Caveolin-1 deficiency protects against mesangial matrix expansion in a mouse model of type 1 diabetic nephropathy.

AIMS/HYPOTHESIS: Glomerular matrix protein accumulation, mediated largely by resident mesangial cells (MCs), is central to the pathogenesis of diabetic nephropathy. We previously showed that caveolin (CAV)-1/caveolae mediate matrix upregulation by MCs in response to high glucose and TGFß, two important pathogenic mediators of diabetic glomerular sclerosis. Here, we evaluated the in vivo role of CAV-1/caveolae in the development of diabetic nephropathy. METHODS: Diabetes was induced in Cav1-knockout (KO) mice and their wild-type (WT) counterparts by streptozotocin injection. After 10 months, kidneys were evaluated for the development of nephropathy, including glomerular sclerosis and upregulation of matrix proteins. Parallel experiments assessing glucose-induced matrix upregulation were carried out in MCs isolated from KO mice. RESULTS: KO diabetic mice developed hyperglycaemia and renal hypertrophy, but were protected from developing albuminuria and glomerular sclerosis compared with WT mice. KO mice were significantly protected from the upregulation of glomerular collagen I, fibronectin, connective tissue growth factor (CTGF) and TGFß. In vitro, glucose induced collagen I A1 promoter activation and collagen I, fibronectin and CTGF protein upregulation in WT but not KO MCs. Re-expression of Cav1 in KO cells restored this response. CONCLUSIONS/INTERPRETATION: Cav1 deletion rendered significant protection from glomerular matrix accumulation and albuminuria in a mouse model of type 1 diabetes. These studies provide a foundation for the development of renal-targeted interference with CAV-1/caveolae as a novel approach to the treatment of diabetic nephropathy.

High glucose-induced RhoA activation requires caveolae and PKCß1-mediated ROS generation.

Glomerular matrix accumulation is a hallmark of diabetic nephropathy. We previously showed that RhoA activation by high glucose in mesangial cells (MC) leads to matrix upregulation (Peng F, Wu D, Gao B, Ingram AJ, Zhang B, Chorneyko K, McKenzie R, Krepinsky JC. Diabetes 57: 1683-1692, 2008). Here, we study the mechanism whereby RhoA is activated. In primary rat MC, RhoA activation required glucose entry and metabolism. Broad PKC inhibitors (PMA, bisindolylmaleimide, Gö6976), as well as specific PKCß blockade with an inhibitor and small interfering RNA (siRNA), prevented RhoA activation by glucose. PKCß inhibition also abrogated reactive oxygen species (ROS) generation by glucose. The ROS scavenger N-acetylcysteine (NAC) or NADPH oxidase inhibitors apocynin and DPI prevented glucose-induced RhoA activation. RhoA and some PKC isoforms localize to caveolae. Chemical disruption of these microdomains prevented RhoA and PKCß1 activation by glucose. In caveolin-1 knockout cells, glucose did not induce RhoA and PKCß1 activation; these responses were rescued by caveolin-1 reexpression. Furthermore, glucose-induced ROS generation was significantly attenuated by chemical disruption of caveolae and in knockout cells. Downstream of RhoA signaling, activator protein-1 (AP-1) activation was also inhibited by disrupting caveolae, was absent in caveolin-1 knockout MC and rescued by caveolin-1 reexpression. Finally, transforming growth factor (TGF)-ß1 upregulation, mediated by AP-1, was prevented by RhoA signaling inhibition and by disruption or absence of caveolae. In conclusion, RhoA activation by glucose is dependent on PKCß1-induced ROS generation, most likely through NADPH oxidase. The activation of PKCß1 and its downstream effects, including upregulation of TGF-ß1, requires caveolae. These microdomains are thus important mediators of the profibrogenic process associated with diabetic nephropathy.

HB-EGF release mediates glucose-induced activation of the epidermal growth factor receptor in mesangial cells.

Glomerular matrix accumulation is a hallmark of diabetic nephropathy. We showed that transactivation of the epidermal growth factor receptor (EGFR) is an important mediator of matrix upregulation in mesangial cells (MC) in response to high glucose (HG). Here, we study the mechanism of EGFR transactivation. In primary MC, EGFR transactivation by 1 h of HG (30 mM) was unaffected by inhibitors of protein kinase C, reactive oxygen species, or the angiotensin II AT1 receptor. However, general metalloprotease inhibition, as well as specific inhibitors of heparin-binding EGF-like growth factor (HB-EGF), prevented both EGFR and downstream Akt activation. HB-EGF was released into the medium by 30 min of HG, and this depended on metalloprotease activity. One of the metalloproteases shown to cleave proHB-EGF is ADAM17 (TACE). HG, but not an osmotic control, activated ADAM17, and its inhibition prevented EGFR and Akt activation and HB-EGF release into the medium. siRNA to either ADAM17 or HB-EGF prevented HG-induced EGFR transactivation. We previously showed that EGFR/Akt signaling increases transforming growth factor (TGF)-ß1 transcription through the transcription factor activator protein (AP)-1. HG-induced AP-1 activation, as assessed by EMSA, was abrogated by inhibitors of metalloproteases, HB-EGF and ADAM17. HB-EGF and ADAM17 siRNA also prevented AP-1 activation. Finally, these inhibitors and siRNA prevented TGF-ß1 upregulation by HG. Thus, HG-induced EGFR transactivation in MC is mediated by the release of HB-EGF, which requires activity of the metalloprotease ADAM17. The mechanism of ADAM17 activation awaits identification. Targeting upstream mediators of EGFR transactivation including HB-EGF or ADAM17 provides novel therapeutic targets for the treatment of diabetic nephropathy.

EGFR-PLCgamma1 signaling mediates high glucose-induced PKCbeta1-Akt activation and collagen I upregulation in mesangial cells.

Glomerular matrix accumulation is a hallmark of diabetic nephropathy. We have recently shown that epidermal growth factor receptor (EGFR) transactivation mediates high glucose (HG)-induced collagen I upregulation through PI3K-PKCbeta1-Akt signaling in mesangial cells (MC). Phospholipase Cgamma1 (PLCgamma1) interacts with activated growth factor receptors and activates classic PKC isoforms. We thus studied its role in HG-induced collagen I upregulation in MC. Primary rat MC were treated with HG (30 mM) or mannitol as osmotic control. Protein kinase activation was assessed by Western blotting and collagen I upregulation by Northern blotting. Diabetes was induced in rats by streptozotocin. HG treatment for 1 h led to PLCgamma1 membrane translocation and Y783 phosphorylation, both indicative of its activation. Mannitol was without effect. PLCgamma1 Y783 phosphorylation was also seen in cortex and glomeruli of diabetic rats. HG induced a physical association between EGFR and PLCgamma1 as identified by coimmunoprecipitation. PLCgamma1 activation required EGFR kinase activity since it was prevented by the EGFR inhibitor AG1478 or overexpression of kinase-inactive EGFR (K721A). Phosphoinositide-3-OH kinase inhibition also prevented PLCgamma1 activation. HG-induced Akt S473 phosphorylation, effected by PKCbeta1, was inhibited by the PLCgamma inhibitor U73122. PLCgamma1 inhibition or downregulation by small interference RNA also prevented HG-induced collagen I upregulation. Our results indicate that EGFR-PLCgamma1 signaling mediates HG-induced PKCbeta1-Akt activation and subsequent collagen I upregulation in MC. Inhibition of EGFR or PLCgamma1 may provide attractive therapeutic targets for the treatment of diabetic nephropathy.

Collagen I induction by high glucose levels is mediated by epidermal growth factor receptor and phosphoinositide 3-kinase/Akt signalling in mesangial cells.

AIMS/HYPOTHESIS: Glomerular matrix accumulation is a hallmark of diabetic nephropathy. Recent data have linked the serine/threonine kinase protein kinase B (Akt) to matrix modulation. Here, we studied its role in high glucose-induced collagen elaboration by mesangial cells. METHODS: Primary rat mesangial cells were treated with high glucose levels (30 mmol/l) or mannitol as osmotic control. Western blots, northern blots, ELISA and immunohistochemistry were used for assessment. Diabetes was induced in rats by streptozotocin. RESULTS: Phosphorylated Akt at S473 (pAktS473), corresponding to Akt activation, was seen in diabetic glomeruli. In mesangial cells, high glucose levels induced pAktS473 by 20 min. This was sustained to 72 h, while mannitol had no effect. Akt activation by kinase assay and phosphorylation on threonine 308 was also observed. Phosphoinositide 3-kinase (PI3K) inhibitors LY294002 (20 micromol/l) and wortmannin (100 nmol/l) prevented pAktS473. Collagen IA1 transcript and collagen I protein upregulation by high glucose levels were inhibited by PI3K blockade, as was collagen I secretion into the medium (ELISA). Dominant-negative Akt overexpression also inhibited high glucose-induced collagen IA1 transcript and collagen I protein production. Since signalling through the epidermal growth factor receptor (EGFR) can activate PI3K-Akt, we studied its activation by high glucose levels. EGFR was correspondingly activated by 10 min; mannitol had no effect. EGFR activation was also seen in glomeruli from diabetic rats and co-localised with collagen IA1 in diabetic glomeruli. Specific EGFR inhibition (AG1478, 5 micromol/l or dominant-negative EGFR) blocked high glucose-induced pAktS473, phosphorylation on threonine 308 and activation of the EGFR downstream target p44 extracellular signal-regulated kinase (Erk) mitogen-activated protein kinase. Finally, EGFR inhibition also blocked high glucose-induced collagen I upregulation at transcriptional and protein levels. CONCLUSIONS/INTERPRETATION: We conclude that EGFR-PI3K-Akt signalling mediates high glucose-induced collagen I upregulation in mesangial cells and that this pathway is activated in diabetic glomeruli. Targeting its components may provide a new therapeutic approach to diabetic kidney disease.

Homocysteine induces mesangial cell apoptosis via activation of p38-mitogen-activated protein kinase.

Hyperhomocysteinemia is prevalent among patients with chronic kidney disease (CKD) and has been linked to progressive kidney and vascular diseases. Increased glomerular mesangial cell (MC) turnover, including proliferation and apoptosis, is a hallmark of CKD. Activation of p38-mitogen-activated protein kinase (p38-MAPK) has been linked to apoptosis in many cell lines. Accordingly, we studied the effect of homocysteine (Hcy) on MC p38-MAPK signalling and apoptosis. Hcy (50 microM/24 h) increased MC apoptosis as determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) and single-stranded DNA (ssDNA) analysis. In addition to increases in pro-caspase-3 protein and caspase-3 activity, cells exposed to Hcy manifested enhanced reactive oxygen species content. Hcy increased p38-MAPK activity (fivefold), with maximal effect at 50 microM and 20 min; p38-MAPK activation was attenuated by N-acetylcysteine (Nac) and catalase (Cat), further indicating that the effect was via oxidative stress. Confocal microscopy revealed activation and nuclear translocation of p38-MAPK that was attenuated by Cat. In addition, Hcy-induced apoptosis as determined by TUNEL and ssDNA assay was abrogated by Nac, Cat, and SB203580 (p38-MAPK inhibitor). We conclude that in MC, Hcy (i) activates p38-MAPK and increases p38MAPK nuclear translocation via an oxidative stress dependent mechanism and (ii) induces DNA damage and apoptosis that is dependent on oxidative stress and p38-MAPK activation.

Utility of ultrasonographic venous assessment prior to forearm arteriovenous fistula creation.

AIM: The purpose of this study was to evaluate the clinical utility of Doppler ultrasound (US) prior to native forearm arteriovenous fistula (AVF) creation. MATERIALS AND METHODS: US mapping was carried out pre-operatively to evaluate the major veins and arteries in the appropriate arm. One hundred and 6 patients were identified retrospectively over 2 years with complete clinical and US data. A failed fistula was defined as an inability to provide blood flow to meet adequacy targets by 6 months (urea reduction ratio > or = 65%). RESULTS: Twenty-nine patients (27.4%) had successful forearm AVFs. The mean minimum forearm cephalic vein diameter (CVD) was 2.51 +/- 0.14 and 2.23 +/- 0.06 mm in successful and failed fistulae, respectively (p = 0.04). This result was primarily due to differences observed in women. A receiver operator curve analysis showed that a cutpoint of 2.6 mm for minimum forearm CVD had the greatest predictive value with a likelihood ratio of 3.94 (95% CI: 1.97 - 7.84) for fistula failure. Multivariate logistic regression analysis determined that male gender and minimum forearm CVD were the only significant predictors for fistula success with odds ratios of 3.90 (95% CI: 1.30 - 11.68) and 2.31 (95% CI: 1.00 - 5.43), respectively. The study is limited by the possibility that US results in patients may have lead to an alternative type of access being attempted. CONCLUSIONS: US mapping prior to forearm AVF creation is of modest benefit. Only male gender and minimum forearm CVD were predictive of AVF success.

The clinical utility of Doppler ultrasound prior to arteriovenous fistula creation.

Arteriovenous fistula (AVF) is the preferred access for long-term hemodialysis, with superior long-term patency rates; however, early failure rates are significant. Recent evidence has brought into question the preferred site of AVF creation in many patient groups. A preoperative test that could reliably predict the outcome of a proposed AVF would be of great benefit. Doppler ultrasound has been the most extensively studied and widely used test to guide access creation. Accurate and validated measurements of internal vessel diameter, both arterial and venous, and blood flow in the upper extremity are obtainable by Doppler ultrasound. Studies evaluating the utility of Doppler ultrasound prior to AVF creation suggest that vessel size and blood flow are predictive of AVF outcome. An AVF created using a cephalic vein and/or radial artery smaller than 1.5-2.0 mm is likely to fail; such preoperative data may indicate that an upper arm AVF should be the primary access attempted. Further prospective studies are needed to evaluate the utility of Doppler ultrasound.

Thrombolysis for restoration of patency to haemodialysis central venous catheters: a systematic review.

Urokinase, previously used to restore patency to thrombosed haemodialysis catheters, is now unavailable in North America. We performed systematic reviews of four questions related to the safety and efficacy of alternative agents for catheter thrombolysis, searching Medline and the Cochrane Controlled Clinical Trials Register. In dialysis patients, large case series have documented that urokinase is safe and effective (>70 % efficacy for catheter instillation, and >80 % for systemic lysis). Experience with streptokinase is limited and allergic complications develop with repeated use. Studies of catheter instillation with 1--2 mg of tPA per lumen reported short-term success in 83--98 % of uses. One non-peer-reviewed study described 44--59 % success using systemic tissue plasminogen activator (tPA), 2.5 mg through each of 2 lumens, over 1 h. Meta-analysis of randomized comparisons of urokinase and tPA as full-dose thrombolytic agents suggested that 1 mg tPA was likely equivalent in thrombolytic potency to 36,000 units urokinase. In nondialysis populations, four case series suggested that catheter instillation with 0.5--2 mg tPA was effective and safe in reestablishing patency, and a randomized controlled trial found 2--4 mg tPA more effective than 5,000--10,000 units urokinase. No complications have been reported in any patient treated with systemic or local tPA for catheter thrombolysis. In studies of fistula thrombolysis with 5--50 mg tPA major complications occurred in one episode in 130 patients treated. This review suggests that 1--2 mg/lumen tPA is a suitable dose for catheter instillation and likely to be more effective than 5000 units/lumen urokinase. Systemic lysis with 5--10 mg tPA is likely to be safe and effective in suitably selected patients. Further studies are needed.

Akt is activated in response to an apoptotic signal.

Akt is a serine-threonine kinase known to exert antiapoptotic effects through several downstream targets. Akt is cleaved during mitochondrial-mediated apoptosis in a caspase-dependent manner. The reason for this is not clear, however, because Akt has not been demonstrated to be activated in response to mitochondrial apoptotic stimuli. Accordingly, we explored whether the well described mitochondrial apoptotic stimuli staurosporine (STS) and etoposide activate Akt and whether such activation impacts apoptosis. Both STS and etoposide activated Akt in NIH 3T3 cells, maximally at 8 and 2 h, respectively, preceding the onset of apoptosis and poly(ADP-ribose) polymerase cleavage. The overexpression of Akt delayed STS-induced apoptosis with an even more pronounced delay observed with overexpression of constitutively active Akt. Akt activation by proapoptotic stimuli lay upstream of mitochondria, because neither caspase inhibitors nor overexpression of Bcl-2 or Bcl-x(L) could prevent it. Activation depended on phosphatidylinositol 3-kinase activity, however. Conversely, inhibition of phosphatidylinositol 3-kinase with wortmannin sensitized cells to apoptosis initiated by STS. These data demonstrate that mitochondrial apoptotic stimuli also activate Akt and such activation modulates apoptosis in this setting.

Nitric oxide modulates stretch activation of mitogen-activated protein kinases in mesangial cells.

BACKGROUND: In vivo, intraglomerular hypertension results in resident cell hypertrophy, proliferation and matrix protein production, leading to glomerulosclerosis. Mesangial cells (MCs) exposed to in vitro stretch also proliferate and produce matrix. We have shown activation of Jun N-terminal kinase/stress-activated protein kinase (SAPK) and p42/44 mitogen-activated protein kinase (MAPK) in stretched MCs and have also demonstrated that L-arginine decreases resident cell proliferation and protects against glomerulosclerosis in remnant kidney glomeruli, presumably by increasing nitric oxide (NO) production. Consequently, we studied whether NO could affect SAPK and p42/44 MAPK activation in stretched MCs. METHODS: MCs (passages 5 to 10) cultured on type 1 collagen-coated, flexible-bottom plates were exposed to 0 to 30 minutes of cyclic strain (60 cycles per minute) by computer-driven generation of vacuum of -27 kPa, inducing 28% elongation in the diameter of the surface. Control MCs were grown on coated, flexible-bottom plates. Protein levels (by Western blot) and activity assays for SAPK/JNK and p42/44 MAPK were performed under these conditions. As maximal activation was at 10 minutes, with decay by 30 minutes, the effect of NO on kinase activation was studied at 0, 2, 5, and 10 minutes by preincubation with 70 micromol/L s-nitroso-n-acetylpenicillamine (SNAP; an NO donor) or 1 mmol/L 8-bromo cyclic guanosine monophosphate (8-bromo-cGMP). Downstream events in response to stretch and NO were studied at the time of maximal response (10 minutes) by examining nuclear translocation of SAPK with immunofluorescence microscopy and transcription factor activator protein-1 nuclear protein binding by gel mobility shift assay. The effect of kinase inhibition by NO donors on MC proliferation was studied by Western blotting for proliferating cell nuclear antigen (PCNA). RESULTS: Cyclic MC stretch led to prompt SAPK and p42/44 MAPK activation, which was maximal at 10 minutes. Preincubation with either SNAP or 8-bromo-cGMP decreased this by 50 and 70%, respectively (N = 4), suggesting that the effect of NO was through cGMP generation. Nuclear translocation of both phosphorylated kinases was seen after 10 minutes of stretch and was largely prevented by 8-bromo-cGMP. Increased DNA binding of activator protein-1 proteins was observed in the nuclei of stretched MCs at 10 minutes by mobility shift assay (N = 4), which was also largely prevented by 8-bromo-cGMP. Stretch increased PCNA expression by MCs, and this was inhibited by 8-bromo-cGMP. CONCLUSIONS: Stretch-induced activation of SAPK and p42/44 MAPK in MCs can be inhibited by NO. The effect of NO is mediated by the generation of cGMP. These mechanisms may be responsible, at least in part, for the protective effect of NO in animal models of glomerular injury characterized by glomerular capillary hypertension.

Stretch activation of jun N-terminal kinase/stress-activated protein kinase in mesangial cells.

BACKGROUND: Mesangial cells (MCs) grown on extracellular matrix (ECM)-coated plates and exposed to cyclic stretch/relaxation proliferate and produce ECM protein, suggesting that this may be a useful in vitro model for MC behavior in response to increased physical forces. The induction of c-fos in response to MC stretch has been shown. Stimuli that lead to c-fos induction pass through mitogen-activated protein (MAP) kinase pathways. We have seen early activation of jun N-terminal kinase/stress-activated protein kinase (SAPK/JNK) in MCs exposed to cyclic stretch. Accordingly, we studied SAPK/JNK activation in stretched MCs and the downstream consequences of this signaling. METHODS: MCs (passages 5 to 10) cultured on type 1 collagen-coated, flexible-bottom plates were exposed to 2 to 60 minutes of cyclic strain (60 cycles per minute) by generation of vacuums of -10 to -27 kPa, inducing approximately 16 to 28% maximum elongation in the diameter of the surfaces. Control MCs were grown on coated rigid bottom plates. Protein levels (by Western blot) and activity assays for SAPK/JNK were performed under these conditions. We observed marked activation at -18 kPa and above and at two minutes, and then we studied activation mechanisms under these conditions. Nuclear protein binding to activator protein-1 (AP-1) consensus sequences was also examined. The role of calcium was studied with EGTA and BAPTA-AM to chelate extra- and intracellular calcium, respectively. Protein kinase C (PKC) was down-regulated by incubation with phorbol ester (PMA) for 24 hours prior to stretch. In unstretched MCs, A23187 was used as a calcium ionophore, and PKC was up-regulated with PMA application for 30 minutes to determine the effects on SAPK/JNK. Nuclear protein binding to AP-1 was also determined under these conditions. The effects of stretch, acute PMA, and A23187 on fibronectin mRNA levels were studied using reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Cyclic strain/relaxation led to increased SAPK/JNK activity only at two minutes and -18 kPa and above. The activation of SAPK/JNK was dependent on intracellular calcium, with BAPTA-AM almost completely abrogating the response to stretch. EGTA was without effect. Down-regulation of PKC also led to a diminution of activity. In static cells, the calcium ionophore A23187 increased SAPK/JNK activity, and this was potentiated by acute PMA. Stretch, acute PMA, and A23187 all increased nuclear protein binding to AP-1 consensus sequences. mRNA levels for fibronectin were increased by stretch in MCs and by PMA and A23187 in static MCs. No change was observed in the amount of SAPK/JNK protein present in stretched MCs by Western blot. CONCLUSIONS: Stretch leads to early activation of SAPK/JNK in MCs. This is dependent on intracellular calcium and PKC and can be replicated by activation of these stimuli in static MCs. A downstream induction of nuclear protein binding to AP-1 consensus sequences was seen in a pattern that was completely concordant with the SAPK/JNK induction.

NO inhibits stretch-induced MAPK activity by cytoskeletal disruption.

Mesangial cells (MC) grown on extracellular matrix protein-coated plates and exposed to cyclic strain/relaxation proliferate and produce extracellular matrix protein, providing an in vitro model of signaling in stretched MC. Intracellular transduction of mechanical strain involves mitogen-activated protein kinases, and we have shown that p42/44 mitogen-activated protein kinase (extracellular signal-regulated kinase (ERK)) is activated by cyclic strain in MC. In vivo studies show that increased production of nitric oxide (NO) in the remnant kidney limits glomerular injury without reducing glomerular capillary pressure, and we have observed that NO attenuates stretch-induced ERK activity in MC via generation of cyclic guanosine monophosphate (cGMP). Accordingly, we sought to determine whether NO affects strain-induced ERK activity after strain and how this is mediated. Strain-induced ERK activity was dependent on time and magnitude of stretch and was maximal after 10 min at -27 kilopascals. Actin cytoskeleton disruption with cytochalasin D abrogated this. The non-metabolizable cGMP analogue 8-bromo cyclic GMP (8-Br-cGMP) dose-dependently attenuated strain-induced ERK activity. Cytoskeletal stabilization with jasplakinolide prevented this inhibitory effect of 8-Br-cGMP. Cyclic strain increased nuclear translocation of phospho-ERK by immunofluorescent microscopy, again attenuated by 8-Br-cGMP. Jasplakinolide prevented the inhibitory effect of 8-Br-cGMP on activated ERK nuclear translocation after strain. Strain increased ERK-dependent AP-1 nuclear protein binding, which was attenuated by cytochalasin D and 8-Br-cGMP. These data indicate that cGMP can inhibit cyclic strain-induced ERK activity, nuclear translocation, and AP-1 nuclear protein binding. Cytoskeletal disruption leads to the same effect, whereas cytoskeleton stabilization reverses the effect of 8-Br-cGMP. Thus, NO inhibits strain-induced ERK activity by cytoskeletal destabilization.

Nitric oxide modulates mechanical strain-induced activation of p38 MAPK in mesangial cells.

Mesangial cells (MC), grown on extracellular matrix (ECM) protein-coated plates and stretched, proliferate and produce ECM, recapitulating in vivo responses to increased glomerular capillary pressure (Pgc). Transduction of strain involves mitogen-activated protein kinases (MAPK), and we have shown that p38 MAPK is activated by strain in MC. Because in vivo studies show that nitric oxide (NO) in the remnant kidney limits glomerular injury without reducing Pgc, we studied whether NO attenuated stretch-induced p38 activation in MC. Increasing p38 activation occurred with increasing stretch, maximally at 10 min at -27-kPa vacuum. Cyclic strain increased nuclear translocation of phosphorylated p38 by immunofluorescent microscopy and nuclear protein binding to nuclear factor-kappaB (NF-kappaB) consensus sequences by mobility shift assay. Both events were largely abrogated by the p38 inhibitor SB-203580. The NO donors 3-morpholinosydnonimine, S-nitroso-N-acetylpenicillamine, and 8-bromoguanosine 3',5'-cyclic monophosphate, a stable cGMP analog, prevented p38 activation and nuclear translocation. Thus strain induces p38 activity and translocation to the nucleus and p38-dependent increases in nuclear protein binding to NF-kappaB. This pathway is attenuated by the NO donors or a cGMP analog.

DNA adducts produced by oils, oil fractions and polycyclic aromatic hydrocarbons in relation to repair processes and skin carcinogenesis.

Ten polycyclic aromatic hydrocarbons (PAHs) mainly with three or four aromatic rings were tested for their ability to induce DNA adduct formation in mouse skin. Four of these were selected to investigate adduct formation and loss over a period of 8 days. Three mineral oils were also examined for their adduct forming ability and one was selected for adduct formation and loss over a period of 8 days. In addition, fractions derived from the same oil containing 2-3- and 4-6-ring aromatic compounds were applied to mouse skin in a non-carcinogenic oil vehicle and adduct levels were observed over an 8-day period. It was found that PAHs that had no mutagenic, initiating or carcinogenic activity and those that had mutagenic activity in bacteria but no initiating activity in mouse skin failed to produce DNA adducts in mouse skin. Two of the three PAHs with initiating activity and both complete carcinogens produced clear evidence of adduct formation, the adduct levels produced by complete carcinogens being 100-1000 times greater than those produced by initiators. Examination of adduct formation and loss with the carcinogenic PAHs benzo[a]pyrene and 5-methylchrysene over an 8-day period showed a peak at 24 h and an apparent two-phase process of adduct loss. It is suggested that the first steep loss was due to DNA repair and that the more gradual subsequent loss was probably due to epidermal hyperplasia and desquamation. With the initiator 1, 4-dimethylphenanthrene (three rings) a peak of adduct formation was seen at 2 days and adduct levels were not reduced much by 8 days. This suggested that, with initiators, adduct formation and repair may be spread over a longer period than with complete carcinogens. With the whole oils, clear evidence of adduct formation was seen with both a carcinogenic non-solvent-refined oil and with a non-carcinogenic residual oil. The level of adduct formation with the residual oil, however, was much lower than with the carcinogenic oil. When adduct formation by the carcinogenic oil was examined over 8 days, the pattern of adduct formation and loss was similar to that of a tumour initiator rather than a complete carcinogen. Peak adduct levels on the diagonal of the thin-layer chromatography (TLC) plates seemed to occur at 1 and 4 days after treatment, with no clear reduction after 8 days. From examination of adducts formed by the 2-3-ring and 4-6-ring aromatic fractions, it appeared that the main adduct spots produced by the carcinogenic oil were due to the 2-3-ring aromatic components of the oil. Adduct spots near the vertical axis of the TLC plates were also seen with the 2-3-ring and 4-6-ring fractions. The relevance of these spots is uncertain, but if they truly represent adducts, the findings suggest that they are due mainly to 4-ring PAHs. The studies suggest that the activity of carcinogenic oils is largely due to substituted 3- and 4-ring polycyclic aromatic compounds and that more attention should be paid to substituted 3-ring compounds in predicting the carcinogenic potential of oils from analytical data.

Prolonged sulfonylurea-induced hypoglycemia in diabetic patients with end-stage renal disease.

Renal impairment is a recognized risk factor for prolonged hypoglycemia, but predisposing characteristics in patients with advanced renal impairment have not been studied. We observed prolonged hypoglycemia in a number of patients with end-stage renal disease (ESRD) and conducted a case-control study at two Canadian centers to identify such risk factors. Through hospital, pharmacy, and dialysis program records, we retrospectively identified 7 case patients and 31 controls with ESRD and type 2 diabetes using oral hypoglycemic monotherapy. Control patients had no history of hospital admission for prolonged hypoglycemia. All case patients and 28 controls were receiving glyburide (glibenclamide in Europe); the remainder were treated with tolbutamide. Duration of intravenous treatment for hypoglycemia ranged from 28 to 256 hours, with 83 g to 2 kg of glucose administered per episode. Preceding treatment with glyburide varied from 2 days to 13 years. Univariate analyses showed a recent decline in oral intake (odds ratio [OR], 81; 95% confidence interval [CI], 3.6 to 1,840), previous hypoglycemic episodes (OR, 15; 95% CI, 0.77 to 297), longer duration of diabetes (22 versus 12 years; P = 0.008), and a history of cerebrovascular disease (OR, 7. 0; 95% CI, 1.0 to 47) to be associated with prolonged hypoglycemia. No association between prolonged hypoglycemia and age, sex, beta blockers, angiotensin-converting enzyme inhibitors, oral hypoglycemic dose, or duration of treatment was identified. This study describes the potentially devastating effect of sulfonylurea-based oral hypoglycemic therapy in ESRD. Patients at greatest risk appear to be those with reduced intake, previous hypoglycemic episodes, and longer duration of diabetes. We describe the mechanisms for observed hypoglycemia and suggest that alternative drugs may be considered in this patient group.

Stress-responsive signal transduction mechanisms in glomerular cells.

Mechanical stresses appear to play a key role in the progression of glomerular diseases that are characterized by increased transcapillary hydraulic pressure. Glomerular mesangial cells proliferate and produce extracellular matrix proteins in vivo in such diseases. Mesangial cell responses to pulsatile mechanical stimuli have been studied extensively in vitro during the past few years. Mechanical signals are sensed at the cell membrane and propagated through the cytoplasm, and result in the activation of transcription factors that elicit production of prosclerotic cytokines and matrix proteins, and cell proliferation. Endothelial cells are exposed to shear and pulsatile stress and show some similar responses in other vascular beds.

Metabolic investigation of recurrent nephrolithiasis: compliance with recommendations.

OBJECTIVES: Nephrolithiasis is a recurrent condition with significant associated morbidity and economic impact. Although urologic intervention addresses symptomatic stone episodes, prevention of recurrences with proven medical therapy is indicated. METHODS: This retrospective study examined 97 patients who presented in 1997 and 1998 with recurrent nephrolithiasis in a large tertiary care center for the presence of an appropriate metabolic investigation as recommended by the National Institutes of Health Consensus Conference. Complete data were abstracted from the hospital and private clinic charts. RESULTS: The average patient age was 50.5 years; 61.9% of patients were men. The mean number of stones per patient was 5.6 (range 2 to 62), with stone analysis performed for 78 patients. Fifty-eight stones (74.4%) were calcium oxalate and/or phosphate, 14 (17.9%) urate, 8 (10.3%) struvite, and 3 (3.8%) cystine. Five patients had two stone types on different occasions. Either lithotripsy or a urologic procedure was required for at least one stone presentation in 89 patients (91.8%). An investigation for stone disease was pending in 54 patients (55.7%). A complete evaluation, satisfying the preset criteria, was performed in 34 patients (35.1%). Six patients who did not undergo evaluation were lost to follow-up. Univariate analysis revealed that referral to a nephrologist (P = 0.001), treatment with medications used for stone disease (P = 0.008), and urate stones (P = 0.005) were associated with a complete investigation. Similarly, these were independently associated with a complete evaluation in regression analysis of 77 complete data sets, with odds ratios of 24.4 (nephrology referral), 4.9 (medication use), and 5.6 (urate stones). CONCLUSIONS: The results of this study demonstrate that a significant proportion of patients with recurrent nephrolithiasis do not undergo appropriate metabolic investigations. Efforts should be made to improve the evaluation of these patients.

Mesangial cell signaling cascades in response to mechanical strain and glucose.

BACKGROUND: Elevated glucose levels and glomerular hypertension (Pgc) are considered to contribute to the elaboration of matrix protein by mesangial cells (MCs) in diabetic glomeruli. MCs grown in 30 mM of glucose produce excessive matrix protein, as do MCs exposed to cyclic strain, and the combination of the two exacerbates this. Tight glucose control or reduction in Pgc clinically delays progression of diabetic nephropathy. MC c-fos is induced in response to either application of strain or high ambient glucose, inducing increases in activated protein-1 transactivational activity and extracellular matrix production. Stimuli that lead to c-fos induction pass through the three mitogen-activated protein (MAP) kinase pathways: p44/42, SAPK/JNK, and p38/HOG. We studied MAP kinase activation in MCs exposed to mechanical strain and a high glucose. METHODS: MCs (passage 5 through 10) cultured for 96 hours on type 1 collagen-coated flexible-bottom plates in either 5.6 or 30 mM glucose were exposed to 5, 10, or 30 minutes of cyclic strain (60 cycles per min) by computer-driven generation of vacuums of -14 kPa, inducing 20% elongation in the diameter of the surface. Control MCs were grown on both coated rigid and flexible-bottom plates. Protein levels (by Western blot) and activity assays for all three kinase cascades were performed at baseline and after 5, 10, and 30 minutes. All experiments were performed in triplicate. RESULTS: MAP kinase signaling was seen in response to stretch, and high ambient glucose affected the pattern of activation. Both p44/42 and p38HOG kinase activities showed small increases to a maximum of 2.5- to 3.5-fold greater than static MCs at 10 minutes. Activity in both kinase cascades was slightly suppressed by 30 mM glucose. In contrast, SAPK/JNK activity was present at a very low level in static MCs and increased markedly by 10 minutes of stretch. Thirty micromolars of glucose augmented this effect by a factor of six over MCs cultured in 5.6 mM glucose after 10 minutes of stretch. Neither glucose concentration nor mechanical strain had any effect on the protein expression of any of the kinases by Western blot. CONCLUSIONS: MAP kinase cascade signaling is seen when physical force is applied to MCs, and glucose affects the pattern of activity. Thirty micromolars of glucose markedly increase the level of SAPK/JNK activation. This may have implications in diabetic signal transduction and matrix protein production.

Activation of mesangial cell signaling cascades in response to mechanical strain.

BACKGROUND: Mesangial cells (MCs) are constantly exposed to pulsatile stretch and relaxation in their role as architectural support for the glomerulus. There is no cell proliferation in normal glomeruli. In contrast, animal models of increased glomerular capillary pressure are characterized by resident glomerular cell proliferation and elaboration of extracellular matrix (ECM) protein, resulting in glomerulosclerosis. This process can be ameliorated by maneuvers, such as angiotensin converting enzyme inhibition, that reduce glomerular capillary pressure. MCs grown on ECM-coated plates and exposed to cyclic stretch/relaxation proliferate and produce ECM protein, suggesting that this may be a useful in vitro model for MC behavior in response to increased physical forces. Previous work has shown induction of c-fos in response to application of mechanical strain to MCs, which may induce increases in AP-1 transcription factor activity, which, in turn, may augment ECM protein and transforming growth factor beta transcription and cell proliferation. Stimuli that lead to c-fos induction pass through mitogen-activated protein kinase (MAPK) pathways. Three MAPK cascades have been characterized in mammalian cells--p44/42 (classic MAPK), the stress-activated protein kinase/Jun terminal kinase (SAPK/JNK) pathway, and p38/HOG--and mechanical strain activates p44/42 and SAPK/JNK in cardiac fibroblasts. However, in contrast to MCs, these cells do not proliferate in response to physical force. Accordingly, we studied activation of the MAPK pathways in MCs exposed to mechanical strain. METHODS: MCs (passages 5 to 10) cultured on type 1 collagen-coated, flexible-bottom plates were exposed to 30, 60, or 120 minutes of cyclic strain (60 cycles/min) by computer-driven generation of vacuums of -14 and -28 kPa, inducing 20% and 29% elongations in the diameter of the surfaces, respectively. Control MCs were grown on coated rigid bottom plates. Proliferation was assessed at 24 hours by 3H-thymidine incorporation. Protein levels (by Western blot) and activity assays for all three kinase cascades were performed at 30, 60, and 120 minutes. RESULTS: Cyclic strain/relaxation lead to an approximate doubling of 3H-thymidine incorporation at 24 hours (N = 3, P < 0.05) only in cultures stretched 29%, but not in cultures stretched 20%. At -29% elongation, the increase in 3H-thymidine incorporation was preceded by early activation of MAPK signaling pathways. p44/42 activity increased to a maximum of eightfold greater than control at 60 minutes. p38/HOG activity was not measurable at baseline but was increased markedly at 30 minutes, which was sustained through to 120 minutes. SAPK/JNK activity was present at a very low level in MCs and was not changed by stretch. However, it was markedly increased by sorbitol. In MCs stretched to 20% elongation, lesser increases in p44/42 were seen with a similar time course, whereas no increases in p38/HOG or SAPK could be detected at the time points studied. No increase in any kinase pathway activity was seen at any time in static cultures. CONCLUSIONS: High-pressure cyclic stretch leads to MC proliferation, preceded by marked activation of p44/42 and p38/HOG MAPKs. Cell proliferation is not seen with low-pressure stretch, and there is only modest p44/42 MAPK activation, suggesting that glomerular capillary hypertension may lead to cell proliferation and injury partly through differential activation of kinase cascades.