A novel mouse model of podocyte depletion.

AIM: The goal of this study was to examine the capacity for glomerular repair after a podocyte-depleting injury. METHODS: We created transgenic (TG) mice expressing the yeast enzyme cytosine deaminase specifically in glomerular podocytes. In these TG animals, the prodrug 5-flucytosine (5-FC) is converted to 5-fluorouracil and promotes cell death. RESULTS: Treatment with increasing dosages of 5-FC caused graded increases in proteinuria 1-2 weeks after treatment, which returned to control levels by the 10-week time point. Light microscopic examination revealed minimal pathology at the 2-week time point, but electron microscopy revealed found foot process effacement as well as focal areas of glomerular basement membrane duplication, and immunohistochemical studies detected podocyte apoptosis and a decrease in the number of Wilms' tumor protein 1 (WT1)-positive cells. By the 10-week time point, however, the number of WT1-positive cells was similar to controls and a few mice had developed focal areas of glomerulosclerosis. Consistent with the effects of 5-FC on podocyte number, expression of the podocyte mRNAs for nephrin, podocin, synaptopodin and podocalyxin were altered in a similar temporal fashion. CONCLUSION: The glomerulus has a significant capacity for repair after a podocyte-depleting injury.

Structure-function of recombinant Na/H exchanger regulatory factor (NHE-RF).

Inhibition of the renal brush border membrane (BBM) Na/H exchanger by cAMP-dependent protein kinase, PKA, requires participation of a recently cloned regulatory cofactor, Na/H exchanger-regulatory factor (NHE-RF). As deduced from the cDNA of this 358-amino acid protein, amino acids 11-101 and amino acids 150-241 of the NHE-RF protein share 74% overall homology suggesting duplication of these PDZ containing domains. The serine residues at amino acid position 289 and 340 are considered to be the most likely sites for PKA mediated phosphorylation. To study the structure- function relation between NHE-RF and PKA mediated inhibition of the rabbit BBM Na/H exchanger, the effect of recombinant proteins representing full-length NHE-RF as well as truncated and mutant forms of NHE-RF were determined using a reconstitution assay. The reconstitution assay employed a fraction of rabbit BBM proteins that contains Na/H exchanger activity that is not regulated by PKA. NHE-RF in the presence of ATP and Mg but not PKA, inhibited Na/H exchange activity in a concentration-dependent manner. In the presence of PKA, there was a significant left shift in the dose-response relation such that 10(-12) M NHE-RF inhibited Na/H exchange transport by 30% in the presence but not in the absence of PKA. A recombinant polypeptide representing amino acids 1-151 (Domain I) did not affect Na/H exchange transport in the presence or absence of PKA. A polypeptide representing amino acids 149-358 (Domain II) in the presence of ATP and Mg but not PKA, inhibited Na/H exchange activity in a concentration-dependent manner. In the presence of PKA, there was a left shift in the dose-response relation. 10(-12) M of Domain II polypeptide inhibited transport by 18% in the presence but not in the absence of PKA. Mutation of serine residues 287, 289, and 290 to alanine did not affect the inhibitory effect in the absence of PKA but abolished the left shift in the dose-response relation elicited by PKA. Mutation of serine residues 339 and 340 to alanine were without effect on PKA dependent regulation of Na/H exchange transport. These studies indicate that NHE-RF inhibits basal rabbit renal BBM Na/H exchange activity-an effect which is augmented by PKA. The amino acid sequences in the polypeptide containing only the NH2-terminal PDZ domain of NHE-RF have no intrinsic activity as an inhibitor but appears to be required for the full-length NHE-RF to express its full inhibitory effect on the BBM Na/H exchanger. One or more of the serine residues at positions 287, 289, and/or 290 represent the critical PKA phosphorylation site(s) on the NHE-RF protein that mediates the physiologic effect of cAMP on the renal BBM Na/H exchanger.

Lambda light chain deposition disease in a renal allograft.

Light chain deposition disease (LCDD) of the kidney is characterized by deposition of monoclonal light chains predominantly in glomeruli and in tubular basement membranes. The disease is frequently associated with a lymphoproliferative disorder, and the majority of cases are caused by deposition of kappa light chains. Although the occurrence of de novo multiple myeloma after renal transplantation is uncommon, there are several reports of LCDD involving renal allografts, either de novo or in patients with a diagnosis of LCDD prior to transplantation. To the best of our knowledge, all previously described cases in allografts have been in patients with kappa chain deposition. The relative importance of intrinsic properties of the kidney in predisposing to either kappa or lambda light chain deposition is not known. We present a case of LCDD caused by deposition of lambda light chains in a patient who received a cadaveric renal transplant.

Differential regulation of receptor-stimulated cyclic adenosine monophosphate production by polyvalent cations in MC3T3-E1 osteoblasts.

Extracellular cations have paradoxical trophic and toxic effects on osteoblast function. In an effort to explain these divergent actions, we investigated in MC3T3-E1 osteoblasts if polyvalent cations differentially modulate the agonist-stimulated cyclic adenosine monophosphate (cAMP) pathway, an important regulator of osteoblastic function. We found that a panel of cations, including gadolinium, aluminum, calcium, and neomycin, inhibited prostaglandin E1 (PGE)-stimulated cAMP accumulation but paradoxically potentiated parathyroid hormone (PTH)-stimulated cAMP production. In contrast, these cations had no effect on forskolin- or cholera toxin-induced increases in cAMP, suggesting actions proximal to adenylate cyclase and possible modulation of receptor interactions with G proteins. Phorbol 12-myristate 13-acetated (PMA) mimicked the effects of cations on PGE1- and PTH-stimulated cAMP accumulation in MC3T3-E1 cells, respectively, diminishing and augmenting the responses. Moreover, down-regulation of protein kinase C (PKC) by overnight treatment with PMA prevented gadolinium (Gd3+) from attenuating PGE1- and enhancing PTH-stimulated cAMP production, indicating involvement of PKC-dependent pathways. Cations, however, activated signal transduction pathways not coupled to phosphatidylinositol-specific phospholipase C (PI-PLC), since there was no corresponding increase in inositol phosphate formation or intracellular calcium concentrations. In addition, pertussis toxin treatment failed to prevent Gd(3+)-mediated suppression of PGE1-stimulated cAMP, suggesting actions independent of Gm. Thus, polyvalent cations may either stimulate or inhibit hormone-mediated cAMP accumulation in osteoblasts. These differential actions provide a potential explanation for the paradoxical trophic and toxic effects of cations on osteoblast function that occur in vivo under different hormonal conditions.

Effect of receptor number on desensitization of the mouse thromboxane receptor.

Desensitization of G-protein coupled receptors limits the physiologic effects of an agonist. Short-term desensitization mechanisms are critically dependent on receptor phosphorylation by protein kinases. The effectiveness of these regulatory mechanisms might be limited by substrate (receptor) availability. To investigate the role of receptor number in the desensitization of G-protein coupled receptors, we transfected a mouse mesangial cell line with a genomic clone encoding the mouse thromboxane A2 (TxA2) receptor and obtained cell lines that expressed low (approximately 250-500 fmol/mg protein) or high (2500-4000 fmol/mg protein) levels of TxA2 receptors. Activation of TxA2 receptors stimulated phosphoinositide (PI) hydrolysis and increased intracellular calcium ([Ca2+]i) levels. Prior exposure to the TxA2 agonist (15S)-hydroxy-11alpha,9alpha-(epoxymethano)prosta-5Z,+ ++13E-dienoic acid (U46619) reduced subsequent (15S)-hydroxy- 11alpha,9alpha-(epoxymethano)prosta-5Z,13E-dieno ic acid-induced increases in inositol trisphosphates and intracellular calcium levels by approximately 50% in clones expressing low numbers of TxA2 receptors, but had little effect on TxA2 receptor responsiveness in clones expressing high receptor numbers. Failure of TxA2 receptors to desensitize caused sustained increases in intracellular calcium levels and phosphoinositide hydrolysis. Thus, homologous desensitization of TxA2 receptors is attenuated in cells expressing high levels of receptors for TxA2. These data suggest that receptor number plays a key role in the short-term regulation of G-protein coupled receptors.

Domains of the parathyroid hormone (PTH) receptor required for regulation by G protein-coupled receptor kinases (GRKs).

To investigate the domains of the parathyroid hormone (PTH) receptor required for regulation by G protein-coupled receptor kinases (GRKs), we created mutant PTH receptors lacking potential GRK-phosphorylation sites. Mutant #1 was truncated at amino acid 544 and, therefore, lacked nine hydroxyl group-containing amino acids at the C-terminus. In mutant #2, we replaced threonines 392 and 399 in the third intracellular loop with glycines. Co-transfection of HEK293 cells with the wild-type receptor and either GRK2, GRK3, or GRK5 inhibited PTH-induced cyclic (cAMP) generation; co-transfection of GRK4 or GRK6 had no effect on PTH receptor responsiveness. GRK2-mediated inhibition of PTH receptor signaling was associated with enhanced phosphorylation receptor proteins. Co-expression of GRK2 similarly reduced PTH-induced cAMP generation by the wild-type receptor and mutant #1, and caused phosphorylation of receptor proteins to a similar extent. Co-expression of GRK2 had little effect on PTH-induced cAMP generation by mutant #2 but enhanced agonist-induced phosphorylation of mutant #2 compared with that of either the wild-type receptor or mutant #1. Enhanced phosphorylation of mutant #2 was associated with a reduction in agonist-induced internalization of mutant #2 compared with the wild-type receptor. Thus, phosphorylation of mutant #2 failed to cause receptor desensitization and inhibited receptor internalization. These data are consistent with the notion that: (a) GRKs contribute to regulating PTH receptor responsiveness, and (b) domains in the third intracellular loop are not required for agonist-induced phosphorylation of PTH receptors, but are critical for both agonist-induced internalization of PTH receptors and GRK2-mediated regulation of PTH receptor signaling.

The Prostaglandin E(1) (PGE(1)) Analog Misoprostol Ameliorates Autoimmune Disease and Depletes T Lymphocytes in MRL-lpr/lpr Mice.

MRL-lpr/lpr mice spontaneously develop an autoimmune disease similar to human systemic lupus erythematosus (SLE). Disease manifestations include anti-DNA autoantibody production, arthritis, vasculitis, and an immune-complex glomerulonephritis. The development of autoimmune disease is associated with massive, generalized lymphadenopathy caused by accumulation of an abnormal T-cell population in peripheral lymphoid organs. In MRL-lpr/lpr mice, treatment with E-series prostaglandins ameliorates renal disease and reduces peripheral lymphadenopathy. Little is known about mechanisms of action of E-series prostaglandins in murine lupus or the effects of these agents on other clinically important manifestations of SLE, such as arthritis and vasculitis. To further investigate the effects of an E-series prostaglandin in murine SLE, we administered the prostaglandin, E(1) (PGE(1)) analog misoprostol (1 mg kg(minus sign1) day(minus sign1)) to MRL-lpr/lpr mice for 8 weeks by twice daily subcutaneous injection. At 20 weeks of age, treatment with misoprostol reduced the severity of renal disease and arthritis but did not affect the extent or severity of vasculitis. The beneficial effects of misoprostol on arthritis and renal disease were associated with a significant decrease in splenic and lymph node weight in mice given the PGE(1) analog. This decrease in lymphoproliferation resulted primarily from a generalized reduction in the number of T cells in peripheral lymphoid organs. Thus, T-cell depletion was associated with beneficial effects on arthritis and nephritis in MRL-lpr/lpr mice, supporting a role for T lymphocytes in these disease processes. The ability of E-series prostaglandins to favorably modify autoimmune disease in this murine model suggests that misoprostol may be a useful adjunct to current therapies for the treatment of patients with SLE.

Immature omental teratoma.

Immature omental teratomas are extremely rare neoplasms. To our knowledge, only two cases have been reported in the literature. In some anatomic locations, the malignant potential of immature teratomas correlates with the presence and quantity of neuroectoderm within the tumor mass. We describe the first immature omental teratoma with prominent neuroectodermal differentiation.

Regulated expression of G protein-coupled receptor kinases (GRK's) and beta-arrestins in osteoblasts.

Desensitization of G-protein coupled receptors (GPCR's) is largely mediated by a family of enzymes and protein co-factors termed GRKs and arrestins, respectively. In the present studies, we investigated expression of GRKs and arrestins in osteoblastic cell lines concentrating on the enzymes (GRK2 and GRK3) and protein co-factors (beta-arrestint 1 and beta-arrestin 2) that play dominant roles in regulating GPCR responsiveness in most tissues and cell types. We found that osteoblastic cells express similar amounts of GRK2 with either undetectable or lesser amounts of GRK3. In contrast, expression of beta-arrestin 1 and beta-arrestin 2 by osteoblastic cells varied between cell lines. To determine if GRK2 or beta-arrestin expression is modulated during osteoblast development, we assessed expression of GRK2 and beta-arrestin proteins during differentiation of the mouse osteoblastic cell line MC3T3-E1 cells over a 21-day period. We found that expression of GRK2 and beta-arrestin 2 increased to maximal levels by day 7 and then decreased 4-fold by day 21. In contrast, expression of beta-arrestin 1 increased to maximal levels by day 14 and then decreased 2-fold by day 21. Over this same time period (days 7-21), PTH/PTHrP receptor number decreased to a greater extent than the decrease in PTH(1-34)-induced cAMP generation, suggesting that responsiveness of individual PTH/PTHrP receptors was enhanced in differentiated cells. We conclude that (1) osteoblastic cell lines differentially express the enzymes and protein co-factors that modulate GPCR responsiveness and (2) expression of both GRK2 and beta-arrestins is temporally regulated during osteoblast development. These data are consistent with the notion that GPCR responsiveness may be differentially regulated in osteoblastic cell lines and during osteoblast development.

Regulation of thromboxane receptor (TP) phosphorylation by protein phosphatase 1 (PP1) and PP2A.

To investigate the protein phosphatases that dephosphorylate TP, human embryonic kidney cells (HEK293 cells) stably transfected with 12CA5-tagged TP were treated with TP agonist, washed, and then allowed to recover in the presence or absence of the cell-permeable PP1 and PP2A inhibitors calyculin or okadaic acid (OKA). After recovery, cells were rechallenged with TP agonist and TP responsiveness was assessed by measuring inositol trisphosphate generation. TP responsiveness recovered over a 20-min time period. Recovery of TP responsiveness was inhibited by calyculin and OKA and was associated with dephosphorylation of receptor proteins. To further identify the TP phosphatase, TP phosphorylated in the intact cell were isolated by immunoprecipitation and were used as substrate for protein phosphatases prepared from HEK293 cells. TP were dephosphorylated by whole-cell homogenates. Dephosphorylation of TP was completely inhibited by the PP1 and PP2A inhibitors calyculin and microcystin-LR, suggesting that the decrease in TP phosphorylation was not due to receptor degradation. TP phosphatase activity was partially blocked by 1) inhibitor 2, a specific protein inhibitor of PP1; and 2) OKA at concentrations (1 nM) that specifically inhibit PP2A. TP phosphatase activity did not have an absolute requirement for divalent cations and was found primarily in cytosolic fractions of the cell. These data suggest that PP1- and PP2A-like protein phosphatases dephosphorylate TP. By regulating the phosphorylation state of TP, protein phosphatases may modulate tissue responsiveness to thromboxane.

Role of C-terminal serines in desensitization and phosphorylation of the mouse thromboxane receptor.

To investigate the role of C-terminal hydroxyamino acids in desensitization of the receptor for thromboxane A2 (TxA2), we created a mutant TxA2 receptor (TP receptor) in which serines at positions 321, 322, and 328 were replaced with either alanine or glycine. Mutant and wild type receptors were expressed in a mesangial cell line, and clones expressing similar numbers of receptors were studied. Affinity and specificity of TxA2 binding to the mutant receptor were identical to wild type receptors. In contrast, TxA2-induced inositol trisphosphate generation by the mutant receptor was enhanced compared with the wild type. Prior treatment with the TxA2 agonist U46619 reduced subsequent U46619-induced increases in inositol trisphosphate generation by both receptors; however, the extent of desensitization was significantly reduced in the receptor mutant. Protein kinase C (PKC) inhibitors attenuated TxA2-induced desensitization of wild type receptors, but had little effect on TxA2-induced desensitization of mutant receptors. Pretreatment with the phorbol ester phorbol 12, 13-dybutyrate (PDBu) (100 nM) decreased subsequent responsiveness of wild type but not mutant TP receptors. -induced desensitization of wild type receptors was associated with enhanced phosphorylation of receptor proteins. This agonist-specific phosphorylation of the TP receptor was largely prevented by inhibitors of PKC. Treatment with 100 nM PDBu increased phosphorylation of both wild type and mutant TP receptors, but the extent of phosphorylation of the receptor mutant was reduced compared with the wild type. Increasing the concentration of PDBu from 100 nM to 1 microM PDBu reduced responsiveness of both mutant and wild type receptors without enhancing phosphorylation of either of the receptor proteins. These data suggest that 1) phosphorylation of C-terminal serines contributes to agonist-specific desensitization of the TP receptor, 2) PKC-induced desensitization of TP receptors is caused, in part, by phosphorylation of C-terminal serines, and 3) desensitization of TP receptors by PKC is complex and involves mechanisms that may not require direct phosphorylation of receptor proteins.

The C-terminus of the thromboxane receptor contributes to coupling and desensitization in a mouse mesangial cell line.

To investigate regulatory domains of the thromboxane A2 (TxA2) receptor, we constructed a truncated form of the mouse TxA2 receptor and expressed it in a mesangial cell line. The mutant receptor lacked 22 amino acids in the C-terminus including four potential phosphorylation sites. Ligand binding of mutant receptors was identical with the wild type. Stimulation with TxA2 agonist induced increases in inositol trisphosphate (IP3) generation and [Ca++]i by both wild-type and mutant receptors. However, the initial increase in IP3 generation by the mutant receptor was only approximately 50% of that seen in the wild type. Exposure of wild-type receptors to TxA2 agonist caused desensitization of IP3 and calcium responses. Pretreatment with TxA2 agonist caused some desensitization of mutant receptors, but the extent of desensitization was reduced compared with the wild type. The protein kinase C inhibitor staurosporine attenuated TxA2-induced desensitization of wild-type receptors, but had little effect on TxA2-induced desensitization of mutant receptors. Pretreatment with low concentrations of the phorbol ester, phorbol 12,13-dibutyrate (100 nM), reduced subsequent responsiveness of wild-type but not mutant TxA2 receptors. In contrast, high-dose phorbol 12,13-dibutyrate (1 microM) produced a similar degree of desensitization of both receptor types. These data suggest that: 1) the C-terminus participates in coupling of the TxA2 receptor to its effector systems; 2) the C-terminus contributes to agonist-specific desensitization of the TxA2 receptor; and 3) protein kinase C-induced desensitization of the TxA2 receptor is complex and depends, in part, on C-terminal domains of the TxA2 receptor.

Acute renal failure in critically ill patients: prognosis for recovery of kidney function after prolonged dialysis support.

OBJECTIVE: To clarify the prognosis for eventual recovery of kidney function in patients who experience prolonged dialysis dependence after acute renal failure (ARF). DESIGN: Retrospective, chart review. SETTING: Inpatients of a large, referral-based hospital. PATIENTS: Twenty-six consecutive survivors of ARF who required greater than 4 wk of dialysis support. RESULTS: All 26 patients were critically ill and developed ARF during treatment in an ICU. The clinical course of these patients was characterized by multiple episodes of renal ischemia or nephrotoxin exposure during dialysis dependence. However, despite multiple renal insults and prolonged dialysis support (mean duration 8.4 +/- 0.7 wk), 23 (88%) of the 26 patients recovered sufficient kidney function to discontinue dialysis. Preexisting renal impairment was associated with a greater risk of irreversible renal failure, and, in patients able to discontinue dialysis, renal recovery was often incomplete. CONCLUSIONS: Despite some renal damage, most critically ill patients who survive ARF requiring prolonged dialysis support recover life-sustaining kidney function.

Physiologic role for enhanced renal thromboxane production in murine lupus nephritis.

To investigate the physiologic significance of enhanced renal thromboxane production in murine lupus nephritis, we measured renal hemodynamics and eicosanoid production in MRL-lpr/lpr mice from 8 to 20 weeks of age. Over this age range, MRL-lpr/lpr mice develop an autoimmune disease with nephritis similar to human systemic lupus erythematosus (SLE). In these studies, glomerular filtration rate (GFR) and PAH clearance (CPAH) decreased progressively with age in MRL-lpr/lpr mice, but not in controls. This impairment of renal hemodynamics was associated with increased renal thromboxane production, as well as increased excretion of both thromboxane B2 (TxB2) and 2,3-dinor TxB2 in urine. There was an inverse correlation between renal thromboxane production in MRL-lpr/lpr mice and both GFR and CPAH. Furthermore, there were positive correlations between thromboxane production by the kidney and both the severity of renal histopathology and serum anti-DNA antibody levels measured in individual animals. Enhanced urinary excretion of TxB2 and the development of renal dysfunction also coincided temporally with the appearance of increased levels of interleukin 1 beta (IL-1 beta) mRNA in renal cortex. Acute administration of the specific thromboxane receptor antagonist GR32191 to MRL-lpr/lpr mice restored GFR to normal in early stages of the autoimmune disease. However, in animals with more advanced nephritis, the effect of acute thromboxane receptor blockade on renal hemodynamics was less marked. We conclude that thromboxane A2 is an important mediator of reversible renal hemodynamic impairment in murine lupus, especially in the early phase of disease.

Fish oil feeding modulates leukotriene production in murine lupus nephritis.

Diets enriched with fish oil (FO) ameliorate kidney disease in the MRL-lpr/lpr murine model of lupus nephritis. Although the mechanisms of this effect are not known, FO is rich in the polyunsaturated fatty acid eicosapentaenoic acid (EPA) which may have profound effects on eicosanoid metabolism. In MRL-lpr/lpr mice, FO feeding reduces renal production of cyclooxygenase metabolites. However, EPA may also affect the metabolism of arachidonate by the 5-lipoxygenase (5-LO) pathway and enhanced production of 5-LO metabolites has been implicated in the pathogenesis of kidney disease in MRL-lpr/lpr mice. We therefore investigated the effects of FO feeding on production of 5-LO metabolites in 20 week old MRL-lpr/lpr mice. After 8 weeks of dietary supplementation with FO, both renal hemodynamic function and glomerular histology were improved compared to safflower oil (SO) controls. Amelioration of kidney disease was associated with alterations in the pattern of leukotriene production by macrophages and kidneys from FO fed mice. There was a significant decrease in the production of leukotriene B4 (LTB4) and tetraene peptidoleukotrienes by peritoneal macrophages isolated from mice given FO compared to control animals. Similarly, dietary supplementation with FO decreased renal production of LTB4. Reduced production of tetraene leukotrienes was accompanied by a modest increase in the production of pentaene leukotrienes by macrophages from FO fed mice. We speculate that this modulation of leukotriene production by FO feeding may have beneficial effects on renal disease in autoimmune nephritis.

Analysis of recombinant Phex: an endopeptidase in search of a substrate.

X-linked hypophosphatemia (XLH) is caused by inactivating mutations of Phex, a phosphate-regulating endopeptidase. Further advances in our knowledge of the pathogenesis of XLH require identification of the biological function of Phex and its physiologically relevant substrates. We evaluated several potential substrates using mouse recombinant wild-type Phex proteins (rPhex-WT) and inactive mutant Phex proteins (rPhex-3'M) lacking the COOH-terminal catalytic domain as controls. By Western blot analysis, we demonstrated that Phex is a membrane-bound 100-kDa glycosylated monomer. Neither casein, a substrate for the related endopeptidase thermolysin, human stanniocalcin 1 (hSTC-1), an osteoblast-derived phosphate-regulating factor, nor FGF-23 peptide (amino acid 172-186), comprising the region mutated in autosomal dominant hypophosphatemia, was cleaved by rPhex-WT. In addition, membranes expressing rPhex-WT, rPhex-3'M, and the empty vector hydrolyzed parathyroid hormone-(1-34), indicating the lack of Phex-specific cleavage of parathyroid hormone. In contrast, rPhex-WT did display an EDTA-dependent cleavage of the neutral endopeptidase substrate [Leu]enkephalin. Further studies with wild-type and mutant rPhex proteins should permit the identification of physiologically relevant substrates involved in the pathogenesis of XLH.

Enhanced renal leukotriene production in murine lupus: role of lipoxygenase metabolites.

To investigate the potential role of leukotrienes in murine lupus, we measured renal hemodynamics and renal leukotriene production in MRL-lpr/lpr mice at 12 and 20 weeks of age. Over this age range, these animals develop overt manifestations of autoimmune disease with nephritis similar to human SLE. In the current study, we demonstrated that glomerular filtration rate (GFR) and PAH clearance (CPAH) deteriorated with age in MRL-lpr/lpr mice, but not in MRL(-)+/+ controls. Impaired renal hemodynamic function in MRL-lpr/lpr mice was associated with enhanced ionophore-stimulated production of both leukotriene B4 (LTB4) and leukotriene C4 (LTC4) by preparations of renal cortex. There was a significant inverse correlation between GFR and in vitro production of both LTC4 and LTB4 in kidneys from MRL-lpr/lpr mice, but not in control animals. In addition, in vitro LTC4 production was correlated with the severity of renal histomorphologic abnormalities. Administration of the specific peptidoleukotriene receptor antagonist SKF104353 to 20 week old MRL-lpr/lpr mice significantly improved both GFR and CPAH, whereas this agent had no effect of renal hemodynamics in MRL(-)+/+ controls. These results suggest that renal production of LTC4 and LTB4 is increased in MRL-lpr/lpr mice with nephritis, and that enhanced production of peptidoleukotrienes causes reversible renal dysfunction. Increased leukotriene production within the kidney may therefore be important in the pathogenesis of lupus nephritis.

Modulation of thromboxane receptor activation in rat glomerular mesangial cells.

Rat glomerular mesangial cells were used to investigate mechanisms of thromboxane A2 (TxA2) receptor regulation in the kidney. Exposure of mesangial cells to the TxA2 agonist U-46619 for 10 min reduced subsequent TxA2-induced increases in inositol phosphates and intracellular Ca2+ levels by approximately 70%. This loss of receptor responsiveness could be blocked by the TxA2 receptor antagonist SQ-29548 and was reversible after removal of agonist from the incubation medium. Radioligand binding studies using the TxA2 agonist [125I]BOP suggested that exposure of mesangial cells to U-46619 for 10 min reduced TxA2 receptor responsiveness without a loss of receptor sites from plasma membrane fractions of the cell, although the density of mesangial cell TxA2 receptors was decreased by approximately 60% after more prolonged exposure of mesangial cells to thromboxane agonists. Both desensitization to U-46619 and loss of TxA2 binding sites could be attenuated by the protein kinase C (PKC) inhibitors staurosporine, sphingosine, or H-7, and TxA2 receptor responsiveness was reduced in cells incubated with phorbol esters before stimulation with thromboxane agonists. We conclude that 1) agonist-specific decreases in TxA2 receptor responsiveness may involve initial uncoupling of the receptor from its effector systems, followed by a loss of TxA2 receptor sites from plasma membrane fractions of the cell, and 2) PKC may be involved in these processes.

Sensing of extracellular cations in CasR-deficient osteoblasts. Evidence for a novel cation-sensing mechanism.

We isolated osteoblastic cell lines from wild-type (CasR(+/+)) and receptor null (CasR(-/-)) mice to investigate whether CasR is present in osteoblasts and accounts for their responses to extracellular cations. Osteoblasts from both CasR(+/+) and CasR(-/-) mice displayed an initial period of cell replication followed by a culture duration-dependent increase in alkaline phosphatase activity, expression of osteocalcin, and mineralization of extracellular matrix. In addition, a panel of extracellular cations, including aluminum and the CasR agonists gadolinium and calcium, stimulated DNA synthesis, activated a transfected serum response element-luciferase reporter construct, and inhibited agonist-induced cAMP in CasR(-/-) osteoblasts. The functional responses to these cations were identical in CasR(+/+) and CasR(-/-) osteoblasts. Thus, the absence of CasR alters neither the maturational profile of isolated osteoblast cultures nor their in vitro responses to extracellular cations. In addition, CasR transcripts could not be detected by reverse transcription-polymerase chain reaction with mouse specific primers in either CasR(+/+) or CasR(-/-) osteoblasts, and immunoblot analysis with a CasR-specific antibody was negative for CasR protein expression in osteoblasts. The presence of a cation-sensing response in osteoblasts from CasR(-/-) mice indicates the existence of a novel osteoblastic extracellular cation-sensing mechanism.

Identification of a rat glomerular mesangial cell mitogenic 5-HT2A receptor.

Previous studies have demonstrated the presence of mitogenic serotonin [i.e., 5-hydroxytryptamine (5-HT)] receptors on glomerular mesangial cells and have linked those receptors to a complicated array of intracellular and autocrine/paracrine signaling pathways [T. Knauss and H. E. Abboud. Am. J. Physiol. 251 (Renal Fluid Electrolyte Physiol. 20): F844-F850, 1986; and N. Takuwa, M. Ganz, Y. Takuwa, R. B. Sterzel, and H. Rasmussen. Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26): F431-F439, 1989]. Those studies suggested that the mesangial subtype of 5-HT receptor is a member of the 5-HT2 receptor family, which consists of three known members, designated as subtypes A, B, and C. The purpose of the current study was to identify the subtype of 5-HT2 receptor present on mesangial cells. Northern blot showed detectable mRNA for a putative 5-HT2A receptor, but not for 5-HT1A or 5-HT2C receptors. Reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides derived from the putative third and sixth transmembrane domains of cloned 5-HT2 receptors yielded a 580-nucleotide (nt) fragment. RT-PCR with primers highly specific for the 5-HT2A receptor and designed to amplify > 95% of its coding block yielded a product of 1,320 nt. Nested PCR reactions yielded products of the predicted sizes for the 5-HT2A receptor. Partial sequence information was obtained, and the sequence corresponded exactly (627/627 nt) to that published for the cloned rat brain 5-HT2A receptor. These studies identify the mesangial cell mitogenic 5-HT receptor as a 5-HT2A receptor subtype.