Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone.

Lower urinary tract infections are among the most common human bacterial infections, but extension to the kidneys is rare. This has been attributed to mechanical forces, such as urine flow, that prevent the ascent of bladder microbes. Here, we show that the regional hypersalinity, required for the kidney's urine-concentrating function, instructs epithelial cells to produce chemokines that localize monocyte-derived mononuclear phagocytes (MNPs) to the medulla. This hypersaline environment also increases the intrinsic bactericidal and neutrophil chemotactic activities of MNPs to generate a zone of defense. Because MNP positioning and function are dynamically regulated by the renal salt gradient, we find that patients with urinary concentrating defects are susceptible to kidney infection. Our work reveals a critical accessory role for the homeostatic function of a vital organ in optimizing tissue defense.

Randall's plaque and calcium oxalate stone formation: role for immunity and inflammation.

Idiopathic calcium oxalate (CaOx) stones often develop attached to Randall's plaque present on kidney papillary surfaces. Similar to the plaques formed during vascular calcification, Randall's plaques consist of calcium phosphate crystals mixed with an organic matrix that is rich in proteins, such as inter-α-trypsin inhibitor, as well as lipids, and includes membrane-bound vesicles or exosomes, collagen fibres and other components of the extracellular matrix. Kidney tissue surrounding Randall's plaques is associated with the presence of classically activated, pro-inflammatory macrophages (also termed M1) and downregulation of alternatively activated, anti-inflammatory macrophages (also termed M2). In animal models, crystal deposition in the kidneys has been associated with the production of reactive oxygen species, inflammasome activation and increased expression of molecules implicated in the inflammatory cascade, including osteopontin, matrix Gla protein and fetuin A (also known as α2-HS-glycoprotein). Many of these molecules, including osteopontin and matrix Gla protein, are well known inhibitors of vascular calcification. We propose that conditions of urine supersaturation promote kidney damage by inducing the production of reactive oxygen species and oxidative stress, and that the ensuing inflammatory immune response promotes Randall's plaque initiation and calcium stone formation.

Mononuclear phagocyte system of the mouse defined by immunohistochemical localization of antigen F4/80. Identification of resident macrophages in renal medullary and cortical interstitium and the juxtaglomerular complex.

Macrophages have been identified in mouse kidney by immunohistochemical localization of the macrophage-specific antigen F4/80. They constitute the majority of the renal medullary interstitial cell population and are also found in contact with cortical distal and proximal tubules and Bowman's capsule. They are a physical component of the juxtaglomerular complex.

Characterization of renal papillary antigen 1 (RPA-1), a biomarker of renal papillary necrosis.

Renal papillary necrosis (RPN) is a relatively common toxicity observed in preclinical drug safety testing. It is also observed in a variety of human diseases. RPN is difficult to diagnose without expensive scanning methods or histopathology. A noninvasive biomarker that could be detected at early stages of kidney damage would be of great value both to preclinical drug safety testing and in the clinic. An antibody raised to an unknown epitope of an antigen in rat kidney papilla was found to be specific for collecting duct cells in the kidney; this was termed renal papillary antigen 1 (RPA-1). In this study, the authors show that RPA-1 is an early biomarker of RPN in two different rat models of toxicity: 2-bromoethanamine (BEA) and N-phenylanthranilic acid (NPAA). RPA-1 can be detected in urine at early stages of toxicity and correlates well with the histopathology observed. We also characterized the biochemical properties of RPA-1 and found that the antigen is a high molecular weight membrane bound glycoprotein, with the epitope likely to be carried on an N-linked carbohydrate structure. This study demonstrates that RPA-1 is an excellent marker of RPN that can be used to detect this toxicity in preclinical safety testing.

Th17/Treg Imbalance Induced by Dietary Salt Variation Indicates Inflammation of Target Organs in Humans.

The functions of T helper 17 (Th17) and regulatory T (Treg) cells are tightly orchestrated through independent differentiation pathways that are involved in the secretion of pro- and anti-inflammatory cytokines induced by high-salt dietary. However, the role of imbalanced Th17/Treg ratio implicated in inflammation and target organ damage remains elusive. Here, by flow cytometry analysis, we demonstrated that switching to a high-salt diet resulted in decreased Th17 cells and reciprocally increased Treg cells, leading to a decreased Th17/Treg ratio. Meanwhile, Th17-related pathway was down-regulated after one day of high salt loading, with the increase in high salt loading as shown by microarray and RT-PCR. Subsequently, blood oxygen level-dependent magnetic resonance imaging (BOLD-MRI) observed hypoxia in the renal medulla (increased R2(*) signal) during high-salt loading, which was regressed to its baseline level in a step-down fashion during low-salt feeding. The flow-mediated vasodilatation (FMD) of the branchial artery was significantly higher on the first day of high salt loading. Collectively, these observations indicate that a short-term increase in dietary salt intake could induce reciprocal switches in Th17/Treg ratio and related cytokines, which might be the underlying cellular mechanism of high-salt dietary induced end organ inflammation and potential atherosclerotic risk.

Production and characterization of mouse ureteric bud cell-specific rat hybridoma antibodies utilizing subtractive immunization and high-throughput screening.

The highly branched collecting system of the kidney arises developmentally from the ureteric bud (UB) by a process of branching morphogenesis. This process is critical for the normal development of the collecting ducts and pelvis of the kidney, and is tightly controlled by the spatial and temporal expression of numerous proteins. To identify cell proteins that are differentially expressed by the UB relative to those expressed by the highly differentiated collecting ducts of the adult kidney, two mouse cell populations derived from either the early UB or the adult inner medullary collecting duct (IMCD) were used. A subtractive immunization strategy was performed in rats to generate monoclonal antibodies that preferentially reacted with antigens on UB, but not IMCD cells. In addition, the technique of antibody printing, a novel high-throughput antibody screening method for determining the specificities of a large number of monoclonal antibodies, is described. The methodologies outlined in this manuscript have broad applicability as they demonstrate that subtractive immunization can be performed in rats with cells derived from mice. Additionally, the high-throughput screening methods should facilitate the use of subtractive immunization for identifying antibodies that can distinguish differences in proteins expressed in closely related cell types.

Medullary nephritis in the diagnosis of acute cellular rejection.

BACKGROUND: The purpose of this study was to understand the role of lymphomononuclear inflammation (nephritis) in the renal allograft medulla of transplant recipients with acute dysfunction, by comparing the immunophenotype of inflammatory cells present in the medulla and cortex of kidney graft biopsies. METHOD: This is a retrospective study of 113 renal allograft needle biopsies, presenting with medullary nephritis, divided into two groups according to the main location of nephritis: in cortical and medullary regions (corticomedullary nephritis) or exclusively in the medullary region (medullary nephritis). We performed immunohistochemistry (IHC) of the cells composing the inflammatory foci, using anti-CD4, CD8, CD20, CD68, and CD138 antibodies, respectively for T-helper cells, cytotoxic T cells, B lymphocytes, macrophages and plasmocytes. The clinical follow-up of the patients was correlated with the morphological findings. RESULTS: The nephritis was corticomedullary in 66 of the 113 cases (58.4%) and exclusively medullary in the remaining 47 cases (41.6%). The immunophenotype of the inflammatory cells was similar in the cortical and medullary compartments and were mainly: cytotoxic T lymphocytes (CD8) and macrophages CD68. The immunosuppressive therapeutic response to acute cellular rejection (ACR), based on decreasing of serum creatinine values, was 81.8% in the patients of the corticomedullary nephritis group and 63.6% in those of the medullary nephritis group. CONCLUSION: Medullary nephritis in renal allograft biopsies may indicate ACR, as could be noted by the immunophenotype, which presented the same cellular mediators of rejection seen in the allograft cortex, and by the positive immunosuppressive therapeutic response observed in most patients.

Major histocompatibility complex antigens in rat kidney, ureter, and bladder. Localization with monoclonal antibodies and demonstration of Ia-positive dendritic cells.

Monoclonal mouse xenoantibodies to the SD and part of the Ia antigen complex of the rat major histocompatibility complex (MHC) were raised, and used to localize MHC antigens on frozen sections of kidney, ureter, and bladder of the DA rat strain. The Ia antigens recognized by our monoclonal antibody were located almost entirely within the cells of some, probably the proximal, convoluted tubules of the kidney. The only other Ia-bearing structures were intensely Ia-positive dendritic cells found predominantly in the renal cortex and in the mucosal connective tissues of the ureter and bladder. The SD antigens were widely distributed in the kidney with a major portion again located within the tubular cells, although in the case of SD antigens all tubular cells, including those of the medulla, were positive. By far the brightest tubules were clusters in the outer medulla, probably representing the thick loops of Henle. The endothelium of arterioles, venules and glomerular and interstitial capillaries all stained very brightly for SD antigens. The glomerular mesangium and the interstitial connective tissues of the kidney, ureter, and bladder all gave diffuse positive staining for SD antigens. Transplantation studies established that the tubular Ia and SD antigens of the kidney are produced by the cells and are not in the process of excretion or reabsorption.

Expression of the progesterone receptor and progesterone- metabolising enzymes in the female and male human kidney.

Due to high binding affinity of progesterone to the human mineralocorticoid receptor (hMR), progesterone competes with the natural ligand aldosterone. In order to analyse how homeostasis can be maintained by mineralocorticoid function of aldosterone at the MR, especially in the presence of elevated progesterone concentrations during the luteal phase and pregnancy, we investigated protective mechanisms such as the decrease of free progesterone by additional binding sites and progesterone metabolism in renal cells. As a prerequisite for sequestration of progesterone by binding to the human progesterone receptor (hPR) we demonstrated the existence of hPR expression in female and male kidney cortex and medulla at the level of transcription and translation. We identified hPR RNA by sequencing the RT-PCR product and characterised the receptor by ligand binding and scatchard plot analysis. The localisation of renal hPR was shown predominantly in individual epithelial cells of distal tubules by immunohistology, and the isoform hPR-B was detected by Western blot analysis. As a precondition for renal progesterone metabolism, we investigated the expression of steroid-metabolising enzymes for conversion of progesterone to metabolites with lower affinity to the hMR. We identified the enzyme 17alpha-hydroxylase for renal 17alpha-hydroxylation of progesterone. For 20alpha-reduction, different hydroxysteroid dehydrogenases (HSDs) such as 20alpha-HSD, 17beta-HSD type 5 (3alpha-HSD type 2) and 3alpha-HSD type 3 were found. Further, we detected the expression of 3beta-HSD type 2 for 3beta-reduction, 5alpha-reductase (Red) type 1 for 5alpha-reduction, and 5beta-Red for 5beta-reduction of progesterone in the human kidney. Therefore metabolism of progesterone and/or binding to hPR could reduce competition with aldosterone at the MR and enable the mineralocorticoid function.

[A new serologic reaction in patients with polymyalgia rheumatica and/or giant cell arteritis]. / Eine neue serologische Reaktion bei Patienten mit Polymyalgia rheumatica und/oder Riesenzellarteriitis.

Fresh sera from patients with active polymyalgia rheumatica and/or giant cell arteritis are fixing the complement components C4 and C3 to structures of the medulla of rat kidney tissue as demonstrated by the indirect immunofluorescent technique. This PMR-associated reaction is also a useful tool in monitoring disease activity, as it becomes negative under effective treatment with steroids.

Urinary antigens as markers of papillary toxicity. I. Identification and characterization of rat kidney papillary antigens with monoclonal antibodies.

Monoclonal antibodies were prepared in an attempt to develop diagnostic tools for the identification of toxic damage to the rat renal papilla. One IgG and five IgM monoclonal antibodies, reacting with antigens localized in the papilla were obtained. Three of the IgM class and the IgG class monoclonal antibodies were found to be specific for antigens localized in collecting ducts, two of them staining papillary collecting ducts more intensely than cortical collecting ducts. The IgG class antibody, termed Pap X 5C10, recognizes an antigen located at high density on the luminal side of papillary collecting duct epithelial cells and at lower density in cortical collecting duct cells. One of the IgM class monoclonal antibodies reacts with an antigen localized in epithelial cells as ascending and descending loops of Henle and of connecting tubules. Another of the IgM class monoclonal antibodies reacts with an antigen localized in the interstices of the inner medulla. All these monoclonal antibodies react with their antigens in native frozen as well as in Bouin-fixed and paraffin-embedded tissue slices. Molecular properties of the Pap X 5C10 antigen have been investigated by gel permeation chromatography, SDS-PAGE, Western blotting, and isoelectric focusing. The results indicate that the antigen in both its tissue-derived and urinary form is of large (150-200 kDa) molecular size and can be separated into two molecular species with isoelectric points of pH 7.2 and 7.3 respectively. In the urine the antigens recognized by the monoclonal antibodies form large complexes with Tamm-Horsfall protein. The antigen-containing complexes can be extracted from urine by adsorption to diatomaceous earth and elution with SDS-containing buffer. Using sandwich ELISA-type assays it is possible to determine the concentration of the antigens. In preliminary experiments we were able to show that at least three of the antigens are detected in the urine following toxic insults to the kidney. The monoclonal antibodies prepared and the tests developed thus may provide direct diagnostic access to the renal papilla and allow, for the first time, early detection of papillary damage.

Distribution of blood group antigen A in normal and pathologic human kidneys.

We tested this distribution with an indirect immunofluorescent technique using purified rabbit anti-A antiserum on 21 whole normal kidneys (a group, N equal to 18; AB group, N equal to 1; O group, N equal to 2) and on 349 kidney biopsy samples (A group, N equal to 140; AB group, N equal to 14; O or B group, N equal to 195) representing a large spectrum of renal diseases. In normal kidneys from A and AB groups, the A antigen was detected in the whole vascular endothelium and in the convoluted distal tubules. In secretors, collecting tubules were brightly positive. Epithelial staining was more diffuse in the inner part than it was in the outer part of the medulla. The basement membrane of the inner collecting tubules was positive in frozen sections but not in paraffin sections. In pathologic kidneys, modifications were obvious: (1) The thickened basement membrane of atrophic convoluted distal tubules was brightly stained. (2) Endothelial staining allowed a precise appreciation of the glomerular and interstitial vasculature. (3) In proliferative changes such as arterial intimal proliferation, proliferative glomerulonephritis, and interstitial cell infiltration, endothelial cells do not proliferate. This routine staining technique of endothelial cells by anti-A antiserum provide information not obtainable with light microscopy.

Depression of the T-lymphocyte response to phytohaemagglutinin by renal cells.

The lymphocytic infiltrate in the renal parenchyma is a consistent histological feature of pyelonephritis, but the role of the lymphocytes in the immunobiology of pyelonephritis is not known. In this investigation the influence of the local environment on the potential function of T lymphocytes in the kidney was investigated. The experiments have demonstrated that the response of rat lymphocytes to stimulation in vitro with phytohaemagglutinin (PHA) can be entirely ablated by normal kidney cells. Even when the number of kidney cells added to cultures of lymphocytes was less than 2% of the cells present some ablation of T-lymphocyte function could be detected. The biological characteristics of the factor causing ablation of the PHA responsiveness of T lymphocytes were partially characterized and the factor appears to have unique features that differentiate it from lymphocyte chalones and other tissue factors influencing lymphocyte function. The results may explain recent findings where T lymphocytes were found to be the predominant lymphocyte in the inflammatory infiltrate but were not responsive to PHA in vitro.

Prostaglandin H synthase immunoreactivity localized by immunoperoxidase technique (PAP) in the small intestine and kidney of rabbit and guinea-pig.

Prostaglandins and inhibitors of prostaglandin synthesis have striking regulatory effects on intestinal muscularis externa. We suggested earlier that a population of macrophage-like cells, located between the external muscle layers might release prostaglandins with a local effect on enveloping interstitial cells of Cajal, postulated pacemaker cells of the gut. To determine cellular production site(s) of prostaglandin we applied monoclonal antibodies against prostaglandin H synthase combined with the PAP technique to sections of rabbit and guinea-pig small intestine and kidney. In rabbit small intestine muscle cells in the circular muscle layer and in the muscularis mucosae were positive, longitudinal muscle negative. Vascular endothelial cells and serosal mesothelial cells were stained. In guinea-pig all muscle layers were unstained but endothelial and mesothelial cells were stained together with unidentified cells in the outermost submucosa. In rabbit kidney, positive staining of collecting ducts, interstitial cells, the parietal layer of Bowman's capsule and arterial endothelial cells was present. Furthermore, we found prostaglandin synthase antigenicity in the epithelial cells lining the loop of Henle, not described before. In guinea-pig medullary collecting ducts were stained and the papilla was lined by stained epithelial cells. The results show a species variation in the distribution of recognizable levels of prostaglandin H synthase. The impressive reaction in the mesothelium must be considered, when enzyme distribution is examined biochemically with fractionated tissue. Our findings do not support our hypothesis that macrophage-like cells are more potent sources of prostaglandins than smooth muscle cells.

Expression of binding sites for Dolichos biflorus agglutinin at the apical aspect of collecting duct cells in rat kidney.

To identify precisely the structural and functional cell type in the collecting duct of the rat kidney expressing binding sites for Dolichos biflorus agglutinin (DBA), we stained serial paraffin sections of kidney with horseradish peroxidase-labeled DBA and with immunocytochemical methods for localizing (Na+ + K+)-ATPase and carbonic anhydrase II (CA II), enzymes found preferentially in principal and intercalated cells, respectively. Most principal cells expressing a strong basolateral staining for (Na+ + K+)-ATPase showed binding sites for DBA at their luminal surfaces. However, a minority of cells rich in CA II and showing morphologic characteristics of intercalated cells also expressed DBA binding sites at their luminal surface and apical cytoplasm. These data suggest that DBA cytochemistry can provide a useful tool for studying the functional polarity of the main cell types of the collecting duct of the rat kidney.

Accelerated immune complex nephritis due to mesangial overloading in spontaneous hypertensive (SHR) rats.

Immune complex nephritis induced by bovine serum albumin (BSA) and horseradish peroxidase (HRP) was superimposed upon spontaneous hypertensive rats (SHR rats) which were pretreated with polyvinyl alcohol (PVA). PVA accumulated predominantly in the mesangium, and the superimposed nephritis developed more accelerated glomerular damage with marked capillary deposition of immune complexes than control animals which were not pretreated with PVA.

Attenuation of renomedullary phospholipase C isozyme, PLC-delta 1, in spontaneously hypertensive rats.

The distributional patterns of PLC isozymes within the kidney were investigated using spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats at 4 and 12 weeks of age. PLC-beta 1, PLC-beta 3 and PLC-delta 1 quantified by Western blot analysis, were present in the highest concentrations in the inner medulla of rats at both 4 and 12 weeks of age. On the other hand, PLC-beta 4, PLC-gamma 1 and PLC-gamma 2 were distributed almost equally among the regions for the rats of both ages. When compared with WKY rats at 12 weeks of age, the amounts of PLC-beta 1, PLC-beta 3, PLC-gamma 1, PLC-gamma 2, and PLC-delta 1 in the inner medulla of SHRs were significantly lower, and the amount of PLC-delta 1 in the inner stripe of the outer medulla was also significantly lower. Even at the prehypertensive stage at 4 weeks of age, the inner medullary concentration of PLC-delta 1 was significantly lower in SHRs than WKY rats. These results suggest that PLC-delta 1 would play an important role in the development of hypertension.