Maternal western diet primes non-alcoholic fatty liver disease in adult mouse offspring.

AIM: Metabolic programming via components of the maternal diet during gestation may play a role in the development of different aspects of the metabolic syndrome. Using a mouse model, we aimed to characterize the role of maternal western-type diet in the development of non-alcoholic fatty liver disease (NAFLD) in the offspring. METHODS: Female mice were fed either a western (W) or low-fat control (L) semisynthetic diet before and during gestation and lactation. At weaning, male offspring were assigned either the W or the L diet, generating four experimental groups: WW, WL, LW and LL offspring. Biochemical, histological and epigenetic indicators were investigated at 29 weeks of age. RESULTS: Male offspring exposed to prenatal and post-weaning western-style diet (WW) showed hepatomegaly combined with accumulation of hepatic cholesterol and triglycerides. This accumulation was associated with up-regulation of de novo lipid synthesis, inflammation and dysregulation of lipid storage. Elevated hepatic transaminases and increased expression of Tnfa, Cd11, Mcp1 and Tgfb underpin the severity of liver injury. Histopathological analysis revealed the presence of advanced steatohepatitis in WW offspring. In addition, alterations in DNA methylation in key metabolic genes (Ppara, Insig, and Fasn) were detected. CONCLUSION: Maternal dietary fat intake during early development programmes susceptibility to liver disease in male offspring, mediated by disturbances in lipid metabolism and inflammatory response. Long-lasting epigenetic changes may underlie this dysregulation.

Pharmacological activation of LXR in utero directly influences ABC transporter expression and function in mice but does not affect adult cholesterol metabolism.

Cholesterol is critical for several cellular functions and essential for normal fetal development. Therefore, its metabolism is tightly controlled during all life stages. The liver X receptors-alpha (LXRalpha; NR1H3) and -beta (LXRbeta; NR1H2) are nuclear receptors that are of key relevance in coordinating cholesterol and fatty acid metabolism. The aim of this study was to elucidate whether fetal cholesterol metabolism can be influenced in utero via pharmacological activation of LXR and whether this would have long-term effects on cholesterol homeostasis. Administration of the LXR agonist T0901317 to pregnant mice via their diet (0.015% wt/wt) led to induced fetal hepatic expression levels of the cholesterol transporter genes Abcg5/g8 and Abca1, higher plasma cholesterol levels, and lower hepatic cholesterol levels compared with controls. These profound changes during fetal development did not affect cholesterol metabolism in adulthood nor did they influence coping with a high-fat/high-cholesterol diet. This study shows that the LXR system is functional in fetal mice and susceptible to pharmacological activation. Despite massive changes in fetal cholesterol metabolism, regulatory mechanisms involved in cholesterol metabolism return to a "normal" state in offspring and allow coping with a high-fat/high-cholesterol diet.

Mice expressing only the mutant APOE3Leiden gene show impaired VLDL secretion.

Apolipoprotein E (apoE)-deficient mice develop hepatic steatosis and show impaired very low density lipoprotein (VLDL)-triglyceride (TG) secretion. These effects are normalized on the introduction of the human APOE3 gene. To assess whether this apoE effect is isoform specific, we studied hepatic lipid metabolism in mice expressing either APOE3 or the mutant APOE3Leiden on apoe-/- or apoe+/- backgrounds. The transgenes were expressed mainly in periportal hepatocytes, as revealed by in situ hybridization. Mice expressing APOE3Leiden, on the apoe-/- and apoe+/- backgrounds, had fatty livers, which were absent in APOE3/apoe-/- mice. APOE3Leiden/apoe-/- mice showed a strongly reduced VLDL-TG secretion compared with APOE3/apoe-/- mice (48+/-14 versus 82+/-10 micromol/kg per hour, respectively). The presence of a single mouse apoe allele increased VLDL-TG secretion in APOE3Leiden/apoe+/- mice (121+/-43 micromol/kg per hour) compared with APOE3Leiden/apoe-/- mice. These results show that APOE3Leiden does not prevent development of a fatty liver and does not normalize VLDL-TG secretion in mice with an apoE-deficient background. The presence of a single mouse apoe allele is sufficient to normalize the APOE3Leiden-associated reduction of VLDL-TG secretion but does not prevent steatosis. We conclude that apoE-mediated stimulation of VLDL secretion is isoform specific.

Acute inhibition of hepatic glucose-6-phosphatase does not affect gluconeogenesis but directs gluconeogenic flux toward glycogen in fasted rats. A pharmacological study with the chlorogenic acid derivative S4048.

Effects of acute inhibition of glucose-6-phosphatase activity by the chlorogenic acid derivative S4048 on hepatic carbohydrate fluxes were examined in isolated rat hepatocytes and in vivo in rats. Fluxes were calculated using tracer dilution techniques and mass isotopomer distribution analysis in plasma glucose and urinary paracetamol-glucuronide after infusion of [U-(13)C]glucose, [2-(13)C]glycerol, [1-(2)H]galactose, and paracetamol. In hepatocytes, glucose-6-phosphate (Glc-6-P) content, net glycogen synthesis, and lactate production from glucose and dihydroxyacetone increased strongly in the presence of S4048 (10 microm). In livers of S4048-treated rats (0.5 mg kg(-1)min(-)); 8 h) Glc-6-P content increased strongly (+440%), and massive glycogen accumulation (+1260%) was observed in periportal areas. Total glucose production was diminished by 50%. The gluconeogenic flux to Glc-6-P was unaffected (i.e. 33.3 +/- 2.0 versus 33.2 +/- 2.9 micromol kg(-1)min(-1)in control and S4048-treated rats, respectively). Newly synthesized Glc-6-P was redistributed from glucose production (62 +/- 1 versus 38 +/- 1%; p < 0.001) to glycogen synthesis (35 +/- 5% versus 65 +/- 5%; p < 0.005) by S4048. This was associated with a strong inhibition (-82%) of the flux through glucokinase and an increase (+83%) of the flux through glycogen synthase, while the flux through glycogen phosphorylase remained unaffected. In livers from S4048-treated rats, mRNA levels of genes encoding Glc-6-P hydrolase (approximately 9-fold), Glc-6-P translocase (approximately 4-fold), glycogen synthase (approximately 7-fold) and L-type pyruvate kinase (approximately 4-fold) were increased, whereas glucokinase expression was almost abolished. In accordance with unaltered gluconeogenic flux, expression of the gene encoding phosphoenolpyruvate carboxykinase was unaffected in the S4048-treated rats. Thus, acute inhibition of glucose-6-phosphatase activity by S4048 elicited 1) a repartitioning of newly synthesized Glc-6-P from glucose production into glycogen synthesis without affecting the gluconeogenic flux to Glc-6-P and 2) a cellular response aimed at maintaining cellular Glc-6-P homeostasis.

Induction of experimental proteinuria in vivo following infusion of human plasma hemopexin.

BACKGROUND: The human plasma constituent hemopexin (Hx), following incubation with renal tissue, is able to induce glomerular alterations in vitro that are similar to those seen in minimal change disease (MCD). Whether this acute phase reactant is also able to induce proteinuria and minimal change-like alterations in vivo is questioned. METHODS: In the first set of experiments, Hx (4.0 mg in 5.0 mL saline) or equal amounts of control fraction, that is, heat-inactivated Hx (HI-Hx), were infused into conscious rats (N = 6) that had been surgically equipped with a cannula inserted into the suprarenal artery (SRA), enabling direct contact of the infusate and the renal microvasculature. Each animal received HI-Hx at day 1 for 15 minutes (flow rate 20.0 mL/h), subsequently followed by saline for seven hours (Flow rate 5.0 mL/h), after which the cannula was disconnected. At day 2, identical infusions in the same rat were carried out, using native Hx. Urine samples collected every 30 minutes during the experiments were monitored for protein content using standard methods. In the second set of experiments, unilateral perfusion was done ex vivo in anesthetized rats with Hx (N = 5) or HI-Hx (N = 3; 1.5 mg/mL; 4.0 mL during 6 min). After reconnection of the circulation, urine samples of both kidneys were collected every 30 minutes during five hours via ureter cannulation. Urinary protein (expressed as the difference in excretion between perfused and nonperfused kidney) was calculated in mg/24 h. In additional experiments, rats were sacrificed two hours after perfusion of Hx or heat-inactivated (control) Hx (first set of experiments) or after five hours (second set of experiments), and kidneys were processed for immunohistochemical and ultrastructural examination. RESULTS: The results of experiment 1 show a significant increase of proteinuria after Hx infusion versus HI-Hx (means +/- SD, 41.91 +/- 16.01 mg/24 h vs. control, 21.22 +/- 5.69 mg/24 h; P

Is 100KF an isoform of hemopexin? Immunochemical characterization of the vasoactive plasma factor 100KF.

The human vasoactive plasma factor 100KF has been proposed to play a role in minimal change disease in relapse. Since preliminary data suggested similarity between 100KF and the human plasma glycoprotein hemopexin (Hx), this study was conducted to compare 100KF with purified Hx for sequence homology, immunostaining properties in Western and dot-blot assays, ability to affect glomerular ecto-ATPase and glomerular polyanions in vitro, as well as their glomerular permeability increasing effect following alternate perfusion into the rat kidney ex vivo. 100KF was purified from normal pooled plasma according to standard chromatographic techniques, and from the same batch Hx was prepared using affinity chromatography. A second batch of Hx was prepared directly from human serum according to a standard protocol. (For comparison, additional Hx samples obtained from other centers were also included in the study.) The results show: (1) 100% homology of 100KF with plasma Hx after internal sequence analysis; (2) positive staining of the eluate with both monoclonal and polyclonal anti-Hx IgG as well as anti-100KF IgG in dot-blot assays, and similar bands on Western blotting using the same antibodies; (3) affection of glomerular polyanions and glomerular ecto-ATPase after incubation of kidney tissue with either 100KF or Hx (1.5 respectively 1.0 mg/ml; 1.0 h, 37 degrees C), as detected by computerized histochemical quantification; and (4) significant enhancement of urinary protein leakage after Hx perfusion followed by diluted rat serum into the rat kidney ex vivo (Hx: 210.65+/-49.79 microg protein leakage per min versus heat-inactivated Hx control: 112.2+/-49.18 microg per min [both n = 6]). From these data and from the observation that both Hx and 100KF activity can be inhibited by serine protease inhibitors but not by broad spectrum collagenase inhibitors, it is concluded that Hx may be closely related or identical to the active moiety of 100KF.

Pregnancy enhances the sensitivity of glomerular ecto-adenosine triphosphate-diphosphohydrolase to products of activated polymorphonuclear leukocytes.

To test the hypothesis that pregnancy enhances the sensitivity of glomerular ecto-adenosine triphosphate-diphosphohydrolase to products of activated polymorphonuclear leukocytes, cryostat-cut kidney sections of pregnant and cycling rats were exposed to activated polymorphonuclear leukocytes and subsequently stained for ecto-adenosine triphosphate-diphosphohydrolase activity. The results show that the levels of ecto-adenosine triphosphate-diphosphohydrolase activity of pregnant rats showed a significantly greater decrease after incubation with activated polymorphonuclear leukocytes than did those of cycling rats.

Oxygen-dependent injury by a human plasma factor associated with minimal change disease.

The mechanism by which a human plasma factor associated with proteinuria is able to cause experimental glomerular albumin leakage is unknown. This factor (called 100KF) is able to induce glomerular alterations in the rat kidney, similar to those seen in minimal change disease, including loss of glomerular sialoglycoproteins and decreased expression of glomerular ecto-ATPase. It was previously shown that 100KF is able to stimulate release of reactive oxygen species in inflammatory cells in vitro. This prompted us to test whether 100KF-induced injury is oxygen dependent. The expression of glomerular sialoglycoproteins and ecto-ATPase was evaluated by standard histochemistry and computerized image analysis and expressed in arbitrary units. Rat kidney sections were incubated with or without 100KF under normal or oxygen-poor, i.e., nitrogen, conditions, or with supplementation of superoxide dismutase (SOD, 100 U/ml). The effect of 100KF on glomerular ecto-ATPase was oxygen dependent (32.98+/-2.14 under air vs. 65.20+/-5.53 under nitrogen, P< or =0.01), in contrast to the 100KF-induced loss of glomerular sialoglycoproteins that was not significantly altered under nitrogen (62.67+/-10.08 under air vs. 61.74+/-26.05 under nitrogen). Supplementation of SOD to 100KF solution under normal incubation conditions also suggested oxygen-dependent impairment of glomerular ecto-ATPase. Alternate perfusion ex vivo of the rat kidney with 100KF followed by diluted plasma showed that enhanced leakage of plasma proteins could be inhibited with SOD, indicating oxygen dependency of this 100KF-induced enhanced permeability (60.25+/-19.32 microg urinary albumin/ml after 100KF perfusion vs. 25.23+/-12.05 microg/ml after 100KF plus SOD, P< or =0.01). We conclude that the action of 100KF upon specific glomerular matrix molecules is oxygen dependent, as is the albumin leakage induced by 100KF in the present ex vivo model.

Aspirin treatment of the low-dose-endotoxin-treated pregnant rat: pathophysiologic and immunohistologic aspects.

In the present study, we evaluated the effect of low-dose aspirin (acetylsalicylic acid (ASA); 1.0 mg/kg daily) on blood pressure, albumin excretion, glomerular fibrinogen deposits, and glomerular (basement) membrane-bound adenosine diphosphatase (ecto-ADPase) activity, as well as on glomerular inflammation in pregnant rats infused with low-dose endotoxin (1.0 mg/kg). Rats (day 14 of pregnancy) were infused with endotoxin (ET rats) or saline (control rats) and received ASA in their drinking water. These rats were compared with non-ASA-treated rats. Blood pressure and albumin excretion were measured from day 15 to day 21, and glomerular fibrinogen and ecto-ADPase activity were measured at day 21. Glomerular inflammation was evaluated at various times after the start of the infusion. The results show that treatment with ASA had a significant beneficial effect on hypertension and inflammation induced by endotoxin in pregnant rats, whereas it reduced albumin excretion and glomerular fibrinogen deposits in some of the rats.

Plasma endothelin-1 and tumor necrosis factor-alpha concentrations in pregnant and cyclic rats after low-dose endotoxin infusion.

Plasma endothelin-1 and tumor necrosis factor-alpha were determined in pregnant and cyclic rats after infusion of either endotoxin (1.0 microgram/kg of body weight) or saline solution. After endotoxin, but not after saline solution, administration there was a transient endothelin-1 response in pregnant rats but not cyclic rats. In both reproductive conditions there was an equally high transient tumor necrosis factor-alpha response after endotoxin.

Modulation of glomerular ECTO-ADPase expression by oestradiol. A histochemical study.

The effect of 17-beta-oestradiol (OE2) upon the activity of the glomerular anti-thrombotic ecto-enzyme ADPase was studied in cyclic and ovariectomized (OVX) Wistar rats. On day 0 (i.e. at the time of ovariectomy or 11 days after ovariectomy) rats received OE2-releasing Silastic implants or empty implants and were sacrificed on day 3, 10 or 21. Cryostat kidney sections were histochemically stained for ecto-ADPase activity using enzyme-histochemistry and glomerular reaction product was quantitatively evaluated by computerized image analysis. Both the histological distribution of reaction product in each glomerulus, as reflected by the relative glomerular area covered with reaction product, as well as enzyme activity, as reflected by staining intensity of the reaction product, were scored. The results show significantly decreased histological distribution after OVX; OVX, however, did not change enzyme activity. It further appeared that OE2 (partly) prevented the decrease of histological distribution in OVX rats, while the enzyme activity was significantly increased by exogenous OE2. In cyclic rats, OE2 did not change histological distribution, although OE2 significantly increased enzyme activity in these rats. It is concluded that glomerular ecto-ADPase expression in the rat kidney is influenced by one or more ovarian factor(s), a very likely candidate being oestradiol. These results may thus point to a dual action of OE2 upon haemostasis: In addition to the known enhancement of procoagulatory plasma factors by OE2, also anti-aggregatory effects may be stimulated by OE2 as reflected by upregulation of vessel wall associated ecto-ADPase activity.

Impairment of endothelial and subendothelial sites by a circulating plasma factor associated with minimal change disease.

BACKGROUND: The pathogenesis of albuminuria in minimal change disease (MCD) is unknown. A human plasma factor (denoted as 100KF) is able to induce minimal change-like glomerular alterations, i.e. loss of glomerular sialoglycoproteins and decreased expression of glomerular ecto-ATPase, following in vitro incubation with kidney tissue. In addition, increased (selective) permeability for plasma proteins occurs after perfusion of 100KF into the rat kidney ex vivo. As in kidney tissue from subjects with minimal change disease, subendothelial injury has also been observed, i.e. reduced anionic sites in the lamina rara interna, the question was raised whether injury induced by the plasma factor 100KF involves vascular endothelium or subendothelial matrix of the glomerular capillary wall. METHODS: Permeability studies were carried out by using confluent endothelial monolayers (HUVEC) cultured on a standard two-compartment system. The permeability for a macromolecular marker (horse radish peroxidase) was tested following incubation of the monolayers with either the native plasma factor 100KF or the control factor (heat-inactivated 100KF), in combination with histochemical evaluation of the cells for ecto-ATPase expression. Also quantification of glomerular anionic sites at the ultrastructural level was carried out, after ex vivo perfusion of 100KF or control factor into rat kidneys. RESULTS: The plasma factor 100KF is able to increase the permeability of human endothelial monolayers for macromolecules in a dose-dependent manner (relative increase 122.4 +/- 24, 178.4 +/- 34 and 236.1 +/- 58% after preincubation with 0.05; 0.5 and 1.5 mg/ml 100KF respectively), concomitant with induction of minimal change-like histochemical alterations such as reduced expression of ecto-ATPase. The number of anionic sites in the lamina rara interna of the glomerular capillary wall is significantly diminished following perfusion with the plasma factor 100KF versus control factor (7.58 +/- 1.60 versus 12.57 +/- 2.05 per 1000 nm; P < or = 0.02); in contrast to the lamina rara externa (22.71 +/- 3.15 versus 22.27 +/- 2.92 per 1000 nm; statistically not significant). CONCLUSIONS: Endothelial cells and subendothelial matrix along the glomerular filtration barrier may be considered as target structures for the plasma factor 100KF, leading to initial minimal change-like alterations associated with glomerular albumin leakage.

The increased endotoxin-sensitivity of pregnant rats, as reflected by glomerular ecto-ADP-ase activity, is not dependent on the presence of decidual cells.

In the present study the possible role of decidual cells in the pregnancy-associated increased sensitivity of glomerular ecto-ADP-ase to endotoxin was investigated. Early (day 5) pregnant (E-Pr; n = 10), pseudopregnant (E-PSP; n = 10), (day 5), pseudopregnant rats with a decidualized uterus (E-DEC; n - 10), as well as late (day 14) pregnant (L-Pr; n = 10), pseudopregnant (L-PSP: n = 10) (day 14), and pseudopregnant rats with a decidualized uterus (E-DEC; n = 10) were infused with either endotoxin (1.0 mg/kg bw) or saline. Three days later rats were killed and specimens of the left kidney were snap-frozen. Cryostat kidney sections (4 microns) were stained for ecto-ADP-ase activity and quantitatively evaluated. The results show that only glomerular ecto-ADP-ase activity of both groups of pregnant rats (E-Pr and L-Pr) was significantly decreased after endotoxin infusion as compared to saline infusion. In the other groups of rats, no significant differences in ecto-ADP-ase activity were observed between saline and endotoxin infusion. It is concluded that decidual cells do not play a role in the increased sensitivity of ecto-ADP-ase to endotoxin during pregnancy.

Glomerular inflammation in pregnant rats after infusion of low dose endotoxin. An immunohistological study in experimental pre-eclampsia.

Increased endotoxin sensitivity during pregnancy occurs in many animals, including rats. The mechanism of this phenomenon is not understood. In the present study it was investigated whether this increased sensitivity is reflected by an altered inflammatory pattern. Inflammatory cell influx, the O2(-)-producing potential of these cells, and expression of adhesion molecules was studied in the glomeruli of pregnant and cyclic rats at various intervals after low dose endotoxin infusion. Kidney sections were stained for monocytes and adhesion molecules (ICAM-1, VCAM-1, LFA-1, and VLA-4) using monoclonals, while potentially O2(-)-producing neutrophils (ie, activated neutrophils) were quantified using immunohistochemical methods. The results show early glomerular influx of activated neutrophils, maximally 4 hours after endotoxin. Both absolute neutrophil counts and relative numbers of activated neutrophils were significantly increased in pregnant versus cyclic rats. In contrast to cyclic rats, showing transient monocyte influx, in pregnant endotoxin-treated rats monocyte influx reaches a maximum at t = 168 hours. These cell kinetics were paralleled by expression of the various adhesion molecules. It was concluded that pregnancy profoundly influences not only the inflammation kinetics after endotoxin, but also the violence of the reaction, reflected by activated neutrophils. This altered glomerular inflammatory pattern may help to explain why low dose endotoxin infusion induces pre-eclamptic-like symptoms (such as an intraglomerular prothrombotic microenvironment and proteinuria) exclusively in the pregnant rat.

A new animal model for human preeclampsia: ultra-low-dose endotoxin infusion in pregnant rats.

OBJECTIVE: An animal model for preeclampsia was developed by means of an ultra-low-dose endotoxin infusion protocol in conscious pregnant rats. STUDY DESIGN: Rats received a permanent jugular vein cannula on day 0 of pregnancy, through which endotoxin (1.0 micrograms/kg body weight) (n = 10) or saline solution (n = 6) was infused during 1 hour on day 14 of pregnancy. Blood pressure, albuminuria, and platelet counts were measured, and histopathologic studies was performed in these rats. RESULTS: A significant increase of blood pressure (p < 0.05) and of urinary albumin excretion (p < 0.05) was observed in endotoxin-treated pregnant animals, in contrast to control pregnant rats receiving saline solution. Platelet coagulopathy was found and glomerular fibrinogen deposits could be detected only in the endotoxin-treated pregnant rats. In addition, the activity of the glomerular antithrombotic enzyme adenosine diphosphatase was decreased in endotoxin-treated pregnant rats compared with saline solution-treated pregnant rats. CONCLUSION: Because histopathologic and clinical events in this model mimic predominant features of human preeclampsia, this model may enable further study into the pathophysiologic mechanisms of this complication of pregnancy.

A modified cerium-based histochemical method for detection of experimentally-induced ATPase impairment in glomeruli of the rat kidney.

We studied glomerular ATPase activity, as detectable at the light microscopic (LM) level in cryostat sections of the rat kidney, after unilateral local X-irradiation. The biochemically detectable reduction in glomerular ATPase activity after X-irradiation could be demonstrated at the LM level by application of a modified cerium-based technique. Results show a clear reduction of reaction product in glomeruli in X-irradiated kidneys as compared with the contralateral control kidney. Technical parameters (i.e., tissue fixation, second thickness, cerium concentration of the incubation mixture, and percentage H2O2 added for the amplification step) were established for optimal reproducibility of the staining results. We show that this modified staining protocol allows detection of differences of ATPase activity in contrast to conventional histochemical methods. Inhibition studies with various phosphatase inhibitors and competitive substrate inhibition experiments revealed that the enzyme is specific for nucleoside di- and triphosphatases. Since reduced glomerular adenine nucleotidase activity has recently been recognized as an early event in (experimental) glomerulonephritis, we feel that the new staining protocol presented here may be highly relevant for routine tissue section screening in nephropathological research.

Attenuation of anti-Thy1 glomerulonephritis in the rat by anti-inflammatory platelet-inhibiting agents.

Although both ecto-ADPase and prostacyclin (PGI2) inhibit platelets and neutrophils, their action in acute glomerulonephritis is unknown. We tested the PGI2 analog Iloprost and 2chloroadenosine (2Cl-ADO), an analog of adenosine, the end product of nucleotidase activities, during anti-Thy1 nephritis. Rats received anti-Thy1 immunoglobulin G (5 mg/kg body weight, intravenously) and subsequently one subcutaneous injection of either 2Cl-ADO (10 mg/kg body weight; (n = 6) or Iloprost (1 mg/kg body weight; n = 6). Control rats received anti-Thy1 immunoglobulin G with saline (n = 6) or saline alone (n = 6). After 24 hours, kidneys were processed for light-microscopical evaluation. Proteinuria was studied in additional rats. Results showed that both drugs inhibited intraglomerular platelet activation (P < 0.005). 2Cl-ADO also reduced intraglomerular O2- production of neutrophils (P < 0.05), in contrast to Iloprost. Intraglomerular immunoglobulin G deposition, complement activation, neutrophil influx, and myeloperoxidase release were not affected by 2Cl-ADO or Iloprost. However, proteinuria was completely prevented by both drugs. It is concluded that PGI2 and nucleotidases are potentially able to attenuate this form of nephritis by inhibiting platelet activity, whereas nucleotidases also inhibit neutrophil activity in vivo.

Modulation of anti-Thy1 nephritis in the rat by adenine nucleotides. Evidence for an anti-inflammatory role for nucleotidases.

Extracellular adenine nucleotides are considered mediators of inflammation because they modulate functions of neutrophils and platelets. Until now, this role for adenine nucleotides has not been studied in vivo. In particular in the rat kidney, where ATP- and ADPase activity is present in the glomerular basement membrane, studies about the role of nucleotides may increase our understanding of the dynamics of glomerulonephritis (GN). Therefore, we examined effects of adenine derivatives ATP gamma S, ADP beta S and 2chloro-adenosine (2chloro-ADO) in vitro and during anti-Thy1 GN. The in vitro results show that ADP beta S and ATP gamma S are not degraded by glomerular nucleotidases but, on the other hand do stimulate O2- production of peritoneal exudate cells (PEC). In contrast, 2chloro-ADO significantly inhibits O2- production of peritoneal exudate cells. For in vivo studies rats were rendered nephritic by intravenous injection of monoclonal anti-Thy1 IgG (5 mg/kg body weight). Subsequently, rats were treated with saline (group 1, N = 10), 2chloro-ADO (group 2, N = 10), ADP beta S (group 3, N = 10) or ATP gamma S (group 4, N = 10). All analogs (10 mg/kg body weight) were administered both at t = 0 and t = 12 hour. After 24 hours, rats were sacrificed and kidneys were examined histochemically. In an additional group of nephritic rats (N = 5) proteinuria was studied after 2-chloro-ADO treatment. Results show increased intraglomerular platelet aggregation in nephritic rats treated with ADP beta S, whereas 2chloro-ADO inhibits aggregation significantly as compared with nephritic rats receiving saline. The percentage of granulocytes producing O2- is significantly increased in glomeruli after treatment of nephritic rats with ATP gamma S, whereas cell influx itself is not changed. In contrast, 2chloro-ADO inhibits intraglomerular O2- production, which is associated with the complete inhibition of proteinuria in the early phase of anti-Thy1 GN. These data demonstrate significant pro-inflammatory activities of extracellular adenine nucleotides during anti-Thy1 GN suggesting an anti-inflammatory role for glomerular ATP/ADPase, which in concert with 5' nucleotidase converts ATP and ADP to antiinflammatory ADO.

Demonstration of antithrombotic activity of glomerular adenosine diphosphatase.

We have demonstrated that reduced glomerular adenosine diphosphatase (ADPase) activity within the rat kidney is associated with an increased thrombotic tendency. To establish a possible causal relationship between these intraglomerular events, experiments were conducted to inhibit adenosine diphosphate (ADP) degradation without influencing other glomerular prothrombotic or antithrombotic mechanisms. Concurrently, we studied intraglomerular platelet aggregation. Two ways of selective inhibition of glomerular ADPase activity were applied: (1) by competitive substrates (ie, uridine diphosphate [UDP]), and (2) by the nondegradable ADP analogue ADP-beta-S. Both strategies were used during ex vivo alternate perfusion of kidneys with platelets and ADP (to test intraglomerular thrombotic tendency). Each group (n = 6) received different substrates or a combination of substrates. A significant increase in platelet aggregation was observed in kidneys after perfusion with platelets and ADP together with the competitive substrate UDP as compared to perfusions with platelets and ADP alone (78.5% +/- 9.8% v 27.9% +/- 11.4% glomeruli staining positive for platelets, P less than .005). In contrast, UDP alone had no effect on platelet aggregation. Other nucleoside polyphosphates (guanosine diphosphate and inosine triphosphate) were also effective as competitive substrates in the ex vivo perfusion model (n = 4). None of these substrates was capable of increasing ADP-induced aggregation when studied in vitro. In addition, ADP- beta-S also increased platelet aggregation in the perfusion model as compared with native ADP (P less than .005). These results show that selective reduction of ADP degradation in intact kidneys strongly promotes the intraglomerular proaggregatory condition. It can be concluded that glomerular ADPase exerts potent antithrombotic activity within the normal rat kidney.

Intraglomerular thrombotic tendency and glomerular ADPase. Unilateral impairment of ADPase elicits a proaggregatory microenvironment in experimental glomerulonephritis.

It has been proposed, predominantly from ex vivo studies, that glomerular ADPase may function as an antithrombotic principle within the rat kidney. Therefore, intraglomerular platelet aggregation was studied in vivo in rats after impairment of glomerular ADPase activity using local X-irradiation (20 Gy). Biochemical assays in suspensions of glomeruli obtained from rats 24 hours after local X-irradiation (group I) demonstrated a significant reduction in ADPase activity as compared to sham treated rats (group II; p less than 0.01). Cytochemical observations at the ultrastructural level showed that this reduction in glomerular enzyme activity represents in particular ADPase activity detectable in the basement membrane. Following X-irradiation, intraglomerular platelet aggregation was quantitatively studied in two groups of rats. Both groups received X-irradiation of the left kidney (20 Gy). Twenty-four hours after X-irradiation, animals received an intravenous injection of either 0.5 ml of saline (group III; N = 6) or 0.5 ml of heterologous nephrotoxic serum (NTS; group IV; N = 6). Subsequently, 24 hours after this injection, platelet aggregation in left kidneys was compared with aggregation in contralateral non-X-irradiated kidneys. The results showed that while X-irradiation per se did not induce intraglomerular platelet aggregation as compared with the contralateral kidney (0.20 +/- 0.08% versus 0.17 +/- 0.06% platelet aggregation/glomerulus), a significant increase in platelet aggregation could be demonstrated in X-irradiated kidneys in the early phase of nephrotoxic serum nephritis as compared with the contralateral nephritic kidney (2.45 +/- 0.66% versus 1.37 +/- 0.35% platelet aggregation per glomerulus; p less than 0.005). A potential effect of altered influx of inflammatory cells after X-irradiation could be excluded since no difference in H2O2 producing cells was observed between left and right kidneys. Thus, while ADPase impairment by X-irradiation does not induce platelet aggregation per se, it is clear that in proaggregatory conditions, like in NTS nephritis, the thrombotic tendency, due to decreased glomerular ADPase, is enhanced. These results demonstrate the functional significance of glomerular ADPase activity as an antithrombotic principle following platelet activation in vivo.