Human Kidney Sections Research Articles

Jouberin localizes to collecting ducts and interacts with nephrocystin-1

Joubert syndrome and related disorders are autosomal recessive multisystem diseases characterized by cerebellar vermis aplasia/hypoplasia, retinal degeneration and cystic kidney disease. There are five known genes; mutations of which give rise to a spectrum of renal cystic diseases the most common of which is nephronophthisis, a disorder characterized by early loss of urinary concentrating ability, renal fibrosis, corticomedullary cyst formation and renal failure. Many of the proteins encoded by these genes interact with one another and are located at adherens junctions or the primary cilia and or basal bodies. Here we characterize Jouberin, a multi-domain protein encoded by the AHI1 gene. Immunohistochemistry with a novel antibody showed that endogenous Jouberin is expressed in brain, kidney and HEK293 cells. In the kidney, Jouberin co-localized with aquaporin-2 in the collecting ducts. We show that Jouberin interacts with nephrocystin-1 as determined by yeast-2-hybrid system and this was confirmed by exogenous and endogenous co-immunoprecipitation in HEK293 cells. Jouberin is expressed at cell-cell junctions, primary cilia and basal body of mIMCD3 cells while a Jouberin-GFP construct localized to centrosomes in subconfluent and dividing MDCK cells. Our results suggest that Jouberin is a protein whose expression pattern supports both the adherens junction and the ciliary hypotheses for abnormalities leading to nephronophthisis.

Osteopontin is histochemically detected by the AgNOR acid-silver staining.

Silver nitrate staining of decalcified bone sections is known to reveal osteocyte canaliculi and cement lines. Nucleolar Organising Regions (NOR) are part of the nucleolus, containing argyrophilic proteins (nucleoclin/C23, nucleophosmin/B23) that can be identified by silver staining at low pH. The aim of this study was to clarify the mechanism explaining why AgNOR staining also reveals osteocyte canaliculi. Human bone and kidney sections were processed for silver staining at light and electron microscopy with a modified method used to identify AgNOR. Sections were processed in parallel for immunohistochemistry with an antibody direct against osteopontin. Protein extraction was done in the renal cortex and decalcified bone and the proteins were separated by western blotting. Purified hOPN was also used as a control. Proteins were electro-transferred on polyvinylidene difluoride membranes and stained for AgNOR proteins. In bone, Ag staining identified AgNOR in cell nuclei, as well as in osteocyte canaliculi, cement and resting lines. In the distal convoluted tubules of the kidney, silver deposits were also observed in cytoplasmic granules on the apical side of the cells. Immunolocalization of osteopontin closely matched with all these locations in bone and kidney. Ag staining of membranes at low pH revealed bands for NOR proteins and 56 KDa (kidney), 60KDa (purified hOPN) and 75 KDa (bone) bands that corresponded to osteopontin. NOR proteins and osteopontin are proteins containing aspartic acid rich regions that can bind Ag. Staining protocols using silver nitrate at low pH can identify these proteins on histological sections or membranes.

Identification and subcellular localization of a new cystinosin isoform

Nephropathic cystinosis is a lysosomal disorder caused by functional defects of cystinosin, which mediates cystine efflux into the cytosol. The protein sequence contains at least two signals that target the protein to the lysosomal compartment, one of which is located at the carboxy terminal tail (GYDQL). We have isolated from a human kidney cDNA library a cystinosin isoform, which is generated by an alternative splicing of exon 12 that removes the GYDQL motif. Based on its last three amino acids, we have termed this protein cystinosin-LKG. Contrary to the lysosomal cystinosin isoform, expression experiments performed by transient transfection of green fluorescent protein fusion plasmids in HK2 cells showed that cystinosin-LKG is expressed in the plasma membrane, in lysosomes, and in other cytosolic structures. This subcellular localization of the protein was confirmed by transmission electron microscopy. In addition, immunogold labeling was observed in the endoplasmic reticulum and in the Golgi apparatus. Expression of the protein in renal tubular structures was also directly demonstrated by immunostaining of normal human kidney sections. The plasma membrane localization of cystinosin-LKG was directly tested by [(35)S]cystine flux experiments in COS-1 cells. In the presence of a proton gradient, a marked enhancement of intracellular cystine transport was observed in cells overexpressing this isoform. These data indicate that the expression of the gene products encoded by the CTNS gene is not restricted to the lysosomal compartment. These finding may help elucidate the mechanisms of cell dysfunction in this disorder.

Factor XI protein in human pancreas and kidney

Dear Sir, Factor XI (FXI) is the zymogen of a plasma protease that contributes to haemostasis through activation of factor IX (1). As with other coagulation proteases, the liver is the primary source of plasma FXI (2–4). However, we have detected FXI mRNA by northern blot in human pancreas and kidney (4). The signal for pancreas was of comparable intensity to that of liver. Subsequently, we isolated cDNAs from a human pancreas library (not shown) with identical nucleotide sequence to the FXI cDNA from liver (5). We now present results of a study demonstrating FXI protein in human pancreas and kidney. Paraffin-imbedded sections of human liver, pancreas and kidney (three specimens for each) were obtained from surgery and autopsy specimens used as normal controls in the Vanderbilt University Hospital Histology Laboratory. Specimens were heated to 60°C for 1 hour (h), fixed sequentially with xylene, 100% ethanol, 3% H2O2 in 100% methanol, and 95% and 75% ethanol, then rinsed with water. Antigen retrieval was with 10 mM citrate pH 6.0 for 1 minute (min), and steaming for 25 min. Slides were incubated with blocking buffer (1.5% horse serum in phosphate-buffered saline [PBS]) for 20 min, then mouse anti-human FXI monoclonal IgG 1A6 (5 µg/ml) in blocking buffer at RT for 1 h. 1A6 binds to the FXI A2 domain, and recognizes a single 160 kDa protein in plasma corresponding to FXI (6). After washing three times with PBS, sections were incubated with biotinylated anti-mouse IgG (Vectastain Elite ABC, Vector Laboratories, Burlingame, CA, USA) in blocking buffer, and developed according to the manufacturer's recommendation. Sections were counterstained with hematoxylin, and treated with 1.2% lithium carbonate to enhance blue color. Representative photomicrographs are shown in Figure 1. In liver, as expected, the cytoplasm of hepatocytes, but not cells in hepatic sinusoids, stained uniformly for FXI (Fig. 1B). Intense staining was detected in pancreatic islets (Fig. 1D and E), but not in acinar cells of the exocrine pancreas. This is surprising, considering the strong signal intensity for FXI in northern blots (4), and the fact that islets make up only 1–2% of pancreatic tissue. FXI was also present in tumor cells from two islet cell carcinomas (insulinomas – Fig. 1G and H). In kidney, FXI was detected in cells of the proximal and distal tubules (Fig. 1J), and the collecting ducts (not seen in Fig. 1J), but not in glomeruli. Identical staining was observed for all tissues with goat polyclonal antihuman FXI IgG (Affinity Biologials, Ancaster, Ontario, Canada – data not shown). FXI protein was not detected in muscle, small bowel, lymph node, or bone marrow (data not shown). Figure 1 Detection of FXI protein in normal human tissues and islet cell tumors This study demonstrates for the first time that FXI protein is synthesized outside of the liver. Patients with congenital FXI deficiency may bleed excessively with trauma, particularly when the urinary tract or oropharynx is involved (1). It is thought that FXI contributes to maintenance of clot integrity in the face of robust fibrinolysis in these tissues. FXI synthesized in the kidney may help maintain local haemostatic balance by countering fibrinolytic activity associated with high local levels of urokinase. A role for FXI in islet cell physiology has not been identified and, at this point, is not obvious. Infusion of pancreatic islets into the portal vein during islet cell transplantation for diabetes mellitus is accompanied by a thrombo-inflammatory response (7) that occasionally causes disseminated intravascular coagulation (8) or portal vein thrombosis (9). High plasma levels of factor XI are a risk factor for venous thromboembolism (10), and it is possible that FXI may contribute to the pro-coagulant state during islet cell transplants. The transcription factor hepatocyte nuclear factor-4α (HNF-4α) is required for liver-specific FXI expression (2). Hepatocytes and pancreatic islet cells share similarities in terms of developmental paradigms and transcriptional control of gene regulation (11). HNF-4α is required for normal glucose-stimulated secretion of insulin by β-cells (12). HNF-4α is also expressed in kidney (13), reinforcing the impression that the tissue distribution of FXI protein synthesis is related to expression of this transcription factor. FXI is structurally similar to plasma prekallikrein (PK) (5). The genes for the two proteins are separated by <10 kilobases, and are clearly products of a duplication event involving an ancestral gene (2). PK mRNA is present in many human tissues, with the highest levels in liver, pancreas and kidney. PK protein was recently demonstrated in hepatocytes, pancreatic islets, and kidney tubules and collecting ducts (14); a distribution strikingly similar to that of FXI. It is not known if HNF-4α is required for PK gene expression, but the results suggest that FXI and PK expression may be coordinated in some tissues.

C4d Immunohistochemistry in glomerulonephritis with different antibodies

The presence of C4d in the kidney is generally detected particularly for the diagnosis of antibody-mediated rejection in renal transplants. In frozen sections of immunofluorescence (IF) staining with anti-C4d monoclonal antibodies (mAbs), we noted intrinsic C4d deposition even in normal glomeruli though their pathogenic or an intrinsic role is unkown. An anti-C4d polyclonal antibody (C4dpAb), which is suitable for paraffin immunoperoxidase (IP) staining, is less used than mAbs, and it has demonstrated that intrinsic C4d is not evident. To establish a stable and reproducible procedure for C4d detection with the C4dpAb and to determine the staining characteristics of it, the present study aimed to test whether the method was comparable with IF with a mAb. We compared the C4dpAb with the mAb in adjacent sections of human diseased kidneys, and then compared IP with IF of C4dpAb. Two ways of antigen retrieval was examined for IP. On comparing the two antibodies for glomerular staining with IF, we found that the pattern and intensity (C4dpAb showed intrinsic C4d with IF) were similar. In addition, C4dpAb staining with IP and IF demonstrated that the intrinsic staining in the normal glomerulus was mostly undetectable by IP, whereas IF showed distinct staining. Likewise, C4d deposition with IP in some cases was apparently weaker than that on IF, suggesting that this deposition is not intrinsic but indicates pathogenic complement activation. The advantage of the C4dpAb for immunohistochemistry is of value for reconsidering the role of C4d in glomerular diseases.

Aquaporin-1 Is not Expressed in Descending Thin Limbs of Short-Loop Nephrons

In mammalian kidneys, aquaporin-1 is responsible for water reabsorption along the proximal tubule and is also thought to be involved in the concentration of urine that occurs in the medulla. It has been suggested, however, that aquaporin-1 is not expressed in the last part of the descending thin limbs of short loop nephrons in rats and mice, and its expression in this region in humans has not been studied. We examined the expression of aquaporin-1 and the urea transporter UT-A2 in serial sections of mouse nephrons in the inner stripe of the outer medulla using immunohistochemistry. In contrast to previous observations, we demonstrate a complete absence of aquaporin-1 along the entire length of descending thin limbs of 90% of short loop nephrons. Conversely, as expected, we identified aquaporin-1 in proximal tubules, descending thin limbs of long loop nephrons, and medullary descending vasa recta. We also observed this abrupt transition from aquaporin-1-positive proximal tubules to aquaporin-1-negative descending thin limbs of short loop nephrons in sections of human and rat kidneys. UT-A2 was restricted to the last 28% to 44% of the descending thin limbs of all short loop nephrons. Because the majority of nephrons are of the short loop variety, our findings suggest that the mechanisms of water transport in the descending thin limbs of short loop nephrons should be reevaluated. Likewise, the roles of aquaporin-1 and UT-A2 in the countercurrent multiplier and water conversation may need to be readdressed.

Osteopontin is an argentophilic protein in the bone matrix and in cells of kidney convoluted tubules

Nucleolar organising regions (NOR) are part of the nucleolus, containing argyrophilic proteins (nucleoclin/C23, nucleophosmin/B23). They are identified by silver staining at low pH. The method also reveals osteocyte canaliculi and cement lines and granules in the cytoplasm of kidney cells in locations that mimic osteopontin distribution. Human bone and kidney sections, benign and lymphomatous pleural effusions were processed for silver staining to identify AgNOR. Sections were processed in parallel for immunohistochemistry with an antibody direct against osteopontin. In pleural effusions, AgNORs were found increased in the nuclei of lymphoma cells. In bone, Ag staining identified AgNOR in cell nuclei, as well as in osteocyte canaliculi, cement and resting lines. In the distal convoluted tubules of the kidney, silver deposits were also observed in cytoplasmic granules on the apical side of the cells. Immunolocalization of osteopontin closely matched with all these locations in bone and kidney. NOR proteins and osteopontin are proteins containing aspartic acid rich repeats that can bind Ag. Staining protocols using silver nitrate at low pH can identify these proteins on histological sections. AgNOR is a useful histochemical method to identify osteopontin in bone sections.

Human Renal Cortical and Medullary UDP-Glucuronosyltransferases (UGTs): Immunohistochemical Localization of UGT2B7 and UGT1A Enzymes and Kinetic Characterization ofS-Naproxen Glucuronidation

There is currently little information regarding the localization of UDP-glucuronosyltransferases (UGTs) in human renal cortex and medulla, and the functional contribution of renal UGTs to drug glucuronidation remains poorly defined. Using human kidney sections and human kidney cortical microsomes (HKCM) and human kidney medullary microsomes (HKMM), we combined immunohistochemistry to investigate UGT1A and UGT2B7 expression with in vitro microsomal studies to determine the kinetics of S-naproxen acyl glucuronidation. With the exception of the glomerulus, Bowman's capsule, and renal vasculature, UGT1A proteins and UGT2B7 were expressed throughout the proximal and distal convoluted tubules, the loops of Henle, and the collecting ducts. Additionally, UGT1A and UGT2B7 expression was demonstrated in the macula densa, supporting a potential role of UGTs in regulating aldosterone. Consistent with the immunohistochemical data, S-naproxen acyl glucuronidation was catalyzed by HKCM and HKMM. Kinetic data were well described by the two-enzyme Michaelis-Menten equation. K(m) values for the high-affinity components were 34 +/- 14 microM (HKCM) and 45 +/- 14 microM (HKMM). Fluconazole inhibited the high-affinity component establishing UGT2B7 as the enzyme responsible for S-naproxen glucuronidation in cortex and medulla. The low-affinity component was relatively unaffected by fluconazole (<15% inhibition), supporting the presence of other UGTs with S-naproxen glucuronidation capacity (e.g., UGT1A6 and UGT1A9) in cortex and medulla. We postulate that the ubiquitous distribution of UGTs in mammalian kidney may buffer physiological responses to endogenous mediators, but at the same time competitive xenobiotic-endobiotic interactions may provide an explanation for the adverse renal effects of drugs, including nonsteroidal anti-inflammatory drugs.

V1 and V0 Domains of the Human H+-ATPase Are Linked by an Interaction between the G and a Subunits

The specialized H(+)-ATPases found in the inner ear and acid-handling cells in the renal collecting duct differ from those at other sites, as they contain tissue-specific subunits, such as a4 and B1, and in the kidney, C2, d2, and G3 as well. These subunits replace the ubiquitously expressed forms. Previously, we have shown that, in major organs of both mouse and man, G3 subunit expression is limited to the kidney. Here we have shown wide-spread transcription of murine G3 in specific segments of microdissected nephron, and demonstrated additional G3 expression in epithelial fragments from human inner ear. We raised a polyclonal G3-specific antibody, which specifically detects G3 from human, mouse, and rat kidney lysates, and displays no cross-reactivity with G1 or G2. However, immunolocalization using this antibody on human and mouse kidney sections was unachievable, suggesting epitope masking. Phage display analysis and subsequent enzyme-linked immunosorbent assay, using the G3 antibody epitope peptide as bait, identified a possible interaction between the G3 subunit and the a4 subunit of the H(+)-ATPase. This interaction was verified by successfully using purified, immobilized full-length G3 to pull down the a4 subunit from human kidney membrane preparations. This confirms that a4 and G3 are component subunits of the same proton pump and explains the observed epitope masking. This interaction was also found to be a more general feature of human H(+)-ATPases, as similar G1/a1, G3/a1, and G1/a4 interactions were also demonstrated. These interactions represent a novel link between the V(1) and V(0) domains in man, which is known to be required for H(+)-ATPase assembly and regulation.

Expression of complement system regulatory molecules in the endometrium of normal ovulatory and hyperstimulated women correlate with menstrual cycle phase

The present study describes the temporal expression of complement regulatory molecules membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1, and CD59 in the human endometrium throughout the normal menstrual cycle and in patients submitted to ovarian hyperstimulation. During its proliferative phase, the endometrium expresses MCP, with increased expression during the secretory phase. Phase-dependent expression also was observed for DAF and CD59, mainly in the secretory phase. Expression of CR1 was not detected. These results suggest the presence of complement system activity during the menstrual cycle, with greater expression of regulatory molecules during the secretory phase to protect the epithelial integrity of human endometrium.

Upregulation of histidine decarboxylase expression in superficial cortical nephrons during pregnancy in mice and women

Mechanisms regulating pregnancy-induced changes in renal function are incompletely understood. Few candidate genes have been identified and data suggest that alternate mechanisms remain to be elucidated. Our objective was to screen thousands of genes expressed in kidneys from mice throughout gestation to identify possible key regulators of renal function during pregnancy. Mouse complementary DNA microarrays were used to screen for differences in expression during pregnancy in C57BL/6 mice. Interesting candidate genes whose expression varied with pregnancy were further analyzed by reverse transcription-PCR and Northern blot. Expression was localized by in situ hybridization and immunohistochemistry. Follow-up immunohistochemical analyses in archival human kidney sections from the fetus, non-pregnant, and pregnant women were also performed. Histidine decarboxylase (HDC), the enzyme that synthesizes histamine, was markedly upregulated in the mouse kidney during pregnancy. HDC expression localized to proximal tubule cells of fetal and adult mice. Females showed strong expression in the juxtamedullary zone before pregnancy and upregulation in the superficial cortical zone (SCZ) by mid-gestation. Histamine colocalized with HDC. Male mice showed only low HDC expression. Similar expression patterns were observed in human kidneys. Our results show that HDC expression and histamine production are increased in the SCZ during pregnancy. If histamine acts as a vasodilator, we speculate that increasing production in the SCZ may increase renal blood flow to this zone and recruit superficial cortical nephrons during pregnancy.

Regulation of mesangial cell function by PKD2

Ca2+ influx through plasma membrane is a major component of MC response to vasoconstrictors. PKD2, the protein product of the gene mutated in type 2 autosomal dominant polycystic kidney disease, has been shown to function as an non-selective cation channel (NSCC) in renal tubular epithelial cells. The present study was performed to test the hypothesis that PKD2 and its binding partner constitute NSCC in MCs and contribute to contractile function of the cells. Western blotting and immunocytochemistry showed PKD2 expression in cultured human MCs. The existence of PKD2 in MCs was further confirmed by immunohistochemsitry in rat and human kidney sections. Co-immunoprecipitation showed a selective interaction of PKD2 with TRPC1 and TRPC4. Cell-attached patch clamp experiments revealed nonselective cationic currents stimulated by Ang II. The Agonist-induced currents were enhanced by over-expressing PKD2, but attenuated by knocking down PKD2. Corresponding to the increase in channel currents, Ang II stimulation increased expression of PKD2 in the cell surface as well as interaction with TRPC4. Furthermore, Ang II-induced MC contraction was reduced in PKD2 knocked out MCs. These data suggest that PKD2 might selectively assemble with specific isoform of TRPC proteins to form NSCC in MCs and this channel complex contributes to contractile function of MCs. (supported by AHA SDG)

Lipoprotein Glomerulopathy: A New Apolipoprotein E Mutation With Enhanced Glomerular Binding

We describe a case of lipoprotein glomerulopathy, the second ever reported from the United States, in a Mexican man with a hitherto undescribed mutation in the apolipoprotein E gene (substitution of proline for arginine at position 147 [Arg147Pro]). In this patient, glomerular basement membranes showed double contours and circumferential mesangial extensions, suggesting that deposition of lipids could be injurious to endothelial cells. Immunofluorescence staining of thrombi was positive for apolipoprotein E and B. To study the reason for lipid deposition in glomeruli, we incubated normal human kidney sections with serum from the patient and a healthy control. Apolipoprotein E from the patient's serum showed binding to the glomerular capillary wall, but the control did not, suggesting enhanced binding of the mutated apolipoprotein E to glomerular capillaries. Apolipoprotein E genotyping by means of restriction endonuclease digestion of polymerase chain reaction-amplified genomic DNA showed it to be of the wild-type E3/E3.

Glomerular Permeability Is Altered by Loss of P0, a Myelin Protein Expressed in Glomerular Epithelial Cells

The myelin protein 0 (MPZ or P0) is a transmembrane glycoprotein that represents the most abundant myelin component. Mutations in the P0 gene are associated with one form of autosomal dominant demyelinating peripheral neuropathy, Charcot-Marie-Tooth disease type 1B (CMT1B). Because CMT1 may be associated with renal involvement, mostly focal segmental glomerulosclerosis, we hypothesized that P0 could be expressed in the kidney. P0 mRNA was detected by reverse transcriptase-PCR in the human and mouse renal cortex. P0 transcripts were identified by in situ hybridization at different stages of the mouse kidney development, especially in embryonic structures that give rise to the glomerulus. P0 protein was also detected by Western blot in human and rat glomerular extracts and in a human podocyte cell line using a monoclonal anti-P0 antibody. Immunofluorescence studies on human kidney sections showed that the podocytes were intensely labeled. Immunogold electron microscopy disclosed a predominant staining of the membranes of intracellular vesicles in podocytes. P0 was also detected in the podocyte cell membrane, including at the foot processes. P0(-/-) mice exhibited mild growth retardation and demyelinating neuropathy similar to the one observed in patients with CMT1B. They also presented mild albuminuria, without significant ultrastructural change of the glomerular basement membrane or the podocytes. These results demonstrate that P0, the major myelin protein, is also expressed during nephrogenesis and in mature kidney, mostly in podocytes. They suggest that P0 gene mutations might be involved in renal diseases.

Localization of α-Dystroglycan on the Podocyte: from Top to Toe

alpha-Dystroglycan (DG) is a negatively charged membrane-associated glycoprotein that links the cytoskeleton to the extracellular matrix. Previously, we described that alpha-DG covers the whole podocyte cell membrane in the rat. However, our finding was challenged by the description of a strictly basolateral localization in human kidney biopsies, using a different antibody against alpha-DG. Therefore, we studied the exact localization of glomerular alpha-DG by using these two antibodies in both species. The studies were performed by using monoclonal antibodies (MoAbs) IIH6 and VIA4.1 in immunofluorescence, confocal microscopy, and immunoelectron microscopy on both rat and human kidney sections, as well as on cultured mouse podocytes. The apical localization of alpha-DG on podocytes was more dominant than the basolateral localization. The basolateral staining with MoAb VIA4.1 was more pronounced than that of MoAb IIH6. With both MoAbs, the staining in rat kidneys was more prominent, in comparison to human kidneys. We conclude that alpha-DG is expressed at both the basolateral and apical sides of the podocyte. This localization suggests that alpha-DG plays a dual role in the maintenance of the unique architecture of podocytes by its binding to the glomerular basement membrane, and in the maintenance of the integrity of the filtration slit, respectively.

Expression of the potassium channel ROMK in adult and fetal human kidney

The renal potassium channel ROMK is a crucial element of K+ recycling and secretion in the distal tubule and the collecting duct system. Mutations in the ROMK gene (KCNJ1) lead to hyperprostaglandin E syndrome/antenatal Bartter syndrome, a life-threatening hypokalemic disorder of the newborn. The localization of ROMK channel protein, however, remains unknown in humans. We generated an affinity-purified specific polyclonal anti-ROMK antibody raised against a C-terminal peptide of human ROMK. Immunoblotting revealed a 45 kDa protein band in both rat and human kidney tissue. In human kidney sections, the antibody showed intense staining of epithelial cells in the cortical and medullary thick ascending limb (TAL), the connecting tubule, and the collecting duct. Moreover, a strong expression of ROMK protein was detected in cells of the macula densa. In epithelial cells of the TAL expression of ROMK protein was mainly restricted to the apical membrane. In human fetal kidney expression of ROMK protein was detected mainly in distal tubules of mature nephrons but not or only marginally in the collecting system. No expression was found in early developmental stages such as comma or S shapes, indicating a differentiation-dependent expression of ROMK protein. In summary, these findings support the proposed role of ROMK channels in potassium recycling and in the regulation of K+ secretion and present a rationale for the phenotype observed in patients with ROMK deficiency.

Identification of LAT4, a Novel Amino Acid Transporter with System L Activity

System L amino acid transporters mediate the movement of bulky neutral amino acids across cell membranes. Until now three proteins that induce system L activity have been identified: LAT1, LAT2, and LAT3. The former two proteins belong to the solute carrier family 7 (SLC7), whereas the latter belongs to SLC43. In the present study we present a new cDNA, designated LAT4, which also mediates system L activity when expressed in Xenopus laevis oocytes. Human LAT4 exhibits 57% identity to human LAT3. Like LAT3, the amino acid transport activity induced by LAT4 is sodium-, chloride- and pH-independent, is not trans-stimulated, and shows two kinetic components. The low affinity component of LAT4 induced activity is sensitive to the sulfhydryl-specific reagent N-ethylmaleimide but not that with high affinity. Mutation in LAT4 of the SLC43 conserved serine 297 to alanine abolishes sensitivity to N-ethylmaleimide. LAT4 activity is detected at the basolateral membrane of PCT kidney cells. In situ hybridization experiments show that LAT4 mRNA is restricted to the epithelial cells of the distal tubule and the collecting duct in the kidney. In the intestine, LAT4 is mainly present in the cells of the crypt.

Expression of functional CCR and CXCR chemokine receptors in podocytes.

Chemokines and their receptors play an important role in the pathogenesis of acute and chronic glomerular inflammation. However, their expression pattern and function in glomerular podocytes, the primary target cells in a variety of glomerulopathies, have not been investigated as of yet. Using RT-PCR, we now demonstrate the expression of CCR4, CCR8, CCR9, CCR10, CXCR1, CXCR3, CXCR4, and CXCR5 in cultured human podocytes. Stimulation of these receptors induced a concentration-dependent biphasic increase of the free cytosolic calcium concentration in podocytes in culture. In addition, we demonstrate that podocytes release IL-8 in the presence of FCS and that IL-8 down-regulates cell surface CXCR1. Chemokine stimulation of the detected CCRs and CXCRs increased activity of NADPH-oxidase, the primary source of superoxide anions in podocytes. Immunohistochemistry studies revealed only diffuse and weak CXCR expression in healthy human glomerula. In contrast, in membranous nephropathy, a characteristic podocyte disorder, the expression of CXCR1, CXCR3, and CXCR5 is up-regulated in podocytes. In conclusion, podocytes in culture and podocytes in human kidney sections express a set of chemokine receptors. The release of oxygen radicals that accompanies the activation of CCRs and CXCRs may contribute to podocyte injury and the development of proteinuria during membranous nephropathy.

Segmental expression of somatostatin receptor subtypes sst(1) and sst(2) in tubules and glomeruli of human kidney.

Somatostatin is known to modulate mesangial and tubular cell function and growth, but the somatostatin receptor (sst) subtypes responsible for these effects have not been defined. There are at least five different sst receptor subtypes (sst(1)-sst(5)). We used RT-PCR to demonstrate that normal human kidney consistently expresses mRNA for sst(1) and sst(2) (9 of 9 donors). Some donors expressed sst(4) or sst(5) mRNA, but none expressed sst(3) mRNA. Expression of sst(1) and sst(2) was further assessed by staining serial sections of normal human kidney with sst(1) and sst(2) antisera, Arachis hypogaea (AH) lectin (to define distal tubule/collecting duct cells), Phaseolus vulgaris lectin (proximal tubules), and Tamm-Horsfall protein (THP) antiserum (thick ascending limb of the loop of Henle). Specificity of antisera was demonstrated by transfection and absorption studies. Sst(2), but not sst(1), was expressed in glomeruli. Intense sst(1) and sst(2) staining localized exclusively to AH+ and THP+ tubules. Thus sst(1) and sst(2) subtype-selective analogs may be useful to beneficially modulate renal cell function in pathological conditions.

Characterization of an Antibody to the Human Melatonin mt1 Receptor

AbstractMelatonin acts via high affinity, G‐protein coupled, seven transmembrane domain receptors. To precisely localize these receptors, antibodies were raised in chickens against a 15 amino acid fragment at the intracellular C‐terminal region of the human melatonin receptor subtype mt1 (DSSNDVADRVKWKPS, mt1338−352). A chimeric form of the receptor with a hydrophilic Flag peptide (DYKDDDDK) in sequence with the extracellular N‐terminus (Flag‐mt1) was generated by polymerase chain reaction and expressed in mammalian cell lines. An IgY antibody (Y31), which gave high antibody titres by enzyme‐linked immunosorbent assay, was used to localize Flag‐mt1 in stably transfected cells by immunofluoresence. Flag‐mt1 localization with Y31 was identical to that obtained with the M5 antibody directed against the Flag epitope and was mainly localized to the Golgi apparatus with some staining at the cell surface. No staining was seen in untransfected cells with either antibody. Y31 staining was abolished using antibody preabsorbed with peptide antigen. Y31 immunofluorescence in fetal human kidney sections was restricted to nephrogenic regions and matched that of 2‐(125I)iodomelatonin binding and mt1 gene expression by in situ hybridization. Y31 was used to immunoprecipitate biotinylated membrane proteins from Flag‐mt1 stably transfected and untransfected CHO cells. Western blotting of immunoprecipitated proteins revealed two major bands specific to stably transfected cells, one at 63 kDa and one at 86 kDa. The first band almost certainly corresponds to the glycosylated form of Flag‐mt1 and the second band to receptor dimers. Thus, Y31 antibody is suitable for use in detecting the human mt1 receptor subtype in tissues and in transfected cells.