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B Kaissling

Publications and source records attributed to B Kaissling.

At least 19 recordsLinked to original sources
PubMed1999

Effects of phosphate intake on distribution of type II Na/Pi cotransporter mRNA in rat kidney.

BACKGROUND: Renal phosphate (Pi) reabsorption is regulated by dietary Pi intake, as well as in other ways. Changes in Pi reabsorption are associated with the modulation of sodium/Pi cotransporter type II (NaPi-2) protein abundance in the brush border membrane (BBM) of proximal tubules (PTs) and of renal NaPi-2 mRNA levels. In this study, we address whether the NaPi-2 protein and NaPi-2 mRNA distribution patterns in the renal cortex vary in parallel with changes of dietary Pi intake. METHODS: We investigated in cryosections of perfusion-fixed rat kidneys by in situ hybridization (ISH) and immunohistochemistry (IHC) the distribution patterns of NaPi-2 mRNA and of NaPi-2 protein one week, two hours, and four hours after changes in dietary Pi intake. RESULTS: NaPi-2 mRNA and NaPi-2 protein were present in PTs exclusively. In rats adapted to one week of high Pi intake, signals for NaPi-2 mRNA and NaPi-2 protein in cortical PTs were weak, except in the convoluted parts of PTs of juxtamedullary nephrons. After one week of low Pi intake, the ISH and IHC signals for NaPi-2 were high in PT segments in all cortical levels. The switch from a chronic high to a low Pi intake within two and four hours induced no increase and a slight increase, respectively, in the NaPi-2 mRNA signal in PTs of midcortical and of superficial nephrons, whereas in the BBM of these nephrons, NaPi-2 protein was markedly up-regulated. Two and four hours after switching from low to high Pi intake, the overall high ISH signal for NaPi-2 mRNA was unchanged, whereas NaPi-2 protein staining was drastically down-regulated in the BBM of PTs from superficial and midcortical nephrons. CONCLUSIONS: The marked changes in NaPi-2 protein abundance in the BBM, following altered dietary Pi intake, precede corresponding changes at the RNA level by several hours. Thus, the early adaptation to altered Pi intake involves mRNA-independent mechanisms. The up- or down-regulation of NaPi-2 protein abundance in the BBM and NaPi-2 mRNA in PT affects mainly midcortical and superficial nephrons.

Animals↗
PubMed1999

Rapid downregulation of rat renal Na/P(i) cotransporter in response to parathyroid hormone involves microtubule rearrangement.

Renal proximal tubule cells express in their apical brush border membrane (BBM) a Na/P(i) cotransporter type IIa that is rapidly downregulated in response to parathyroid hormone (PTH). We used the rat renal Na/P(i) cotransporter type IIa (NaPi-2) as an in vivo model to assess early cellular events in the rapid downregulation of this transporter. When rats were treated with PTH for 15 minutes, NaPi-2 abundance in the BBM was decreased. In parallel, transporter accumulated in intracellular vesicles. Concomitantly, microtubules (MTs) were found to form dense bundles of apical-to-basal orientation. After 60 minutes of PTH action, the cells were vastly depleted of NaPi-2, whereas their microtubular cytoskeleton had returned to its normal appearance. Prevention of MT rearrangement by taxol resulted in accumulation of NaPi-2 in the subapical cell portion after 15 minutes and a strong delay in depletion of intracellular transporter after 60 minutes of PTH action. Furthermore, the subapical accumulation of NaPi-2 was associated with the expansion of dense apical tubules of the subapical endocytic apparatus (SEA). Depolymerization of MTs by colchicine likewise caused a retardation of intracellular NaPi-2 depletion. These results suggest that NaPi-2 is downregulated in response to PTH through a rapid endocytic process in 2 separate steps: (a) internalization of the transporter into the SEA, and (b) its delivery to degradative organelles by a trafficking mechanism whose efficiency depends on a taxol-sensitive rearrangement of MTs.

Animals↗
PubMed1999

Distribution of the sodium/phosphate transporter during postnatal ontogeny of the rat kidney.

Renal phosphate reabsorption via the type II sodium/ phosphate cotransporter (NaPi-2) in the brush border membrane (BBM) of proximal tubules underlies alterations during aging. The ontogeny of NaPi-2 in kidneys from newborn to 6-wk-old rats was investigated. NaPi-2 protein distribution in the kidneys of neonatal, 13-d-old, 22-d-old, and 6-wk-old rats was immunohistochemically analyzed, and NaPi-2 mRNA distribution in neonatal and 6-wk-old rats was analyzed by in situ hybridization. In kidneys of newborn rats, the appearance of NaPi-2 protein and mRNA coincided with the development of the brush border (assessed by actin staining) on proximal tubular cells. NaPi-2 was not detectable in the nephrogenic zone or in the outgrowing straight sections of proximal tubules, which lack a brush border. In 13-d-old suckling rats, strong NaPi-2 staining was seen in the BBM of convoluted proximal tubules of all nephron generations. In contrast, in 22-d-old weaned rats, NaPi-2 staining in the BBM of superficial nephrons was weaker than that in the BBM of juxtamedullary nephrons. Western blotting demonstrated that the overall abundance of NaPi-2 protein in the BBM of 22-d-old rats was decreased to approximately 70% of that in 13-d-old rats. In kidneys of 6-wk-old rats, the internephron gradient for NaPi-2 abundance in the BBM corresponded to that in adult rats. The data suggest that the NaPi-2 system in the kidney is fully functional and possesses the capacity for regulation as soon as nephrogenesis is completed. The manifestation of NaPi-2 internephron heterogeneity immediately after weaning might be related to the change in dietary inorganic phosphate content.

Animals↗
PubMed1998

Postnatal maturation of renal cortical peritubular fibroblasts in the rat.

The stromal cells in the renal cortex and medulla of adult rats reveal different phenotypes. Cortical peritubular fibroblasts are ecto-5'nucleotidase (5'NT)-positive and lack alpha-smooth muscle actin (alphaSMA) and vimentin immunoreactivity, whereas medullary fibroblasts are 5'NT-negative and vimentin-positive. We have studied by immunohistochemistry the postnatal (neonatal up to 8 weeks) development of renal cortical stromal cells with respect to 5'NT and to the cytoskeletal proteins alphaSMA and vimentin. Both alphaSMA and vimentin are characteristic for the renal myofibroblasts that replace stromal fibroblasts in interstitial nephritis. In new-born and 1-week-old rats, stromal cells in the cortex and medulla display alphaSMA and vimentin, but lack 5'NT. During the second postnatal week, alphaSMA and vimentin immunoreactivity in cortical interstitial cells gradually declines, whereas 5'NT reactivity becomes progressively apparent between the convoluted tubules in the juxtamedullary labyrinth. For a short time, all three proteins are found to be coexpressed in the same cells. At the end of the third week, interstitial 5'NT-immunoreactivity becomes evident also in the superficial cortical labyrinth, and alphaSMA and vimentin are no longer detectable in cortical peritubular cells. From the fourth week on, the distribution pattern and phenotype of 5'NT-positive cortical fibroblasts correspond to that in adult rats. The temporal pattern of maturation of cortical peritubular fibroblasts seems to parallel the functional maturation of cortical tubules. It is suggested that the local phenotype of peritubular fibroblasts in healthy and possibly also in injured kidneys may be controlled, at least in part, by the local tubular environment, conditioned by tubular metabolism and function.

5'-Nucleotidase↗
PubMed1998

Parathyroid hormone and dietary phosphate provoke a lysosomal routing of the proximal tubular Na/Pi-cotransporter type II.

BACKGROUND: A decrease of proximal tubular reabsorption of phosphate (Pi), which can be provoked by parathyroid hormone (PTH) or by a high Pi-diet, has been shown to correlate with a decrease of the number of type II Na/Pi-cotransporters residing in the brush border membrane. While both PTH and a high Pi-diet lead to an internalization of type II cotransporters, the further cellular routing of internalized cotransporters has not been established unequivocally. METHODS: To prevent lysosomal degradation, rats were treated with leupeptin prior to the injection of PTH or feeding acutely with a high Pi-diet. Kidney cortex were recovered and used for immunohistochemistry. In parallel, brush border membranes and lysosomes were isolated and analyzed by Western blotting. RESULTS: Under both conditions (PTH and high Pi-diet), a strong overlap of internalized type II cotransporters with the late endosomes/lysosomes was observed by immunohistochemistry. In agreement, the content of type II Na/Pi-cotransporters was increased in lysosomes isolated from the corresponding tissues. CONCLUSIONS: These results suggest that in proximal tubular cells type II Na/Pi-cotransporters internalized due to the action of PTH and acute high Pi-diet are routed to the lysosomes, and likely do not enter a recycling compartment.

Animals↗
PubMed1998

Immunocytochemical localization of alpha2,3(N)-sialyltransferase (ST3Gal III) in cell lines and rat kidney tissue sections: evidence for golgi and post-golgi localization.

Sialylation is a biosynthetic process occurring in the trans compartments of the Golgi apparatus. Corresponding evidence is based on localization and biochemical studies of alpha2, 6(N)-sialyltransferase (ST6Gal I) as previously reported. Here we describe generation and characterization of polyclonal antibodies to recombinant rat alpha2,3(N)-sialyltransferase (ST3Gal III) expressed as a soluble enzyme in Sf9 cells or as a beta-galactosidase-human-ST3Gal III fusion-protein from E.coli , respectively. These antibodies were used to localize ST3Gal III by immunofluorescence in various cell lines and rat kidney tissue sections. In transiently transfected COS cells the antibodies directed to soluble sialyltransferase or the sialyltransferase portion of the fusion-protein only recognized the recombinant antigen retained in the endoplasmic reticulum. However, an antibody fraction crossreactive with beta-galactosidase recognized natively expressed ST3Gal III which was found to be colocalized with beta1, 4-galactosyltransferase in the Golgi apparatus of several cultured cell lines. Antibodies affinity purified on the beta-galactosidase-ST3Gal III fusion-protein column derived from both antisera have then been used to localize the enzyme in perfusion-fixed rat kidney sections. We found strong staining of the Golgi apparatus of tubular epithelia and a brush-border-associated staining which colocalized with cytochemical staining of the H+ATPase. This subcellular localization was not observed for ST6Gal I which localized to the Golgi apparatus. These data show colocalization in the Golgi apparatus and different post-Golgi distributions of the two sialyltransferases.

Animals↗
PubMed1998

Renal Na/H exchanger NHE-3 and Na-PO4 cotransporter NaPi-2 protein expression in glucocorticoid excess and deficient states.

Administration of pharmacologic doses of glucocorticoid in vivo increases renal proximal tubule apical membrane Na/H exchange and decreases Na/PO4 cotransport activity (1). Current data suggest that the NHE-3 and NaPi-2 proteins mediate significant fractions of proximal tubule apical membrane Na/H exchange and Na/PO4 cotransport, respectively. This study examines whether glucocorticoid excess or deficiency affects NHE-3 and NaPi-2 protein abundance and the intrarenal distribution of these transporters. Protein abundance of NHE-3 and NaPi-2 in control rats was compared to rats rendered glucocorticoid-deficient by bilateral adrenalectomy, and to rats receiving pharmacologic doses of dexamethasone using immunoblots and immunohistochemistry. Adrenalectomy had modest effects on NHE-3 protein abundance, but dexamethasone administration to either adrenalectomized or sham-operated rats significantly increased NHE-3 protein abundance in both the proximal tubule and thick ascending limb, but not the thin descending limb. Adrenalectomy increased NaPi-2 protein abundance in the proximal tubule, whereas dexamethasone administration dramatically suppressed NaPi-2 protein on the apical membrane in both adrenalectomized and sham-operated animals. No significant reciprocal increase in subapical NaPi-2 staining was seen in the dexamethasone-treated rats. The present study shows that glucocorticoids regulate proximal tubule apical membrane Na/H exchange and NaPi cotransport by changes in protein abundance of NHE-3 and NaPi-2, respectively.

Adaptor Protein Complex 3↗
PubMed1998

Enhanced osteopontin expression and macrophage infiltration in MRL-Fas(lpr) mice with lupus nephritis.

MRL-Fas(lpr) mice spontaneously develop a chronic lupus-like renal disease, characterized by immune complex-mediated glomerulonephritis and abundant mononuclear cell infiltration in the interstitium. In the present study we have examined whether the macrophage chemoattractant osteopontin (Opn) could be important in the recruitment of macrophages in this murine model of autoimmune renal injury. We have examined the expression of Opn in the kidney of MRL-Fas(lpr) mice and have correlated Opn synthesis with the degree of macrophage infiltration. Immunofluorescence staining revealed prominent expression of Opn by proximal tubules in MRL-Fas(lpr) mice but not in MRL-++ control mice. Northern blot analysis demonstrated that steady-state transcript levels for Opn mRNA were also significantly increased in MRL-Fas(lpr) kidneys compared with control kidneys. Furthermore, in situ hybridization showed massive Opn mRNA transcripts in proximal tubules in MRL-Fas(lpr) mice but not in controls. The diffuse macrophage infiltration in the kidney of MRL-Fas(lpr) correlated with the enhanced Opn expression. Opn secretion in vitro by cultured renal tubular epithelial cells was upregulated by TNF-alpha and 1,25(OH)2-vitamin D3, whereas no regulation was observed in a control macrophage cell line. We conclude that the enhanced expression of the chemotactic molecule Opn by tubular cells is a prominent feature of murine lupus nephritis and might be promoted by the proinflammatory cytokine environment in MRL-Fas(lpr). The chronic upregulation of Opn could participate in the recruitment of monocytes in the kidney of MRL-Fas(lpr) mice, thereby contributing to the pathogenesis of autoimmune renal disease.

Animals↗
PubMed1998

Cellular/molecular control of renal Na/Pi-cotransport.

A type II Na/Pi-cotransporter located in the brush border membrane is the rate limiting and physiologically regulated step in proximal tubular phosphate (Pi) reabsorption. In states of altered Pi-reabsorption [for example, in response to parathyroid hormone (PTH) and to altered dietary intake of Pi or as a consequence of genetic abnormalities], brush border expression of the type II Na/Pi-cotransporter is accordingly modified. PTH initiates a regulatory cascade leading to membrane retrieval, followed by lysosomal degradation of this transporter; recovery from inhibition requires its de novo synthesis. Pi-deprivation leads to an increased brush border expression of transporters that does not appear to require de novo synthesis in the short term. Pi-overload leads to membrane retrieval and degradation of transporters. Finally, in animals with genetically altered Pi-handling (Hyp; Gy) the brush border membrane expression of the type II Na/Pi-cotransporter is also reduced, suggesting that a genetically altered protein (such as PEX in Hyp) controls the expression of this transporter.

Animals↗
PubMed1997

Role of microtubules in the rapid regulation of renal phosphate transport in response to acute alterations in dietary phosphate content.

Renal proximal tubular response to acute administration of a low Pi diet is characterized by a rapid adaptive increase in apical brush border membrane (BBM) Na-Pi cotransport activity and Na-Pi cotransporter protein abundance, independent of a change in Na-Pi cotransporter mRNA levels (Levi, M., M. Lötscher, V. Sorribas, M. Custer, M. Arar, B. Kaissling, H. Murer, and J. Biber. 1994. Am. J. Physiol. 267: F900-F908). The purposes of the present study were to determine if the acute adaptive response occurs independent of de novo protein synthesis, and if microtubules play a role in the rapid upregulation of the Na-Pi cotransporters at the apical BBM. We found that inhibition of transcription by actinomycin D and translation by cycloheximide did not prevent the rapid adaptive response. In addition, in spite of a 3.3-fold increase in apical BBM Na-Pi cotransporter protein abundance, there was no change in cortical homogenate Na-Pi cotransporter protein abundance. Pretreatment with colchicine, which resulted in almost complete disruption of the microtubular network, abolished the adaptive increases in BBM Na-Pi cotransport activity and Na-Pi cotransporter protein abundance. In contrast, colchicine had no effect on the rapid downregulation of Na-Pi cotransport in response to acute administration of a high Pi diet. We conclude that the rapid adaptive increase in renal proximal tubular apical BBM Na-Pi cotransport activity and Na-Pi cotransporter abundance is independent of de novo protein synthesis, and is mediated by microtubule-dependent translocation of presynthesized Na-Pi cotransporter protein to the apical BBM.

Adaptation, Physiological↗
PubMed1997

Beta-adrenergic stimulation of renin expression in vivo.

OBJECTIVE: To characterize the effect of beta-adrenergic activation on renal production of renin in the intact organism. DESIGN AND METHODS: Renal expression of renin [renin messenger RNA (mRNA)], plasma renin activity (PRA), inactive renin level, intrarenal renin distribution (immunohistochemistry), and the time course of activation of renin as well as hemodynamic parameters were determined during the subcutaneous infusion of isoproterenol (ISO) into rats. To examine whether beta-adrenergic activation of the renin system is modulated by the rate of salt intake rats were fed diets with normal, low and high salt contents. RESULTS: Systolic blood pressure was not altered. PRA was elevated as much as fourfold after 40 h of ISO infusion. Although renal renin mRNA levels were elevated dose-dependently up to 4.2-fold, no significant recruitment of renin-containing cells could be detected. The time course of PRA revealed a marked transient rise of PRA during 6 h of ISO infusion with a subsequent decline. Inactive renin level was elevated during 3 to 18 h of ISO infusion. In contrast, renin mRNA level increased steadily with a lag phase of 3 h. Infusion of ISO increased PRA and renin mRNA level under a high-salt diet, but had no additional effect either on PRA or on renin mRNA level under low-salt diet. CONCLUSION: Activation of beta-adrenergic receptors is a powerful stimulus of renin secretion and renin gene expression in juxtaglomerular cells in vivo, albeit the kinetics of upregulation of renin secretion and renin expression are markedly different. Therefore, the sympathetic tone might be a major factor determining the activity of the renin system in vivo. The ability of adrenergic agonists to stimulate the renin system appears to be modulated by the steady-state level of salt intake.

Adrenergic beta-Antagonists↗
PubMed1996

Morphology of interstitial cells in the healthy kidney.

Renal interstitial cells play an important role in renal function and renal diseases. We describe the morphology of renal interstitial cells in the healthy kidney. We distinguish within the renal interstitium (1) renal fibroblasts and (2) cells of the immune system. Fibroblasts are in the majority and constitute the scaffold of the kidney; they are interconnected by junctions, and are attached to tubules and vessels. Although the phenotype of fibroblasts shows some variation depending on their location in the kidney and on their functional stage, their recognition as fibroblasts is possible on account of structural features. Among the cell types of the second group, antigen-presenting dendritic cells are the most abundant in in the peritubular interstitial spaces of healthy kidneys. Their incidence is highest in the inner stripe of the outer medulla. They share some morphological features with fibroblasts but lack others--junctional complexes, morphologically defined connections with tubules and vessels, and the prominent layer of actin filaments under the plasma membrane--that are characteristic for fibroblasts. Dendritic cells in healthy kidneys are morphologically different from macrophages, which are characterized by abundant primary and secondary lysosomes. In healthy kidneys macrophages are restricted to the connective tissue of the renal capsule and the pelvic wall, and to the periarterial connective tissue. Lymphocytes are rare in healthy kidneys. The distinction of cell types by morphology is supported by differences of membrane proteins. Among all interstitial cells in the renal cortex, fibroblasts alone exhibit ecto-5'-nucleotidase. Dendritic cells constitutively have a high abundance of MHC class II protein. Both proteins are mutually exclusive. Rat macrophages display the membrane antigen ED 2 and lymphocytes exhibit specific surface antigens, depending on their type and functional stage, e.g., CD4 or CD8.

5'-Nucleotidase↗
PubMed1996

Immunolocalization of Na/SO4-cotransport (NaSi-1) in rat kidney.

The proximal tubule is the major site for renal reabsorption of sulphate. A sodium-dependent transport system for sulphate (NaSi-1) has recently been identified from a rat kidney cortex cDNA library. Recent work demonstrated that NaSi-1 mRNA is expressed predominantly in proximal tubules. In the present work expression along the nephron of the Na/SO4-cotransporter NaSi-1 was studied by immunofluorescence. A polyclonal antibody was raised in rabbits against a fusion protein containing a 53-amino-acid polypeptide specific for the NaSi-1 sequence. The anti-NaSi-1 polyclonal antibody specifically detected a 68-kDa protein on Western blots and, by immunofluorescence specific staining, was observed in MDCK cells transfected with the NaSi-1 cotransporter. Using rat kidney cortex slices specific NaSi-1-related immunoreactivity was detected in proximal tubules and was restricted to the apical membrane. No immunoreactivity was observed in the other nephron segments. This was confirmed by Western blot analysis using proximal tubular apical and basolateral membranes isolated by free-flow electrophoresis. The results indicate that the Na/SO4-cotransporter NaSi-1 is expressed in the apical membrane of proximal tubular cells and is therefore likely to be involved in proximal reabsorption of sulphate.

Animals↗
PubMed1996

Renal Na/Pi-cotransporters.

Two non-homologous proximal tubular apical Na/Pi-cotransport systems (type I and type II) have been identified thus far by expression cloning. Subsequent studies provided evidence that the type II Na/Pi-cotransporter represents a target for the physiological and pathophysiological regulation of proximal reabsorption of phosphate. The exact role of the type I Na/Pi-cotransporter in proximal Pi-reabsorption and eventually also in the renal handling of other substrates, such as organic anions, is currently less clear and needs further investigation. Evidence was obtained that acute changes of brush border membrane Na/Pi-cotransport involves endo- and exocytic movement of type II Na/Pi-cotransporters. In particular, we elucidated if and how phosphorylation reactions are involved and defined the intracellular structures of the endo/exocytic apparatus involved. At the level of the gene it will be necessary to elucidate its organization in order to understand the mechanisms involved in chronic regulations of Na/Pi-cotransport related to the type II Na/Pi-cotransporter. Furthermore, for structural investigations of these integral membrane proteins, they have to be isolated in sufficient quantities. Thus far the type II cotransporter (NaPi-2) has been expressed in Sf9 insect cells [20], which may eventually allow a purification of this protein.

Animals↗
PubMed1996

Renal brush border membrane Na/Pi-cotransport: molecular aspects in PTH-dependent and dietary regulation.

Inorganic phosphate (Pi) is reabsorbed in renal proximal tubules in a sodium (Na)-dependent manner involving brush border Na/Pi-cotransporter(s). Regulation of renal Pi-reabsorption, such as by parathyroid hormone (PTH) and/or by dietary Pi-deprivation, involves alterations in the rate of Na/Pi-cotransport. Two structurally different Na/Pi-cotransporters have been identified: type I-transporter and type II-transporter. The related mRNAs and proteins are located in the proximal tubule and in the brush border membrane. In heterologous expression systems type I and type II Na/Pi-cotransporters mediate Na/Pi-cotransport. Characterization of the transport properties suggested that the type II transporter is "responsible' for brush border membrane Na/Pi-cotransport (as observed in isolated vesicles). Administration of PTH to rats resulted in an inhibition of brush border membrane Na/Pi-cotransport (vesicles) and in a reduced brush border membrane content of the type II transporter. Feeding low Pi-diets resulted in an up-regulation of Na/Pi-cotransport (vesicles) and of type II transporter content; only after a prolonged exposure to low Pi-diets (more than 4 hr) was an increase in specific mRNA content observed. Refeeding high Pi diets had the opposite effects on Na/Pi-cotransport activity and on type II transporter protein. It is currently the task of future experiments to define the specific mechanisms leading to protein-synthesis-independent (PTH, acute Pi-deprivation, Pi-refeeding) and to protein-synthesis-dependent (prolonged Pi-deprivation) regulation of the type II Na/Pi-cotransporter.

Animals↗