Comparison of patients with and without digital ulcers in systemic sclerosis: detection of possible risk factors.

BACKGROUND: Digital ulcers (DU) are a major complication in the course of systemic sclerosis (SSc). In recent years, efficacious, but expensive therapies (e.g. iloprost, sildenafil, bosentan) have been shown to improve healing or to reduce the recurrence of DU. For optimal management it would be useful to identify the risk factors for DU. Such statistical analyses have been rare because they require a high number of patients. OBJECTIVES: To identify potential risk factors for DU in patients with SSc. METHODS: We used the registry of the German Network for Systemic Scleroderma and evaluated the data of 1881 patients included by August 2007. We assessed potential risk factors for DU by comparing patients with (24.1%) and without active DU at time of entry (75.9%). RESULTS: Multivariate analysis revealed that male sex, presence of pulmonary arterial hypertension (PAH), involvement of the oesophagus, diffuse skin sclerosis (only when PAH was present), anti-Scl70 antibodies, young age at onset of Raynaud's phenomenon (RP), and elevated erythrocyte sedimentation rate (ESR) significantly impacted on the appearance of DU. Certain combinations increased the patients' probability of presenting with DU, with the highest probability (88%) for male patients with early onset of RP, ESR>30 mm h(-1), anti-Scl70 antibodies and PAH. Patients with DU developed RP, skin sclerosis and organ involvement approximately 2-3 years earlier than patients without DU. CONCLUSIONS: The results reveal possible risk factors for the occurrence of DU in SSc. As DU are prone to local complications, prophylactic vasoactive treatment for patients presenting with these factors may be justified.

Skin sclerosis is only of limited value to identify SSc patients with severe manifestations--an analysis of a distinct patient subgroup of the German Systemic Sclerosis Network (DNSS) Register.

OBJECTIVES: In SSc, diagnosis and classification is based mainly on skin sclerosis. Herein, we investigated in a large multicentre cohort, to what extent skin sclerosis reflects organ involvement and additional clinical symptoms. METHODS: A total of 1200 SSc patients from the register of the German Systemic Sclerosis Network (DNSS), classified as either lcSSc or dcSSc, were analysed for their serological characteristics, clinical symptoms and organ manifestations in relation to skin involvement measured by the modified Rodnan skin score (mRSS). RESULTS: SSc patients with different mRSS did not differ significantly in their disease duration and in most of the clinical symptoms. They showed a similar distribution of most organ manifestations such as pulmonary arterial hypertension as well as cardiac, renal and nervous system involvement. More severe skin thickening was found to be associated with pulmonary fibrosis and gastrointestinal symptoms, as well as with digital ulcers and musculoskeletal involvement. CONCLUSIONS: In patients with SSc, potentially life-threatening complications and clinical symptoms with high impact on the quality of life occur independently from the extent of skin sclerosis. The diagnosis in SSc patients with a low mRSS could be missed or they could be insufficiently treated.

Intermediate molecular weight proteinuria and albuminuria identify scleroderma patients with increased morbidity.

BACKGROUND: Renal involvement and systemic vascular damage have been shown to significantly affect prognosis in systemic sclerosis (SSc). However, it is often difficult to assess damage to the renal and systemic vasculature in SSc patients. METHODS: Using detailed urinary protein analysis we sought to detect scleroderma renal disease at an early stage and to assess systemic vasculopathy due to SSc. We examined 80 patients with SSc as well as 18 healthy control subjects using urinary protein analysis including determination of urinary albumin excretion rate and urinary total protein excretion as well as urinary polyacrylamide gel electrophoresis. RESULTS: Albuminuria was found in 25% (20/80) of the SSc patients (2.5% macroalbuminuria, 22.5% microalbuminuria), increased total protein excretion in 17.5% (14/80), and intermediate molecular weight proteinuria (IMWP) as determined by urine electrophoresis in 31.3% (25/80). None of these abnormalities was found in the control group (0/18). Presence of IMWP correlated with the diffuse type of SSc (p < 0.01), gastrointestinal involvement (p < 0.05) and increased systolic blood pressure (p < 0.01). Increased total protein excretion was found to correlate with pulmonary involvement (p < 0.05). Albuminuria was associated with prolonged duration of the disease (> 4 years) (p < 0.05) and increased systolic blood pressure (p < 0.01). CONCLUSIONS: Leakage of proteins into the urine in patients with SSc appears to indicate not only renal involvement but also systemic vasculopathy which is associated with increased morbidity. Patients with SSc should be regularly examined using sensitive urinary protein tests such as albumin assays or urine electrophoresis, to detect kidney involvement at an early stage.

The registry of the German Network for Systemic Scleroderma: frequency of disease subsets and patterns of organ involvement.

OBJECTIVE: Systemic sclerosis (SSc) is a rare, heterogeneous disease, which affects different organs and therefore requires interdisciplinary diagnostic and therapeutic management. To improve the detection and follow-up of patients presenting with different disease manifestations, an interdisciplinary registry was founded with contributions from different subspecialties involved in the care of patients with SSc. METHODS: A questionnaire was developed to collect a core set of clinical data to determine the current disease status. Patients were grouped into five descriptive disease subsets, i.e. lcSSc, dcSSc, SSc sine scleroderma, overlap-syndrome and UCTD with scleroderma features. RESULTS: Of the 1483 patients, 45.5% of patients had lcSSc and 32.7% dcSSc. Overlap syndrome was diagnosed in 10.9% of patients, while 8.8% had an undifferentiated form. SSc sine scleroderma was present in 1.5% of patients. Organ involvement was markedly different between subsets; pulmonary fibrosis for instance was significantly more frequent in dcSSc (56.1%) than in overlap syndrome (30.6%) or lcSSc (20.8%). Pulmonary hypertension was more common in dcSSc (18.5%) compared with lcSSc (14.9%), overlap syndrome (8.2%) and undifferentiated disease (4.1%). Musculoskeletal involvement was typical for overlap syndromes (67.6%). A family history of rheumatic disease was reported in 17.2% of patients and was associated with early disease onset (P < 0.005). CONCLUSION: In this nationwide register, a descriptive classification of patients with disease manifestations characteristic of SSc in five groups allows to include a broader spectrum of patients with features of SSc.

[Organ-specific diagnosis in patients with systemic sclerosis: Recommendations of the German Network for Systemic Sclerosis (DNSS)]. / Organspezifische Diagnostik von Patienten mit systemischer Sklerodermie : Empfehlungen des Deutschen Netzwerkes für Systemische Sklerodermie (DNSS).

The diagnosis and therapy of systemic sclerosis (SSc) is demanding due to its nature as a multisystem disease and its chronic, severe course. To date, there are no generally accepted recommendations for diagnostic work-up either for the time of initial disease diagnosis or for the regular follow-up clinical examinations and diagnostic procedures required. However, due to recent advances, e.g. in the therapy of pulmonary arterial hypertension, regular examinations may contribute to early recognition and treatment of developing organ involvement. This manuscript describes the recommendations for initial and follow-up organ-specific clinical examinations and diagnostic work-up as compiled and carried out by the German Network for Systemic Scleroderma [Deutsche Netzwerk für systemische Sklerodermie (DNSS)].

Mechanisms of everolimus-induced glomerulosclerosis after glomerular injury in the rat.

Despite the lack of nephrotoxicity, adverse effects of the new antiproliferative immunosuppressant everolimus have been reported. By varying time point and dose of everolimus treatment as well as the degree of glomerular injury, the specific conditions and potential mechanisms leading to adverse actions in the anti-Thy1 model have been determined. Only the combination of early and high-dose everolimus treatment (1-3 mg/kg bw) with a severe glomerular lesion ('full-dose' anti-Thy1 model) caused adverse effects with a high mortality rate, progressive apoptosis, crescent formation and glomerulosclerosis. In contrast, either later start or low-dose (0.3 mg/kg bw) therapy or treatment of a less severe lesion ('reduced dose' anti-Thy1 model) appeared to be relatively safe for the glomerular architecture. The adverse effects of everolimus were linked to its marked inhibition of endothelial cell, but not necessarily mesangial cell proliferation. In addition, everolimus markedly inhibited the angiogenic cytokine vascular endothelial growth factor in nephritic glomeruli in vivo. These experimental results suggest special caution regarding the use of everolimus in all situations of severe glomerular cell injury requiring extensive capillary repair, where at least adaption to a low dose needs to be considered.

[A rare case of birenal malacoplakia with renal failure]. / Seltener Fall einer birenalen Malakoplakie mit Nierenversagen.

HISTORY AND ADMISSION FINDINGS: A 45 year old man was admitted to our hospital because of fever, loss of appetite, and deterioration of general health. For two weeks the patient suffered from diarrhea which had resulted in moderate volume depletion. In addition, he complained of bilateral flank pain at the time of admission. Furthermore, the patient had a history of heavily drinking alcohol as well as cigarette smoking for many years. He had never attended a medical doctor before. INVESTIGATIONS: The patient presented with the clinical picture of acute renal failure and urosepticaemia which was caused by Escherichia coli. The kidneys were found to be at the upper limit of normal by sonography. Magnetic resonance imaging revealed signal-alterations in both kidneys with hyper- and hypointense zones in the renal parenchyma. DIAGNOSIS: To clarify the cause of rapid deterioration of renal function, we performed a renal biopsy. The histology of the renal specimen revealed an unusual type acute bacterial interstitial nephritis most likely due to an infection with E. coli. The clinical picture, the laboratory findings and renal histology, lead to the diagnosis of birenal malakoplakia. TREATMENT AND COURSE: After intravenous and subsequent oral antibiotic therapy the fever and the clinical signs of urosepticaemia subsided and renal function gradually improved. Antibiotic therapy and supplementation with vitamins were continued for 20 weeks. Five years after initial diagnosis, renal function was stable at a glomerular filtration rate of approximately 45 ml/min. CONCLUSIONS: Malakoplakia of the kidney is a rare form of bacterial interstitial nephritis and requires long-term antibiotic therapy.

[Early diagnosis of chronic kidney diseases]. / Frühdiagnostik von chronischen Nierenerkrankungen.

Chronic kidney diseases frequently progress to end-stage renal failure over a period of several years. Ultimately, the glomerular filtration apparatus and tubulus are destroyed to such an extent that renal replacement therapy is required. This process may last longer than 10 years. To avoid loss of renal function, chronic kidney diseases should be diagnosed as early as possible so that therapeutic measures are initiated in time. Medications which prevent or delay progression to end-stage renal failure, e.g. ACE inhibitors and AT(1) blockers, are more effective if started early in the course of renal disease. A significant number of patients with chronic kidney disease are still diagnosed too late and are without adequate therapy so that chances for prevention and/or delay of chronic kidney disease are missed.

Positron emission tomography reveals a leiomyosarcoma causing proteinuria.

Obstruction of the renal veins may result in proteinuria and is frequently caused by thrombosis or tumorous processes. Since thrombosis and malignancy may occur simultaneously in the venous outflow of the kidneys, search for an underlying intraluminal tumor may be impeded by extensive thrombosis in the lumen of renal and caval veins. We report the case of a 30-year-old man with moderate proteinuria which was caused by an obstructing process of the vena cava inferior and the renal veins. While the obstructive mass was initially misdiagnosed as thrombosis, positron emission tomography helped to reveal the tumorous character of the lesion and fine-needle biopsy allowed rapid diagnosis of a leiomyosarcoma originating from the caval or renal veins. We conclude that undelayed diagnosis of the cause of renal and caval vein obstruction is facilitated by early positron emission tomography and subsequent fine-needle biopsy to identify possible tumorous lesions.

Regulation of mesangial cell function by vasodilatory signaling molecules.

Proliferation of mesangial cells and expansion of mesangial matrix is a hallmark of glomerular disease leading to end-stage renal failure and requiring renal replacement therapy. Independently from the type of injury, e.g. in glomerulonephritis or diabetic nephropathy, the response to injury is remarkably uniform. Chronic glomerular disease is frequently associated with increases in systemic blood pressure and altered intraglomerular hemodynamics. Furthermore, reduction of systemic blood pressure and inhibition of the vasoconstrictor peptide angiotensin II have been shown to delay end-stage renal failure in various types of human kidney disease. Since vasoconstrictors of mesangial cells and efferent glomerular arterioli, such as angiotensin II, are thought to be detrimental for the progression of chronic glomerular disease, we propose that vasodilatory factors which antagonize the effects of angiotensin II, might have beneficial effects during the course of progressive kidney disease. To support this concept we will summarize currently available data on the role of vasodilatory signaling molecules such as natriuretic peptides (ANP, BNP and CNP), nitric oxide (NO), the prostaglandines PGE2 and prostacycline, and the purine mediator adenosine in the regulation of mesangial function.

Thrombospondin peptides are potent inhibitors of mesangial and glomerular endothelial cell proliferation in vitro and in vivo.

BACKGROUND: Thrombospondin 1 (TSP1), a multifunctional, matricellular glycoprotein, is expressed de novo in many inflammatory disease processes, including glomerular disease. Short peptide fragments derived from the type I properdin repeats of the TSP1 molecule mimic anti-angiogenic and/or transforming growth factor-beta (TGF-beta)-activating properties of the whole TSP1 glycoprotein. We investigated the effects of D-reverse peptides derived from the type I domain of TSP1 in experimental mesangial proliferative glomerulonephritis in the rat (anti-Thy1 model), as well as their effects on cultured mesangial and glomerular endothelial cells. METHODS: Effects of TSP peptides on proliferation of mesangial or glomerular endothelial cells in culture after growth arrest or growth factor stimulation (fibroblast growth factor-2, platelet-derived growth factor-BB, 10% fetal calf serum) were measured by [3H]thymidine incorporation assay. Adhesion of rat mesangial cells (MCs) to a TSP-peptide matrix was assayed using an attachment-hexosaminidase assay. TSP peptides were intraperitoneally injected daily in rats that had received an intravenous injection of polyclonal anti-Thy1 antibody to induce mesangial proliferative glomerulonephritis. On biopsies from days 2, 5, and 8 of anti-Thy1 disease, mesangial and glomerular endothelial proliferation, matrix expansion, mesangial activation, and microaneurysm formation were assessed. Functional parameters such as blood pressure and proteinuria were also measured. RESULTS: An 18-amino acid peptide (type I peptide) with anti-angiogenic and TGF-beta-activating sequences decreased mesangial and glomerular endothelial cell proliferation in vitro and in vivo and reduced microaneurysm formation and proteinuria in experimental glomerulonephritis. Analogues lacking the TGF-beta-activating sequence mimicked most effects of the type I peptide. The mechanism of action of these peptides may include antagonism of fibroblast growth factor-2 and alteration of MC adhesion. The TGF-beta-activating sequence alone did not have significant effects on mesangial or glomerular endothelial cells in vitro or in experimental kidney disease in vivo. CONCLUSION: Peptides from TSP1 may be promising therapeutics in treating glomerular disease with mesangial and endothelial cell injury.

Extracellular ATP causes apoptosis and necrosis of cultured mesangial cells via P2Z/P2X7 receptors.

Mesangial cells undergo cell death both by apoptosis and necrosis during glomerular disease. Since nucleotides are released from injured and destroyed cells in the glomerulus, we examined whether extracellular ATP and its receptors may regulate cell death of cultured mesangial cells. Addition of extracellular ATP (300 microM to 5 mM) to cultured rat mesangial cells for 90 min caused a 5. 8-fold increase in DNA fragmentation (terminal deoxynucleotidyl transferase assay) and a 4.2-fold increase in protein levels of the tumor suppressor p53, which is thought to regulate apoptosis. Apoptotic DNA fragmentation was confirmed by the diphenylamine assay and by staining with the DNA-specific fluorochrome Hoechst 33258. The necrotic markers, release of lactate dehydrogenase and uptake of trypan blue, were not positive before 3 h of ATP addition. The effects of ATP on DNA fragmentation and p53 expression were reproduced by the purinergic P2Z/P2X7 receptor agonist, 3'-O-(4-benzoylbenzoyl)-ATP, and inhibited by the P2Z/P2X7 receptor blocker, oxidized ATP. Transcripts encoding the P2Z/P2X7 receptor were expressed by cultured mesangial cells as determined by Northern blot analysis. P2Z/P2X7 receptor-associated pore formation in the plasma membrane was demonstrated by the Lucifer yellow assay. We conclude that activation of P2Z/P2X7 receptors by extracellular ATP causes apoptosis and necrosis of cultured mesangial cells. Activation of purinergic P2Z/P2X7 receptors may play a role in causing death of mesangial cells during glomerular disease.

Activation of purinergic P2Y2 receptors inhibits inducible NO synthase in cultured rat mesangial cells.

Cytokine-induced nitric oxide (NO) is produced on glomerular inflammation. Glomerular injury and thrombocyte aggregation result in the release of nucleotides, which may regulate induced NO synthesis in cultured rat mesangial cells (MCs). ATP (10(-3) M) inhibited 24-h nitrite production induced by lipopolysaccharide (LPS, 10 microg/ml)/interferon-gamma (IFN-gamma, 100 U/ml) by 48.2 +/- 6. 3%, as well as induction of inducible NOS (iNOS) protein and mRNA. Also, coincubation with either 10(-4) M of UTP, ATP, or ATPgammaS inhibited LPS/IFN-gamma-induced nitrite production by 29.9 +/- 5.8, 36.4 +/- 4.3, and 50.3 +/- 6.5%, respectively, indicating involvement of purinergic P2Y2 receptors. Correspondingly, cultured MCs expressed P2Y2 receptor mRNA. Agonists for other purinergic receptors [alpha,beta-methylene-ATP, 3'-O-(4-benzoyl)-benzoyl-ATP, 2-methylthio-ATP, ADP, UDP, adenosine] were ineffective. Treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 10(-8) M) reproduced the inhibitory effect of ATP on iNOS protein expression and nitrite inhibition (by 46.6 +/- 10. 4%). The effect of ATP or PMA was reversed by the PKC inhibitors Ro-31-8220 (10(-8) M) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (10(-5) M), indicating that suppression of iNOS is mediated via activation of PKC through stimulated P2Y2 receptors. In conclusion, the release of purine mediators may play a critical role for iNOS expression and synthesis of NO during glomerular inflammatory disorders.

Adenine dinucleotides: a novel class of signalling molecules.

1. Adenine dinucleotides (Ap3A, Ap4A, Ap5A, Ap6A) are stored in secretory granules of thrombocytes, chromaffin cells and neuronal cells. After release into the extracellular space, the dinucleotides exhibit divergent biological effects on a variety of target cells and organs. The dinucleotides are metabolized by soluble enzymes in the blood plasma as well as by membrane-bound ectoenzymes of endothelial cells, smooth muscle cells, and other cell types. 2. The enzymatic cleavage of the dinucleotides plays a dual role for their biological function: (a) termination of the signal; and (b) generation of purinergically active products such as ATP, ADP and finally adenosine. In contrast to ATP the dinucleotides are long-lived purine nucleotides in the blood. 3. The potential role of the dinucleotides as signalling molecules has been demonstrated in several systems. The adenosine polyphosphates have autocrine function for thrombocytes. Ap3A at low concentration reversibly activates isolated platelets. The mechanism of activation has been elucidated by showing a continuous cleavage of Ap3A, leading to the formation of ADP which is a known agonist of the P2T receptor on thrombocytes. Ap4A and other dinucleotides act as antagonists and inhibit platelet activation. 4. The vasotone of perfused isolated arteries as well as of resistance vessels in the beating heart is differentially influenced by adenine dinucleotides. While Ap3A and Ap4A exhibit relaxing effects at micromolar concentrations, Ap5A and Ap6A elicit vasoconstriction in these vessels. 5. In rat kidney mesangial cells adenine dinucleotides efficiently promote growth. Stimulation of DNA synthesis by various growth factors is enhanced synergistically. ApnA significantly increase the expression of the early growth response gene Egr-1. 6. The specificity and, in some tissues, the uniqueness of effects evoked by dinucleotides may be mediated by genuine dinucleotide receptors (P4) or by specialized P2 receptors (P2D).

Extracellular nucleotides as signalling molecules for renal mesangial cells.

1. Glomerular diseases frequently cause chronic renal failure which ultimately requires dialysis and kidney transplantation. The events leading to destruction of the glomerular filtration apparatus include injury of glomerular cells, aggregation of thrombocytes and infiltration of immune cells into the glomerulus. 2. Nucleotides (e.g. ATP and UTP) are present in all glomerular cell types as well as in thrombocytes. The release of nucleotides into the extracellular space occurs after damage of glomerular cells and aggregation of thrombocytes. Several in vitro and in vivo findings indicate that extracellular nucleotides may play a role as pro-inflammatory mediators in glomerulonephritis. 3. A hallmark finding in kidney biopsies from patients with glomerulonephritis is proliferation of glomerular mesangial cells. Cell culture studies demonstrated that extracellular ATP (10-300 microM) stimulated growth of mesangial cells. The mitogenic effect of ATP was potentiated in the presence of multiple growth factors. 4. Nucleotide-induced signalling in mesangial cells included an increase of intracellular calcium, activation of phosphatidylinositol-specific phospholipase C and phospholipase D, inhibition of adenylylcyclase, stimulation of mitogen-activated protein kinase and increased expression of the immediate early genes, c-fos, c-jun and Egr-1. 5. In previous studies of experimental mesangioproliferative glomerulonephritis, exogenously given ADP beta S and ATP gamma S have been shown to aggravate the course of the disease, while 2-chloroadenosine had beneficial effects. 6. Taken together, these findings support the concept that nucleotides may function as proinflammatory mediators in glomerulonephritis while adenosine may have antiinflammatory effects.

Glomerular basement membrane-derived perlecan inhibits mesangial cell adhesion to fibronectin.

To explore whether the heparan sulfate proteoglycan, periecan, may act as a regulator of glomerular cell behavior, we examined the effects of perlecan on adhesive properties of rat and human mesangial cells in culture. We observed that mesangial cells did not adhere to purified perlecan. Mesangial cell adhesion to fibronectin, but not to laminin, was inhibited when perlecan was present in the substratum, reaching complete inhibition at 20 micrograms/ml perlecan. Similarly, perlecan reduced mesangial cell adhesion to the 120 kDa fragment of fibronectin and to bovine serum albumin-coupled RGD peptides both lacking the fibronectin heparin-binding domains, indicating that the inhibitory effect of perlecan does not require interaction of its heparan sulfate chains with fibronectin. The core protein of perlecan seems to be relevant, since perlecan retained most of its anti-adhesive potency after total deglycosylation. It is unlikely that perlecan causes steric inhibition of the cell binding sites of fibronectin by direct binding, since the 120 kDa fragment of fibronectin which includes the cell binding site, did not bind to intact perlecan. In the presence of submaximal inhibitory concentrations of perlecan, soluble RGD peptides (100 micrograms/ml), by themselves without effect, completely inhibited cell adhesion to fibronectin. Using immunocytochemistry, we examined the effects of perlecan on the organization of fibronectin-specific, RGD-dependent beta 1 integrins in focal contacts. When perlecan was added to the substratum, human mesangial cells adhered to fibronectin only in the presence of collagen I. Under these conditions, perlecan did not significantly alter the organization of alpha 5 beta 1 integrin into focal contacts of mesangial cells which adhered to a substratum composed of collagen I plus fibronectin. These data suggest that perlecan exerts its anti-adhesive effects on mesangial cells via a core protein-dependent mechanism which reduces the avidity of the fibronectin receptor. The resulting avidity level is too low for mediating cell binding but sufficient to induce integrin organization into focal contacts.

Vasoactive diadenosine polyphosphates promote growth of cultured renal mesangial cells.

Diadenosine polyphosphates (diadenosine triphosphate, Ap3A; diadenosine tetraphosphate, Ap4A; diadenosine pentaphosphate, Ap5A; diadenosine hexaphosphate, Ap6A) are potent vasoactive molecules stored and released by platelets. We examined whether these dinucleotides might contribute to the glomerular inflammatory response by stimulating the proliferation of mesangial cells. In cultured rat mesangial cells all four tested dinucleotides (10 to 100 mumol/L) significantly stimulated DNA synthesis as measured by [3H]thymidine uptake at 48 hours (x-fold increase compared with unstimulated control cells: Ap3A, 1.5; Ap4A, 1.8; Ap5A, 1.6; Ap6A, 1.6). In combination with the platelet products platelet-derived growth factor, epidermal growth factor, and serotonin, the dinucleotides synergistically increased DNA synthesis. Dinucleotides by themselves increased cell counts by 23% to 43% at day 2 and augmented mesangial cell growth induced by platelet-derived growth factor, epidermal growth factor, and serotonin. Furthermore, dinucleotides (100 mumol/L) rapidly induced a modest increase in expression of the early growth response gene Egr-1 at 30 minutes (x-fold increase over baseline control: Ap3A, 1.9; Ap4A, 2.8; Ap5A, 2.2; Ap6A, 2.1). We found that extracellular Ap4A was metabolized by mesangial cell ectoenzymes to mononucleotides and adenosine, which also have been shown to be mitogenic for mesangial cells. The combination of Ap4A with mononucleotides or adenosine failed to cause additive stimulation of DNA synthesis in mesangial cells. We conclude that diadenosine polyphosphates stimulate proliferation of cultured mesangial cells and augment mesangial cell growth induced by other mitogens released from platelets. Different molecular mechanisms may be involved in dinucleotide-induced mitogenesis of mesangial cells. Direct effects of dinucleotides on cultured mesangial cells. Direct effects of dinucleotides on cultured mesangial cells appear to play a role because dinucleotides rapidly caused activation of Egr-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular ATP augments mesangial cell growth induced by multiple growth factors.

BACKGROUND: ATP is released into the extracellular milieu during tissue injury and platelet aggregation and has a modest mitogenic effect on cultured rat glomerular mesangial cells (MCs). In this study we investigated the interaction of ATP with multiple growth factors during MC mitogenesis. METHODS: Replication of MCs was determined by direct cell counting and DNA synthesis was measured by 3H-thymidine uptake. Activity of phosphatidylinositol-specific phospholipase C (PI-PLC) was assessed by measuring formation of inositol phosphates from 3H-labelled precursors. RESULTS: Extracellular ATP (1-100 microM) exerted a powerful synergistic effect on DNA synthesis of MCs when used simultaneously with various mitogens, including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB), endothelin-1 (ET-1), arginine vasopressin (AVP), thrombin (Thr), serotonin (5-HT), and interleukin-1 beta (IL-1 beta). ATP synergized with these factors by increasing the maximum of DNA synthesis, without changing the half-maximal concentration of the growth factor. In addition cell counts revealed that ATP significantly augments MC growth induced by EGF, bFGF, PDGF-BB, ET-1, AVP, Thr, 5-HT, and IL-1 beta. ATP caused activation of PI-PLC, but did not synergize with any of the tested growth factors in this respect. ATP-induced PI-PLC activation was inhibited by preincubation with pertussis toxin by 40-93%. This treatment did not suppress DNA synthesis induced by ATP alone, by PDGF, or by PDGF plus ATP. The calcium antagonists, TMB-8 and verapamil, as well as the inhibitors of protein kinase C, calphostin C and H7, had no effect on DNA synthesis induced by ATP or PDGF plus ATP. Also, ATP synergistically stimulated DNA synthesis in combination with the direct activator of protein kinase C, phorbol myristate acetate (PMA), and with the reaction product of phospholipase D, phosphatidic acid. Finally, ATP was mitogenic in an MC line of higher passage which did not respond with PI-PLC activation. CONCLUSIONS: Extracellular ATP synergistically augments MC growth induced by multiple growth factors. While the ATP-induced mitogenic signal is presently unclear, our observations support the concept that ATP may play a role during the course of glomerulonephritis when multiple growth factors are released from glomerular and inflammatory cells.

P2U-purinergic receptor activation mediates inhibition of cAMP accumulation in cultured renal mesangial cells.

Extracellular ATP has been reported to exert mitogenic and contractile effects on cultured renal mesangial cells (MCs). Since it is possible that these actions involve changes in the cAMP second messenger system, we examined the effect of extracellular nucleotides on the accumulation of cAMP in rat MCs. ATP, UTP and adenosine 5'-0-(3-thio)triphosphate (ATP gamma S) (100 microM) had no significant effects on baseline cAMP levels, but inhibited forskolin-stimulated accumulation of cAMP by 21-75% in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Maximal inhibitory effects were observed at 100 microM of ATP gamma S with a threshold dose of 1 microM. ATP gamma S, ATP and UTP were the most potent inhibitors indicating stimulation of the P2u receptor. The P2x agonists adenosine 5'-(alpha, beta-methylene) triphosphate and adenosine 5'-(beta, gamma-methylene) triphosphate, and the P2y agonist 2-methylthio-ATP did not affect cAMP accumulation. Treatment with the P2 receptor antagonist suramin (200 microM) reduced the inhibition by 58%. The inhibitory effects of the nucleotides were significantly attenuated by preincubation with pertussis toxin (10-100 ng/ml). Inhibition of phospholipase C and protein kinase C did not prevent the inhibitory effect of the nucleotides. Inhibitors of forskolin-stimulated cAMP accumulation had different effects on DNA synthesis in cultured MCs as measured by 3H-thymidine uptake at 48 h: ATP, ATP gamma S and the inhibitor of adenylyl cyclase, SQ 22536, stimulated DNA synthesis in MCs, while UTP showed no significant mitogenic effect. Agents which increased baseline levels of intracellular cAMP (forskolin, IBMX, dibutyryl-cAMP) significantly diminished DNA synthesis in MCs. The results indicate that the P2u-purinergic receptor mediates inhibition of forskolin-induced cAMP accumulation which is likely due to inhibition of adenylyl cyclase. This effect appears to be partially mediated by PTX-sensitive G proteins. While the increase in cAMP accumulation is anti-mitogenic, inhibition of cAMP accumulation by P2u receptors is not correlated with MC growth control. Thus, additional mechanisms other than inhibition of cAMP accumulation by P2u receptors are likely to be involved in the mitogenesis of extracellular ATP.