific function of this proteinase.     

Iron Accumulates in Huntington’s Disease Neurons: Protection by Deferoxamine

Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by a polyglutamine-encoding CAG expansion in the huntingtin gene. Iron accumulates in the brains of HD patients and mouse disease models. However, the cellular and subcellular sites of iron accumulation, as well as significance to disease progression are not well understood. We used independent approaches to investigate the location of brain iron accumulation. In R6/2 HD mouse brain, synchotron x-ray fluorescence analysis revealed iron accumulation as discrete puncta in the perinuclear cytoplasm of striatal neurons. Further, perfusion Turnbull’s staining for ferrous iron (II) combined with transmission electron microscope ultra-structural analysis revealed increased staining in membrane bound peri-nuclear vesicles in R6/2 HD striatal neurons. Analysis of iron homeostatic proteins in R6/2 HD mice revealed decreased levels of the iron response proteins (IRPs 1 and 2) and accordingly decreased expression of iron uptake transferrin receptor (TfR) and increased levels of neuronal iron export protein ferroportin (FPN). Finally, we show that intra-ventricular delivery of the iron chelator deferoxamine results in an improvement of the motor phenotype in R6/2 HD mice. Our data supports accumulation of redox-active ferrous iron in the endocytic / lysosomal compartment in mouse HD neurons. Expression changes of IRPs, TfR and FPN are consistent with a compensatory response to an increased intra-neuronal labile iron pool leading to increased susceptibility to iron-associated oxidative stress. These findings, together with protection by deferoxamine, support a potentiating role of neuronal iron accumulation in HD.     

The alpha macroglobulins of rat serum.

Cortex of rat kidney was homogenized and fractions enriched in plasma membrane, endoplasmic reticulum or brush border were prepared by several techniques of differential centrifugation. The identity and homogeneity of the membrane fragments were investigated by assaying marker enzymes and by transmission and scanning electron microscopy. Kallikrein was present in both plasma-membrane- and endoplasmic-reticulum-enriched fractions isolated by two fractionation procedures. Kallikrein was highly concentrated in a plasma-membrane fraction but was absent from the brush-border membrane of proximal tubular cells. Cells of transplanted renal tumours of the rat, originating from the proximal tubule, had no kallikrein activity. Kininase activity, angiotensin I-converting enzyme (kininase II) and angiotensinase were found in a plasma-membrane-enriched fraction and especially in the fraction containing isolated brush border. It is suggested that after renal kallikrein is synthesized on endoplasmic reticulum, it is subsequently reoriented to a surface membrane for activation and release. Renal kallikrein may enter the tubular filtrate distal to the proximal tubules. The brush-border membrane of proximal tubule is the major site of inactivation of kinins and angiotensin II..     

The amelioration of renal damage in Skp2-deficient mice canceled by p27 Kip1 deficiency in Skp2-/- p27-/- mice

SCF-Skp2 E3 ubiquitin ligase (Skp2 hereafter) targets several cell cycle regulatory proteins for degradation via the ubiquitin-dependent pathway. However, the target-specific physiological functions of Skp2 have not been fully elucidated in kidney diseases. We previously reported an increase in Skp2 in progressive nephropathy and amelioration of unilateral ureteral obstruction (UUO) renal injury associated with renal accumulation of p27 in Skp2(-/-) mice. However, it remains unclear whether the amelioration of renal injury in Skp2(-/-) mice is solely caused by p27 accumulation, since Skp2 targets several other proteins. Using Skp2(-/-)p27(-/-) mice, we investigated whether Skp2 specifically targets p27 in the progressive nephropathy mediated by UUO. In contrast to the marked suppression of UUO renal injury in Skp2(-/-) mice, progression of tubular dilatation associated with tubular epithelial cell proliferation and tubulointerstitial fibrosis with increased expression of collagen and α-smooth muscle actin were observed in the obstructed kidneys in Skp2(-/-)p27(-/-) mice. No significant increases in other Skp2 target proteins including p57, p130, TOB1, cyclin A and cyclin D1 were noted in the UUO kidney in Skp2(-/-) mice, while p21, c-Myc, b-Myb and cyclin E were slightly increased. Contrary to the ameliorated UUO renal injure by Skp2-deficiency, the amelioration was canceled by the additional p27-deficiency in Skp2(-/-)p27(-/-) mice. These findings suggest a pathogenic role of the reduction in p27 targeted by Skp2 in the progression of nephropathy in UUO mice.     

Endocannabinoids and the renal proximal tubule: An emerging role in diabetic nephropathy

Diabetic nephropathy is a leading cause for the development of end-stage renal disease. In diabetes mellitus, a number of structural changes occur within the kidney which leads to a decline in renal function. Damage to the renal proximal tubule cells (PTCs) in diabetic nephropathy includes thickening of the basement membrane, tubular fibrosis, tubular lesions and hypertrophy. A clearer understanding of the molecular mechanisms involved in the development of diabetic kidney disease is essential for the understanding of the role cellular pathways play in its pathophysiology. The endocannabinoid system is an endogenous lipid signalling system which is involved in lipogenesis, adipogenesis, inflammation and glucose metabolism. Recent studies have demonstrated that in diabetic nephropathy, there is altered expression of the endocannabinoid system. Future investigations should clarify the role of the endocannabinoid system in the development of diabetic nephropathy and within this system, identify potential therapeutics to reduce the burden of this disease.     

Intrarenal Renin Angiotensin System Revisited: ROLE OF MEGALIN-DEPENDENT ENDOCYTOSIS ALONG THE PROXIMAL NEPHRON

The existence of a local renin angiotensin system (RAS) of the kidney has been established. Angiotensinogen (AGT), renin, angiotensin-converting enzyme (ACE), angiotensin receptors, and high concentrations of luminal angiotensin II have been found in the proximal tubule. Although functional data have documented the relevance of a local RAS, the dualism between biosynthesis and endocytotic uptake of its components and their cellular processing has been incompletely understood. To resolve this, we have selectively analyzed their distribution, endocytosis, transcytosis, and biosynthesis in the proximal tubule. The presence of immunoreactive AGT, restricted to the early proximal tubule, was due to its retrieval from the ultrafiltrate and storage in endosomal and lysosomal compartments. Cellular uptake was demonstrated by autoradiography of radiolabeled AGT and depended on intact endocytosis. AGT was identified as a ligand of the multiple ligand-binding repeats of megalin. AGT biosynthesis was restricted to the proximal straight tubule, revealing substantial AGT mRNA expression. Transgenic AGT overexpression under the control of an endogenous promoter was also restricted to the late proximal tubule. Proximal handling of renin largely followed the patterns of AGT, whereas its local biosynthesis was not significant. Transcytotic transport of AGT in a proximal cell line revealed a 5% recovery rate after 1 h. ACE was expressed along late proximal brush-border membrane, whereas ACE2 was present along the entire segment. Surface expression of ACE and ACE2 differed as a function of endocytosis. Our data on the localization and cellular processing of RAS components provide new aspects of the functional concept of a “self-contained” renal RAS.     

Roles of LAMP-1 and LAMP-2 in lysosome biogenesis and autophagy

The lysosomal membrane proteins LAMP-1 and LAMP-2 are estimated to contribute to about 50% of all proteins of the lysosome membrane. Surprisingly, mice deficient in either LAMP-1 or LAMP-2 are viable and fertile. However, mice deficient in both LAMP-1 and LAMP-2 have an embryonic lethal phenotype. These results show that these two major lysosomal membrane proteins share common functions in vivo. However, LAMP-2 seems to have more specific functions since LAMP-2 single deficiency has more severe consequences than LAMP-1 single deficiency. Mutations in LAMP-2 gene cause a lysosomal glycogen storage disease, Danon disease, in humans. LAMP-2 deficient mice replicate the symptoms found in Danon patients including accumulation of autophagic vacuoles in heart and skeletal muscle. In embryonic fibroblasts, mutual disruption of both LAMPs is associated with an increased accumulation of autophagic vacuoles and unesterified cholesterol, while protein degradation rates are not affected. These results clearly show that the LAMP proteins fulfil functions far beyond the initially suggested roles in maintaining the structural integrity of the lysosomal compartment.     

Protein localization of SLC26A2 (DTDST) in rat kidney

The SLC26 family represents a group of integral membrane anion transport proteins. Mutations in one member of this protein family, SLC26A2 (DTDST or diastrophic dysplasia sulfate transporter), result in various chondrodysplasias due to undersulfation of proteoglycans in chondrocytes, a major site of DTDST protein expression. DTDST mRNA has been detected in the kidney, but protein expression has not been characterized. Our objective for this study was to determine the protein localization of this sulfate transporter in the kidney. We used immunofluorescence (IMF) techniques with an anti-DTDST monoclonal antibody to examine kidneys harvested from adult rats. Double labeling was performed with antibodies directed against megalin, which is found in the microvillus membrane and coated pits of the proximal tubule. IMF analysis indicated that DTDST protein expression was limited to the microvillus membrane of proximal tubule cells in the renal cortex but absent in glomeruli and other nephron segments. DTDST was also detected in isolated rat kidney proximal tubule microvillus membranes by Western blot analysis, confirming the immunofluorescent localization of the DTDST transporter to this nephron segment. The functional role of the DTDST protein in the kidney is unknown, but it may play a role in proximal tubule sulfate transport.     

Experimental evidence for a direct cytotoxicity of Loxosceles intermedia (brown spider) venom in renal tissue.

Brown spider (Loxosceles genus) venom causes necrotic lesions often accompanied by fever, hemolysis, thrombocytopenia, and acute renal failure. Using mice exposed to Loxosceles intermedia venom, we aimed to show whether the venom directly induces renal damage. The experimental groups were composed of 50 mice as controls and 50 mice that received the venom. Light microscopic analysis of renal biopsy specimens showed alterations including hyalinization of proximal and distal tubules, erythrocytes in Bowman's space, glomerular collapse, tubule epithelial cell blebs and vacuoles, interstitial edema, and deposition of eosinophilic material in the tubule lumen. Electron microscopic findings indicated changes including glomerular epithelial and endothelial cell cytotoxicity as well as disorders of the basement membrane. Tubule alterations include epithelial cell cytotoxicity with cytoplasmic membrane blebs, mitochondrial changes, increase in smooth endoplasmic reticulum, presence of autophagosomes, and deposits of amorphous material in the tubules. We also found that the venom caused azotemia with elevation of blood urea levels but did not decrease C3 complement concentration or cause hemolysis in vivo. Confocal microscopy with antibodies against venom proteins showed direct binding of toxins to renal structures, confirmed by competition assays. Double-staining immunofluorescence reactions with antibodies against type IV collagen or laminin, antibodies to venom toxins, and fluorescent cytochemistry with DAPI revealed deposition of toxins in glomerular and tubule epithelial cells and in renal basement membranes. Two-dimensional electrophoresis showed venom rich in low molecular mass and cationic toxins. By immunoblotting with antibodies to venom toxins on renal extracts from venom-treated mice, we detected a renal binding toxin at 30 kD. The data provide experimental evidence that L. intermedia venom is directly involved in nephrotoxicity.