Exploring the Motivational Drivers of Young Adults with Diabetes for Participation in Kidney Research.

Understanding motivational drivers and barriers to patient participation in diabetes research are important to ensure research is relevant and valuable. Young adults with type 1 diabetes (T1D) completed a 31-question qualitative survey evaluating participant experience, understanding, and motivators and barriers to research involvement. A total of 35 participants, 19-28 years of age, 60% female, completed the survey. Motivating factors included personal benefit, relationship with the study team, curiosity, financial compensation, altruism, and nostalgia. Older participants (>22 years) reported higher levels of trust in the study team (p = 0.02) and their relationship with the study team positively influenced their decision to participate (p = 0.03). Financial compensation was a strong motivator for participants with higher education (p = 0.02). Age, sex, education level, and trust in the study team influenced participants' understanding. Barriers included logistics and lack of familial support. Important motivational drivers and barriers to participation in research by young adults with T1D must be considered to increase research engagement and facilitate the discovery of new knowledge.

Plasma uric acid effects on glomerular haemodynamic profile of patients with uncomplicated Type 1 diabetes mellitus.

AIMS: Increased plasma uric acid (PUA) levels are associated with impaired renal function in patients with Type 1 diabetes, but the mechanisms are not well understood. Our aim was to evaluate whether higher PUA levels are associated with increased afferent arteriolar resistance in patients with Type 1 diabetes vs. healthy controls, thereby influencing renal function. METHODS: PUA, GFR (inulin) and effective renal plasma flow (ERPF; para-aminohippurate) were measured in 70 otherwise healthy patients with Type 1 diabetes and 60 healthy controls. Gomez's equations were used to estimate afferent (RA ) and efferent (RE ) arteriolar resistances, glomerular hydrostatic pressure (PGLO ) and filtration pressure (ΔPF ). The relationships between PUA and glomerular haemodynamic parameters were evaluated by univariable linear regression correlation coefficients. RESULTS: In patients with Type 1 diabetes, higher PUA correlated with lower PGLO (P = 0.002) and ΔPF (P = 0.0007), with higher RA (P = 0.001), but not with RE (P = 0.55). These associations were accompanied by correlations between higher PUA with lower GFR (P = 0.0007), ERPF (P = 0.008), RBF (P = 0.047) and higher RVR (P = 0.021). There were no significant correlations between PUA and renal haemodynamic parameters in the healthy controls. CONCLUSIONS: The association between higher PUA with lower GFR and lower ERPF in patients with Type 1 diabetes is driven by alterations in the estimated RA . PUA-mediated RA may be caused by increased tone or thickening of the afferent renal arteriole, which might potentiate renal injury by causing ischaemia to the renal microcirculation.

NADPH oxidase inhibition prevents beta cell dysfunction induced by prolonged elevation of oleate in rodents.

AIMS/HYPOTHESIS: The activation of NADPH oxidase has been implicated in NEFA-induced beta cell dysfunction. However, the causal role of this activation in vivo remains unclear. Here, using rodents, we investigated whether pharmacological or genetic inhibition of NADPH oxidase could prevent NEFA-induced beta cell dysfunction in vivo. METHODS: Normal rats were infused for 48 h with saline or oleate with or without the NADPH oxidase inhibitor apocynin. In addition, NADPH oxidase subunit p47(phox)-null mice and wild-type littermate controls were infused with saline or oleate for 48 h. This was followed by measurement of NADPH oxidase activity, reactive oxygen species (ROS) and superoxide imaging and assessment of beta cell function in isolated islets and hyperglycaemic clamps. RESULTS: Oleate infusion in rats increased NADPH oxidase activity, consistent with increased total but not mitochondrial superoxide in islets and impaired beta cell function in isolated islets and during hyperglycaemic clamps. Co-infusion of apocynin with oleate normalised NADPH oxidase activity and total superoxide levels and prevented beta cell dysfunction. Similarly, 48 h NEFA elevation in wild-type mice increased total but not mitochondrial superoxide and impaired beta cell function in isolated islets. p47(phox)-null mice were protected against these effects when subjected to 48 h oleate infusion. Finally, oleate increased the levels of total ROS, in both models, whereas inhibition of NADPH oxidase prevented this increase, suggesting that NADPH oxidase is the main source of ROS in this model. CONCLUSIONS/INTERPRETATION: These data show that NADPH-oxidase-derived cytosolic superoxide is increased in islets upon oleate infusion in vivo; and whole-body NADPH-oxidase inhibition decreases superoxide in concert with restoration of islet function.

The effect of renal hyperfiltration on urinary inflammatory cytokines/chemokines in patients with uncomplicated type 1 diabetes mellitus.

AIMS/HYPOTHESIS: High intraglomerular pressure causes renal inflammation in experimental models of diabetes. Our objective was to determine whether renal hyperfiltration, a surrogate for intraglomerular hypertension, is associated with increased excretion of urinary cytokines/chemokines in patients with type 1 diabetes mellitus. METHODS: Blood pressure, renal haemodynamic function (inulin and para-aminohippurate clearances for glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), respectively) and urine samples were obtained during clamped euglycaemia in individuals with type 1 diabetes with either hyperfiltration (GFR determined using inulin [GFRINULIN] ≥ 135 ml min⁻¹ 1.73 m⁻², n = 28) or normofiltration (n = 21) and healthy control individuals (n = 18). RESULTS: Baseline clinical characteristics, dietary sodium and protein intake and blood pressure levels were similar in the diabetic and healthy control groups. In addition, HbA1c levels were similar in the two diabetic groups. As expected baseline GFR was higher in hyperfilterers than either normofiltering diabetic patients or healthy control patients (165 ± 9 vs 113 ± 2 and 116 ± 4 ml min⁻¹ 1.73 m⁻², respectively, p < 0.01). ERPF and renal blood flow were also comparatively higher and renal vascular resistance was lower in hyperfiltering patients (p < 0.01). Hyperfiltering diabetic patients had higher excretion rates for eotaxin, IFNα2, macrophage-derived chemokine, platelet-derived growth factor (PDGF)-AA, PDGF-AB/BB and granulocyte-macrophage colony-stimulating factor (p ≤ 0.01). Urinary monocyte chemoattractant protein (MCP)-1 and RANTES (regulated on activation, normal T expressed and secreted) excretion was also higher in hyperfiltering vs normofiltering diabetic individuals (p < 0.01) and fibroblast growth factor-2, MCP-3 and CD40K excretion was elevated in hyperfiltering diabetic individuals vs healthy controls (p < 0.01). CONCLUSIONS/INTERPRETATION: Renal hyperfiltration is associated with increased urinary excretion of inflammatory cytokines/chemokines in patients with uncomplicated type 1 diabetes.

Deletion of p47phox attenuates the progression of diabetic nephropathy and reduces the severity of diabetes in the Akita mouse.

AIMS/HYPOTHESIS: Reactive oxygen species (ROS) contribute to diabetes-induced glomerular injury and endoplasmic reticulum (ER) stress-induced beta cell dysfunction, but the source of ROS has not been fully elucidated. Our aim was to determine whether p47(phox)-dependent activation of NADPH oxidase is responsible for hyperglycaemia-induced glomerular injury in the Akita mouse, a model of type 1 diabetes mellitus resulting from ER stress-induced beta cell dysfunction. METHODS: We examined the effect of deleting p47 (phox) (also known as Ncf1), the gene for the NADPH oxidase subunit, on diabetic nephropathy in the Akita mouse (Ins2 (WT/C96Y)) by studying four groups of mice: (1) non-diabetic mice (Ins2 (WT/WT)/p47 (phox+/+)); (2) non-diabetic p47 (phox)-null mice (Ins2 (WT/WT)/p47 (phox-/-)); (3) diabetic mice: (Ins2 (WT/C96Y)/p47 (phox+/+)); and (4) diabetic p47 (phox)-null mice (Ins2 (WT/C96Y)/p47 (phox-/-)). We measured the urinary albumin excretion rate, oxidative stress, mesangial matrix expansion, and plasma and pancreatic insulin concentrations in 16-week-old mice; we also measured glucose tolerance and insulin sensitivity, islet and glomerular NADPH oxidase activity and subunit expression, and pro-fibrotic gene expression in 8-week-old mice. In addition, we measured NADPH oxidase activity, subunit expression and pro-fibrotic gene expression in high glucose-treated murine mesangial cells. RESULTS: Deletion of p47 (phox) reduced kidney hypertrophy, oxidative stress and mesangial matrix expansion, and also reduced hyperglycaemia by increasing pancreatic and circulating insulin concentrations. p47 (phox-/-) mice exhibited improved glucose tolerance, but modestly decreased insulin sensitivity. Deletion of p47 (phox) attenuated high glucose-induced activation of NADPH oxidase and pro-fibrotic gene expression in glomeruli and mesangial cells. CONCLUSIONS/INTERPRETATION: Deletion of p47 (phox) attenuates diabetes-induced glomerular injury and beta cell dysfunction in the Akita mouse.

Urinary markers of renal inflammation in adolescents with Type 1 diabetes mellitus and normoalbuminuria.

AIMS: Patients with the highest albumin:creatinine ratio within the normal range are at an increased risk for developing microalbuminuria. The mechanistic basis for this is unknown, but may be related to renal inflammation. Our goal was to characterize the urinary excretion of cytokines/chemokines in normoalbuminuric adolescents with Type 1 diabetes to determine whether higher range normoalbuminuria is associated with evidence of renal inflammation. METHODS: Forty-two urinary cytokines/chemokines were measured in subjects who were screened for the Adolescent Type 1 Diabetes Cardio-Renal Intervention Trial. Urinary cytokines/chemokines were compared across low (n = 50), middle (n = 50) or high (n = 50) albumin:creatinine ratio tertile groups. RESULTS: At baseline, participants in the upper tertile were younger and had shorter diabetes duration compared with the other groups. Other clinical characteristics were similar. Urinary levels of interleukin 6, interleukin 8, platelet-derived growth factor-AA and RANTES differed across albumin:creatinine ratio tertiles, with higher values in patients in the middle and high tertiles compared with the lower tertile (ANCOVA P ≤ 0.01). CONCLUSIONS: Within the normal albumin:creatinine ratio range, higher urinary albumin excretion is associated with elevated urinary levels of inflammatory markers. Ultimately, this may provide mechanistic insights into disease pathophysiology and stratify the risk of nephropathy in Type 1 diabetes.

The effect of oral contraceptives on the nitric oxide system and renal function.

We have demonstrated that oral contraceptive (OC) users exhibit elevated angiotensin II levels and angiotensin II type 1 receptor expression, indicative of renin-angiotensin system (RAS) activation, yet the renal and systemic consequences are minimal, suggesting that there is increased vasodilatory activity, counteracting the effect of RAS activation. We hypothesized that the nitric oxide (NO) system would be upregulated in OC users and that this would be reflected by a blunted hemodynamic response to l-arginine infusion. All subjects were studied after a 7-day controlled sodium and protein diet. Inulin and para-aminohippurate clearance techniques were used to assess renal function. l-Arginine was infused at 100, 250, and 500 mg/kg, each over 30 min. Skin endothelial NO synthase mRNA expression was assessed by real-time PCR. While OC nonusers exhibited significant increases in effective renal plasma flow (670.8 +/- 35.6 to 816.2 +/- 59.7 ml.min(-1).1.73 m(-2)) and glomerular filtration rate (133.4 +/- 4.3 to 151.0 +/- 5.7 ml.min(-1).1.73 m(-2), P = 0.04) and declines in renal vascular resistance (81.1 +/- 6.1 to 63.5 +/- 6.2 mmHg.ml(-1).min, P = 0.001) at the lower l-arginine infusion rates, the responses in OC users were blunted. While l-arginine reduced mean arterial pressure at the 250 and 500 mg/kg doses in OC nonusers, OC users only exhibited a decrease in mean arterial pressure at the highest infusion rate. In contrast, tissue endothelial NO synthase mRNA levels were higher in the OC users (P = 0.04). In summary, these findings suggest that the NO system is upregulated by OC use in young, healthy women. Increased activity of the NO pathway may modulate the hemodynamic effects of RAS activation in OC users.

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.

Angiotensin II activates the GFAT promoter in mesangial cells.

Expression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme for glucose entry into the hexosamine pathway, is transcriptionally regulated. Immunohistochemical studies of human kidney biopsies demonstrate increased GFAT expression in diabetic glomeruli, but the mechanism responsible for this overexpression is unknown. Given the role of ANG II in diabetic kidney disease, we chose to study the effect of ANG II on GFAT promoter activity in mesangial cells (MC). Exposure of MC to ANG II (10(-7) M) increased GFAT promoter activity (2.5-fold), mRNA (3-fold), and protein (1.6-fold). ANG II-mediated GFAT promoter activation was inhibited by the ANG II type I receptor antagonist candesartan (10(-8) M) but was unaffected by the ANG II type II receptor antagonist PD-123319 (10(-8) M). The intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (10(-6) M), protein kinase C (PKC) inhibitors bisindoylmaleimide-4 (10(-6) M) and calphostin C (10(-7) M), protein tyrosine kinase (PTK) inhibitor genistein (10(-4) M), Src family kinase inhibitor PP2 (2.5 x 10(-7) M), p42/44 mitogen-activated protein kinase (MAPK) inhibitor PD-98059 (10(-5) M), and the epidermal growth factor (EGF) inhibitor AG-1478 all attenuated GFAT promoter activation by ANG II. We conclude that the GFAT promoter is activated by ANG II via the AT1 receptor. Promoter activation is calcium dependent and PKC dependent but also involves PTK signaling pathways including Src, the EGF receptor, and p42/44 MAPK.

Effect of oral contraceptives on the renin angiotensin system and renal function.

We examined the effect of oral contraceptive (OC) usage on the renin angiotensin system (RAS) in two related experiments. In the first experiment, subjects were 34 healthy, normotensive, premenopausal women, 15 OC users and 19 OC nonusers, mean age 25 +/- 1 yr, ingesting a controlled sodium diet. We assessed arterial pressure, glomerular filtration rate, effective renal plasma flow, renal vascular resistance (RVR), and filtration fraction (FF) using inulin and p-aminohippurate clearance techniques, both at baseline and in response to the ANG II receptor blocker losartan. In the second experiment, in similar subjects, 10 OC users and 10 nonusers, we examined circulating RAS components [angiotensinogen, ANG II, aldosterone, plasma renin activity (PRA), and active renin] in response to incremental lower body negative pressure (LBNP), to determine whether renin secretion is suppressed by OC usage. OC users exhibited elevations in systolic blood pressure, RVR, and FF compared with nonusers, which were partially corrected by losartan. In the LBNP phase of the study, baseline measures of PRA, angiotensinogen, ANG II, and aldosterone were all increased in the OC group compared with the control group. Active renin levels did not differ between groups. Incremental LBNP resulted in increased circulating levels of RAS components in both groups. We conclude that the RAS is activated in women using OCs. There was no evidence that decreases in renin secretion result in normalization of the RAS as a whole.

Angiotensin II type 1 receptor gene polymorphisms in humans: physiology and pathophysiology of the genotypes.

Many studies have attempted to relate genetic variants of components of the renin-angiotensin system to complex diseases such as essential hypertension, cardiovascular disease and progressive renal failure. The angiotensin II type 1 receptor (AT1R) gene is an important example of this approach. Many polymorphisms of the AT1R gene have been identified, but the A1166-->C polymorphism has been the most extensively studied. The physiological significance of this polymorphism is uncertain because of its location in the 3'-untranslated region of the gene. The present review summarizes association studies of the AT1R gene, focusing on clinical end-points and physiological responses.

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.

Effects of diabetes and hypertension on glomerular transforming growth factor-beta receptor expression.

BACKGROUND: Several studies have suggested that transforming growth factor-beta1 (TGF-beta1) is an important determinant of diabetic glomerular injury. TGF-beta1 forms a heteromeric complex with two cellular receptor subtypes, designated TGF-beta RII and TGF-beta RI, but the effects of diabetes mellitus on glomerular TGF-beta receptor expression have not been completely elucidated. We first compared the effect of experimental type I diabetes mellitus and uninephrectomy on glomerular TGF-beta receptor expression in spontaneously hypertensive rats (SHRs), and then sought to determine whether changes in TGF-beta receptor expression were strain specific by studying normotensive Wistar-Kyoto (WKY) rats. METHODS: Five groups of male SHRs were studied. The first group received streptozotocin (60 mg/kg IV) and was studied after one week. The second group received streptozotocin and was studied after two weeks. The third group received streptozotocin (60 mg/kg IV) but received insulin to maintain euglycemia. The fourth group of age-matched SHRs served as the control group, while a fifth group of SHRs underwent uninephrectomy. Four groups of male WKY rats were also studied. The first group of WKY rats served as the age-matched control group. The second group of WKY rats received streptozotocin, while a third group of WKY rats underwent uninephrectomy. The fourth group underwent uninephrectomy and received streptozotocin. At each time point, glomeruli were isolated for protein extraction, and the protein was subjected to Western blot analysis of TGF-beta RII and TGF-beta RI expression. RESULTS: Basal expression of both TGF-beta receptors per microgram of glomerular protein was similar in normotensive WKY rats and hypertensive SHRs. Hyperglycemia (blood glucose level, 17.8 +/- 2.9 mmol/L) led to an early twofold increase in TGF-beta RII protein expression and a fourfold increase in TGF-beta RI protein expression in the glomeruli of hypertensive diabetic SHRs compared with euglycemic SHRs (blood glucose level, 5.8 +/- 0.8 mmol/L), which was sustained after two weeks. Insulin treatment (blood glucose level, 5. 2 +/- 0.9 mmol/L) normalized both TGF-beta RII and TGF-beta RI expression in the glomeruli of SHRs that received streptozotocin. Glomerular capillary hypertension in the uninephrectomized SHRs led to a twofold increase in glomerular TGF-beta RII protein expression, but did not reproduce the effect of diabetes mellitus on TGF-beta RI expression. In contrast to the findings in SHRs, neither hyperglycemia (blood glucose level, 15.5 +/- 2.1 mmol/L), uninephrectomy, nor hyperglycemia (blood glucose level, 16.8 +/- 3.0 mmol/L) and uninephrectomy altered TGF-beta receptor expression in the glomeruli of normotensive WKY rats. CONCLUSION: These studies support the hypothesis that hemodynamic factors and metabolic factors influence glomerular TGF-beta receptor in vivo in the SHRs.

Stretch-induced mesangial cell ERK1/ERK2 activation is enhanced in high glucose by decreased dephosphorylation.

Glomerular hypertension and hyperglycemia are major determinants of diabetic nephropathy. We sought to identify the mechanisms whereby stretch-induced activation of mesangial cell extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2) is enhanced in high glucose (HG). Mesangial cells cultured on fibronectin Flex I plates in normal glucose (NG; 5.6 mM) or HG (30 mM), were stretched by 15% elongation at 60 cycles/min for up to 60 min. In HG, a 5-min stretch increased ERK1/ERK2 phosphorylation by 6.4 +/- 0.4/4.3 +/- 0.3-fold (P < 0.05 vs. NG stretch). In contrast, p38 phosphorylation was increased identically by stretch in NG and HG. Unlike many effects of HG, augmentation of ERK activity by HG was not dependent on protein kinase C (PKC) as indicated by downregulation of PKC with 24-h phorbol ester or inhibition with bisindolylmaleimide IV. In both NG and HG, pretreatment with arginine-glycine-aspartic acid peptide (0.5 mg/ml) to inhibit integrin binding or with cytochalasin D (100 ng/ml) to disassemble filamentous (F) actin, significantly reduced phosphorylation of ERK1/ERK2 and p38. To determine whether the rate of mitogen-activated protein kinase dephosphorylation is affected by HG, cellular kinase activity was inhibited by depleting ATP. Post-ATP depletion, phosphorylation of ERK1/ERK2 was reduced to 36 +/- 9/51 +/- 14% vs. 9 +/- 5/7 +/- 6% in NG (P < 0.05, n = 5). Thus stretch-induced ERK1/ERK2 and p38 activation in both NG and HG is beta(1)-integrin and F-actin dependent. Stretch-induced ERK1/ERK2 is enhanced in high glucose by diminished dephosphorylation, suggesting reduced phosphatase activity in the diabetic milieu. Enhanced mesangial cell ERK1/ERK2 signaling in response to the combined effects of mechanical stretch and HG may contribute to the pathogenesis of diabetic nephropathy.

Overexpression of GFAT activates PAI-1 promoter in mesangial cells.

Effects of hyperglycemia on glomerular cells may be mediated by glucose entry into the hexosamine pathway, and mesangial cell (MC) expression of the hexosamine pathway rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT) is increased in diabetic glomerulosclerosis. We hypothesized that GFAT activity would be an important determinant of gene expression in glomerular MC. When overexpressed in primary MC, GFAT produced a two- to threefold increase in the activity of plasminogen activator inhibitor-1 (PAI-1) promoter. There was a 1.4-fold increase in PAI-1 promoter activity in cells exposed to high glucose (20 mM), whereas in MC overexpressing GFAT, exposure to high glucose caused a 3.5- to 4-fold increase in promoter activity. PAI-1 promoter activation was dependent on GFAT enzyme activity because o-diazoacetyly-L-serine and 6-diazo-5-oxonorleucine, inhibitors of GFAT enzyme activity, abrogated the activation of PAI-1 promoter in MC overexpressing GFAT. Glucosamine, which is downstream of GFAT in the hexosamine pathway, produced a 2.5-fold increase in the PAI-1 promoter activity. In addition to increasing the mRNA levels for transforming growth factor-beta1 (TGF-beta1), GFAT overexpression also increased mRNA levels for the TGF-beta type I and type II receptors. TGF-beta-neutralizing antibody did not normalize PAI-1 promoter activity in MC exposed to glucosamine or those overexpressing GFAT. We conclude that GFAT expression and activity are important determinants of gene expression in MC and that flux through the hexosamine pathway activates expression of genes implicated in vascular injury pathways.

Angiotensin II type 1 receptor gene polymorphism and the response to hyperglycemia in early type 1 diabetes.

Recent studies suggest that there is an association between the A1166-->C polymorphism of the angiotensin II type 1 receptor (AGT1R), glycemic control, and the risk of diabetic nephropathy in subjects with type 1 diabetes. Because hypertension and renal hemodynamic function are also related to the risk of diabetic nephropathy and because hyperglycemia can activate the renin angiotensin system, we sought to determine if there is an association between the AGT1R polymorphism, baseline renal and peripheral hemodynamic function, and pressor response to high glucose in subjects with early uncomplicated type 1 diabetes. There were 39 diabetic subjects genotyped for the AGT1R polymorphism by polymerase chain reaction and segregated into 2 groups: those with and those without the C1166 allele (AA and AC/CC). The average age was 27 +/- 1 years, and the mean duration of diabetes was 3.5 +/- 0.6 years. HbA(1c) values were <10% in all subjects and were similar in the 2 groups (8.2 +/- 0.3 vs. 9.1 +/- 0.4%). After a 7-day controlled diet (150 mmol sodium, 1.5-2.0 g x kg(-1) x day(-1) protein), renal hemodynamic function was assessed by inulin and para-aminohippurate clearance during clamped euglycemic conditions (4-6 mmol/l). Mean values for glomerular filtration rates did not differ between groups during euglycemia. In contrast, mean values for renal plasma flow and renal blood flow were significantly greater in the AC/CC group compared with the AA group. Values for mean arterial pressure were similar in the 2 groups, whereas renal vascular resistance was significantly reduced in the AC/CC group. In 20 subjects (10 from each genotype subgroup), hemodynamic function was assessed on a second occasion during controlled clamped hyperglycemia (9-11 mmol/l) after a similar preparatory period. In response to high glucose, plasma renin activity increased in both genotype groups to the same extent, but a pressor response was noted only in subjects with the C1166 allele. Mean arterial pressure increased significantly in the AC/CC subgroup and remained unchanged in the AA subgroup. We conclude that there is an association between the AGT1R A1166-->C polymorphism and renal hemodynamic function in early type 1 diabetes. But more importantly, the pressor response to hyperglycemia is augmented in those diabetic patients with the C1166 allele and may represent a factor that predisposes them to renal injury during periods of inadequate glucose control.

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 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.

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.

Renal vascular morphology and haemodynamics in Dahl salt-sensitive rats on high salt-low potassium diet: neural and genetic influences.

OBJECTIVE: A dietary combination of high salt and low potassium (HS-LK) exacerbates hypertension in Dahl salt-sensitive (DS) rats and renders Dahl salt-resistant (DR) rats hypertensive. In both strains, the hypertension is accompanied by remodelling of the renal resistance vasculature, and is attenuated by peripheral chemical sympathectomy. In the current study, we sought to determine whether the sympathetic nervous system is causally involved in mediating the renal vascular and haemodynamic alterations associated with HS-LK feeding in Dahl rats. DESIGN: Two groups each of DS and DR rats were maintained on HS-LK diet (8% NaCl, 0.2% KCl) for 8 weeks. One group of DS (n = 9) and DR (n = 8) were treated with 6-hydroxydopamine (6-OHDA) in 0.001 N HCl vehicle to chemically ablate peripheral sympathetic nerve terminals. The two remaining groups (n = 8 each) received equivalent injections of vehicle. METHODS: At the end of the dietary regimen, arterial blood pressure (ABP), glomerular filtration rate (GFR) and renal blood flow (RBF) were measured, and the structure of intra-renal resistance vessels was examined by planar morphometric analysis of coronal sections prepared from perfusion-fixed kidneys. RESULTS: Both 6-OHDA-treated and untreated DS rats presented a greater degree of intra-renal vessel remodelling characterized by reduced lumen diameter in the absence (eutrophic) or presence (hypertrophic) of cross-sectional area expansion, higher renal vascular resistance (RVR) and lower GFR and RBF than DR rats. Chemical sympathectomy increased lumen diameters and reduced vascular wall expansion, resulting in a decrease in RVR and a concomitant increase in RBF and GFR in both strains; however, the effect was more prominent in the DS rats. CONCLUSIONS: We conclude that HS-LK-induced changes in intra-renal vessel structure and renal haemodynamic function in Dahl rats are, at least in part, dependent on the activity of the sympathetic nervous system.