Thin basement membrane disease with heavy proteinuria or nephrotic syndrome at presentation.

Thin basement membrane disease (TBMD) is a condition originally defined as diffuse thinning of the glomerular basement membrane (GBM) associated with hematuria in all patients. Although proteinuria has been described in up to 60% of patients with TBMD, it is almost always mild, with a 24-hour excretion mostly of less than 500 mg. We describe eight patients (four men and four women between 32 and 66 years of age) with TBMD who presented with heavy proteinuria or nephrotic syndrome. Among the seven cases with family history, hematuria was noted in five. All patients had a long history of microscopic hematuria, with episodic gross hematuria in two. Renal biopsies showed diffuse thinning of the GBM in each patient (mean between 185.3 x 29.8 nm and 232.6 x 34.5 nm versus control between 325 x 35 nm and 350 x 15 nm). Three cases showed thinning of GBM only (group I); the remaining five cases showed thinning of GBM associated with focal segmental glomerulosclerosis. All three patients of group I presented with nephrotic syndrome and normal renal function. Treatment with steroids resulted in remission of nephrotic syndrome in two, whereas nephrotic syndrome persisted in the untreated patient. Among the five patients in group II, nephrotic syndrome and normal renal function at presentation were noted in two, whereas the other three had heavy proteinuria (2.2, 2. 5, and 2.6 g/d, respectively) associated with mildly decreased renal function (serum creatinine 1.8, 1.3, and 1.5 mg/dL, respectively). At last follow-up, although the renal function was stable in all five, only the three who received steroid treatment had remission or marked improvement of proteinuria. Hematuria, however, persisted in all eight patients of both groups. Whether specific gene mutations are translated into structural changes responsible for both excessive GBM thinning and increased transcapillary permeability remains to be elucidated. Alternatively, the heavy proteinuria/nephrotic syndrome may not be related to TBMD, but rather is the manifestation of associated glomerular diseases. Follow-up, including a response to steroids, supports the latter hypothesis.

Urinary obstruction causes irreversible renal failure by inducing chronic tubulointerstitial nephritis.

Obstruction of urinary tract is a frequent cause of transient renal dysfunction, which, in the majority of cases, is reversible. Urinary obstruction can occasionally cause chronic renal failure, in spite of successful relief of the obstruction. Sonographic and pathologic studies show renal atrophy, interstitial fibrosis, and interstitial inflammation, which explain the loss of renal function. Herein, we report two cases of bilateral obstructive nephropathy due to prostatic hyperplasia, in which renal ultrasound demonstrated hydronephrosis and normal cortical thickness. In both cases, the renal failure persisted even after urinary obstruction was relieved by continuous catheter drainage. Renal biopsy showed that in each case the cause of the irreversible renal failure was chronic tubulointerstitial nephritis. It is concluded that renal biopsy can be of diagnostic, as well as of prognostic value in obstructive nephropathy, especially when relief of the obstruction is not followed by restoration of the renal function.

Calcium-regulated protein tyrosine phosphorylation is required for endothelin-1 to induce prostaglandin endoperoxide synthase-2 mRNA expression and protein synthesis in mesangial cells.

The role of endothelin (ET)-1-mediated cytosolic calcium ([Ca2+]i) elevation in regulating ET-1-induced prostaglandin endoperoxide synthase, prostaglandin G/H synthase (PGHS)-2 mRNA expression and protein synthesis was investigated in mesangial cells (MC). Ionomycin, a calcium ionophore, and thapsigargin, an inhibitor of calcium ATPase, mimicked the ET-1-stimulated PGHS-2 mRNA and protein induction. Inhibition of [Ca2+]i increases with (2-¿C2-bis-(carboxymethyl)-amino-5 methylphenoxy]methyl¿-6-methoxy-8-bis-(carboxymethyl)-aminoquinoline tetra-(acetoxymethyl)ester (Quin/AM), a calcium chelator, or with the combined presence of [8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, HCl] (TMB), an inhibitor of intracellular calcium stores release, and ethyleneglycol-bis-(beta-aminoethyl)- N,N,N',N'-tetra-acetic acid (EGTA) suppressed ET-1, as well as ionomycin and thapsigargin-mediated PGHS-2 mRNA and protein formation. Also, the ET-1-, ionomycin-, and thapsigargin-induced PGHS-2 mRNA expression and protein formation was inhibited in MC pretreated with inhibitors of calcium calmodulin kinase. In contrast, these conditions did not inhibit interleukin (IL)-1-induced PGHS-2 mRNA expression and protein synthesis. Pretreatment with tyrosine kinase inhibitors abolished the ET-1-, ionomycin-, thapsigargin-, and IL-1-mediated PGHS-2 mRNA and protein induction. ET-1-, ionomycin-, and thapsigargin- induced protein tyrosine phosphorylation, but not IL-1-induced protein tyrosine phosphorylation, was suppressed by inhibiting either [Ca2+]i elevation or calcium calmodulin kinase activation. It was concluded that elevation of [Ca2+]i and activation of calcium calmodulin kinases are upstream mediators of ET-1-induced PGHS-2 gene expression through activation of non-receptor-linked protein tyrosine kinase in MC.

Focal segmental glomerulosclerosis with acute renal failure associated with alpha-interferon therapy.

Both alpha- and beta- interferons (IFNs) have been increasingly used for the treatment of many types of malignancy, and recently the use of alpha-IFN in the management of hepatitis C virus (HCV) infection with or without renal involvement has been emphasized. We report a patient who developed nephrotic syndrome and acute renal failure while being treated with alpha-IFN for HCV infection. The renal biopsy showed focal segmental glomerulosclerosis and tubulointerstitial nephritis. Although several renal lesions have been described in association with either alpha-IFN treatment or HCV infection, focal segmental glomerulosclerosis has not been emphasized in either instance. This case also indicates that when renal abnormalities occur during treatment of HCV infection with alpha-IFN, kidney biopsy may define the responsible renal lesions, suggest possible association with other conditions, and serve as a guideline for proper management.

Constitutive expression of prostaglandin endoperoxide G/H synthetase (PGHS)-2 but not PGHS-1 in hum an tracheal epithelial cells in vitro.

Primary cultures of human tracheal epithelial (HTE) cells cultured in vitro, in defined serum-free media, express prostaglandin endoperoxide G/H synthase (PGHS) activity and produce prostaglandin E2 (PGE2). In contrast to every other cell type studied to date, HTE cells appear to constitutively express PGHS-2, the 'inducible' form of the enzyme, while expressing little or no PGHS-1, the 'housekeeping' isoenzyme in vitro. Prostaglandin synthesis in HTE cells was reduced by a selective PGHS-2 inhibitor, N-[2-cyclohexyloyl-4-nitrophenyl] methane-sulfonamide (NS398), with an IC50 of approximately 1 microM. Immunoblotting and immunoprecipitation of enzymatic activity with isozyme-specific antisera revealed only the PGHS-2 isoform. Full length human cDNA probes detected only PGHS-2 message in Northern blots. Neither PGHS-2 activity nor mRNA levels were dependent on, nor stimulated by peptide growth factors present in the defined serum-free growth medium, or by serum. Prolonged maintenance in the absence of retinoic acid, however, lead to a decline in PGHS activity. Phorbol-myristate acetate (PMA) induced PGHS-2 activity and mRNA and neither PMA-induced, nor constitutive PGHS-2 expression was suppressed by corticosteroids. Actinomycin D-treatment for six hours reduced the PGHS-2 activity and mRNA to only 50% that of untreated cells, suggesting that PGHS-2 mRNA is extremely stable in these cells. HTE cells, at least in vitro, appear unique among prostaglandin-producing cells in that they express PGHS-2, constitutively, independent of regulation by growth factors, serum, or corticosteroids and fail to express PGHS-1 under any culture condition studied.

Immunochemical detection of arylamine N-acetyltransferase in normal and neoplastic bladder.

The N-acetyltransferase (NAT) phenotype is an important determinant of individual susceptibility to occupational bladder cancer. N-Acetyltransferases arc known to metabolize aromatic amine bladder carcinogens, but the functional significance of NAT expression in the target organ is unclear. To resolve this issue, polygonal antisera against purified recombinant enzymes and C-terminal peptides of human NAT Type 1 (NAT1) and Type 2 (NAT2) were generated. Western blot analysis of exfoliated cells from human urine, pig bladder homogenate, and human bladder tumor-derived cell lines showed that NAT1 was expressed in all three systems, whereas NAT2 did not appear to be expressed in the bladder. Immunohistochemical analysis of human bladder tumor sections indicated that well-differentiated tumor cells expressed NAT1, with the highest level of expression being found in the umbrella cells that line the bladder lumen. Poorly differentiated tumor regions appeared to express NAT1 at lower levels than did well-differentiated areas. These findings support the hypothesis that aromatic amines are metabolized in the bladder epithelium by NAT1.

Sphingolipid metabolites differentially regulate extracellular signal-regulated kinase and stress-activated protein kinase cascades.

The mitogen-activated protein kinase (MAPK) signalling pathway serves to translocate information from activated plasma-membrane receptors to initiate nuclear transcriptional events. This cascade has recently been subdivided into two analogous pathways: the extracellular signal-regulated kinase (ERK) cascade, which preferentially signals mitogenesis, and the stress-activated protein kinase (SAPK) cascade, which is linked to growth arrest and/or cellular inflammation. In concurrent experiments utilizing rat glomerular mesangial cells (MCs), we demonstrate that growth factors or sphingosine activate ERK but not SAPK. In contrast, inflammatory cytokines or cell-permeable ceramide analogues activate SAPK but not ERK. Ceramide, but not sphingosine, induces interleukin-6 secretion, a marker of an inflamed phenotype. Moreover, ceramide can suppress growth factor- or sphingosine-induced ERK activation as well as proliferation. These studies implicate sphingolipid metabolites as opposing regulators of cell proliferation and inflammation through activation of separate kinase cascades.

Differential regulation of sphingomyelinase and ceramidase activities by growth factors and cytokines. Implications for cellular proliferation and differentiation.

Sphingosine is a product of sphingolipid metabolism that has been linked to a protein kinase C-independent mitogenic response. In previously published data, utilizing an in vitro model system for platelet-derived growth factor (PDGF)-induced vascular smooth muscle proliferation, we have demonstrated that sphingosine is increased at the expense of a concomitant decrease in ceramide formation, implicating an altered ceramidase activity. To explore mechanisms of growth factor-stimulated sphingosine formation, we have developed and investigated a cell-free model system assessing ceramidase activity. We now report that an alkaline, membrane-associated, ceramidase activity in the rat glomerular mesangial cell, a smooth muscle-like pericyte, is up-regulated by growth factors, apparently via a tyrosine kinase phosphorylation mechanism. PDGF also stimulated sphingomyelinase activity which generates sufficient substrate to drive the subsequent ceramidase reaction. Inflammatory cytokines, including interleukin-1, and tumor necrosis factor-alpha, stimulated sphingomyelinase but not ceramidase activity, a result consistent with the cellular accumulation of the ceramide, apoptidic, differentiating second messenger. Mitogenic vasoconstrictor peptides such as endothelin-1 stimulated neither sphingomyelinase nor ceramidase activities. An inhibitor of ceramidase activity, N-oleoylethanolamine, reduced PDGF- but not endothelin-1-stimulated proliferation. Thus, we conclude that, in mesangial cells, growth factors but not vasoconstrictor peptides or cytokines induce mitogenesis, in part, through ceramidase-mediated sphingosine formation.

Interleukin-1 and endothelin stimulate distinct species of diglycerides that differentially regulate protein kinase C in mesangial cells.

Diglycerides are phospholipid-derived second messengers that serve as cofactors for protein kinase C activation. We have previously shown that, in rat glomerular mesangial cells, the cytokine, interleukin-1 alpha, and the vasoactive peptide, endothelin, generate diglycerides from unique phospholipid precursors. However, neither the molecular species of these diglycerides nor their biological actions were determined. It is now hypothesized that interleukin-1- and endothelin-treated mesangial cells form distinct molecular species of diglycerides which may serve different roles as intracellular signaling molecules. Diglyceride molecular species were resolved and quantified by TLC and high performance liquid chromatography as diglyceride-[14C]acetate derivatives. Endothelin stimulates predominantly ester-linked species (diacylglycerols) in contrast to interleukin-1 which stimulates only ether-linked species (alkyl, acyl- and alkenyl,acylglycerols). In support of these data, interleukin-1-treated mesangial cells hydrolyze ethanolamine plasmalogens, vinyl ether-linked phospholipids. It has been reported that ether-linked, in contrast to ester-linked, diglyceride species do not activate protein kinase C activity. Thus, we next assessed membrane protein kinase C activity in endothelin- or interleukin-1-treated mesangial cells. Even though interleukin-1 has no effect upon basal protein kinase C activity, this cytokine, through the formation of ether-linked diglyceride second messengers, inhibits endothelin, platelet-activating factor, or arginine vasopressin-stimulated protein kinase C activity. We further demonstrate that ester-linked diacylglycerols but not alkyl,acyl- or alkenyl,acylglycerols substitute for phorbol esters in a cell-free protein kinase C assay. In addition, alkenyl,acylglycerols inhibit diacylglycerol-stimulated immunoprecipitated protein kinase C alpha activity in vitro and total protein kinase C activity in permeabilized mesangial cells ex vivo. Taken together, these data suggest that interleukin-1-induced formation of ether-linked diglycerides may physiologically serve to down-regulate receptor-mediated protein kinase C activity and that individual molecular species of diglycerides may serve different roles as intracellular signaling molecules.

Interactions of the vasoconstrictor peptides, angiotensin II and endothelin-1, with vasodilatory prostaglandins.

Vasoconstrictor peptides, such as angiotensin II (Ang II) and endothelin-1 (ET-1), stimulate the synthesis and release of vasodilatory prostaglandins from multiple tissues and diverse cellular types. The synthesis of PGE2 and PGI2 acts as a negative feedback loop to antagonize the contractile actions of Ang II and ET-1. Inhibition of prostaglandin synthesis with nonsteroidal anti-inflammatory drugs (NSAIDs) enhances the constrictor actions of Ang II and ET-1 on the vasculature of the kidney and on the glomerulus. The enhanced production of prostaglandins, in response to constrictor peptides, is both short- and long-term. Prostaglandin synthesis is regulated at multiple steps, including: (1) phospholipase A2, which releases arachidonic acid from membrane phospholipids; (2) PGH synthase (PGHS), which converts arachidonic acid to the endoperoxides PGG2 and PGH2; and (3) PG synthases convert the endoperoxides to PGI2, PGE2, and others. ET-1 acutely activates phospholipase A2 through phosphorylation and acute increases of intracellular calcium. ET-1 also chronically enhances phospholipase A2 activity by transcriptional induction of this enzyme. There are no known acute effects of ET-1 to acutely enhance PGHS activity through posttranslational modification of the molecule. Chronically, however, cellular content of PGHS-2 can be induced through transcriptional induction of the enzyme in response to ET-1. Hence, ET-1 short- and long-term activates a modulatory feedback pathway that depends on upregulation of arachidonic acid release through phospholipase A2 and enhanced synthesis of prostaglandin endoperoxides through PGHS-2.

Endothelin regulates PGE2 formation in rat mesangial cells through induction of prostaglandin endoperoxide synthase-2.

Endothelin-1 stimulates vascular smooth muscle and mesangial cells to release prostaglandin E2 which attenuates the vasoconstrictor and mitogenic effects of endothelin. The role of endothelin-1 to regulate prostaglandin endoperoxide synthase (PGHS)-1 and -2 gene expression and protein synthesis was evaluated in cultured mesangial cells. Endothelin induced mRNA and protein expression for PGHS-2 but not for PGHS-1. A direct correlation was observed between the mass of immunoprecipitated PGHS protein and PGE2 synthetic enzymatic activity.

Endothelin stimulates prostaglandin endoperoxide synthase-2 mRNA expression and protein synthesis through a tyrosine kinase-signaling pathway in rat mesangial cells.

We evaluated the role of endothelin-1 (ET-1), a vasoconstrictor peptide, to regulate prostaglandin endoperoxide synthase (PGHS)-1 and -2 gene expression and protein synthesis in cultured rat mesangial cells (MC). ET-1 induced mRNA for PGHS-2 but not PGHS-1 and also stimulated protein accumulation of PGHS-2 but not PGHS-1 in MC. ET-1 induction of PGHS-2 protein was accompanied by a sustained enhancement of enzymatic activity assessed by conversion of arachidonic acid to prostaglandin E2. The ET-1-stimulated PGHS-2 expression was reduced with protein tyrosine kinase but not with protein kinase C inhibitors or in protein kinase C-depleted cells. Both dexamethasone and heparin reduced ET-1-activated PGHS-2 mRNA expression and protein formation. We conclude that in MC, ET-1 induces PGHS-2 through a protein tyrosine kinase-dependent pathway.

Arylamine N-acetyltransferase activity in human cultured cell lines.

Many arylamine and hydrazine drugs and xenobiotics are acetylated by liver N-acetyltransferase (NAT; EC 2.3.1.5). Two loci, mnat and pnat, encode the enzymes designated monomorphic and polymorphic NAT (mNAT and pNAT) respectively. These isoenzymes have different substrate specificities. In addition, at the polymorphic locus a diversity of alleles is found, which differ by specific point mutations that may or may not result in amino acid substitutions. These point mutations result in the 'slow' acetylation of substrates of pNAT. The substrates for NAT include carcinogenic arylamines. Susceptibility to bladder cancer has been related to slow acetylation. NAT has been characterized in immortalized human cell lines to assess their use in studies of the metabolism or arylamines in vitro. A monocytic cell line (U937) and two hepatoma cell lines of parenchymal lineage (HepG2 and Hep3B) have been shown to catalyse acetylation of substrates of mNAT but do not acetylate sulphamethazine, a substrate specific for pNAT. Using PCR to amplify the alleles of pNAT, followed by restriction-enzyme digestion of the product, the cell lines have been genotyped: U937 cells are homozygous slow acetylators (S1a/S1a) and HepG2 cells are heterozygous slow acetylators (S1a/S2). Transcription of pnat was confirmed in the hepatoma cell lines, by amplification of cDNA generated from these cells. In addition, splicing of mRNA specific for pNAT has been demonstrated by using a primer which anneals to a region in the 5' promoter region. Unlike the hepatoma cell lines, in U937 cells the pNAT gene is not transcribed. However, transcription of mnat was shown to occur in all three cell lines.

Drug metabolising N-acetyltransferase activity in human cell lines.

Many arylamine and hydrazine drugs and xenobiotics are acetylated by N-acetyltransferase (NAT), a cytosolic enzymic activity which has a wide tissue distribution. Humans can be classified as either fast or slow acetylators on the basis of their ability to metabolise isoniazid or sulphamethazine. These are termed polymorphic substrates. The acetylation of other compounds does not vary amongst individuals, e.g., p-aminobenzoic acid, and are termed monomorphic substrates. NAT from human hepatic and non-hepatic tissues, viz., (i) liver, (ii) the hepatoma cell line HepG2, (iii) tonsil lymphocytes and (iv) the monocytic cell line U937 have been compared with respect to substrate specificity towards polymorphic and monomorphic substrates. The chromatographic and centrifugation behaviour of NAT from these sources has also been investigated. NAT from liver shows 2-fold greater activity towards sulphamethazine than towards p-aminobenzoic acid as substrate. All other cell types tested show at least 70-fold greater activity with p-aminobenzoic as substrate compared to sulphamethazine. NAT from HepG2 cells, U937 cells and tonsil lymphocytes migrates as a single peak during ion-exchange chromatography, whereas the liver NAT activity is separated into two peaks. NAT in HepG2 cells resembles extra-hepatic tissue NAT rather than NAT in liver. HepG2 cells do not therefore represent a good in vitro model for investigation of human metabolism of arylamines or hydrazines. The molecular weight of NAT from U937 cells has been determined by a combination of sucrose density gradient centrifugation and gel filtration to be 31,600 +/- 1200 daltons.