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

Effects of diabetes and hypertension on glomerular transforming growth factor-β receptor expression.BackgroundSeveral studies have suggested that transforming growth factor-β1 (TGF-β1) is an important determinant of diabetic glomerular injury. TGF-β1 forms a heteromeric complex with two cellular receptor subtypes, designated TGF-β RII and TGF-β RI, but the effects of diabetes mellitus on glomerular TGF-β receptor expression have not been completely elucidated. We first compared the effect of experimental type I diabetes mellitus and uninephrectomy on glomerular TGF-β receptor expression in spontaneously hypertensive rats (SHRs), and then sought to determine whether changes in TGF-β receptor expression were strain specific by studying normotensive Wistar-Kyoto (WKY) rats.MethodsFive 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-β RII and TGF-β RI expression.ResultsBasal expression of both TGF-β 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-β RII protein expression and a fourfold increase in TGF-β 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-β RII and TGF-β 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-β RII protein expression, but did not reproduce the effect of diabetes mellitus on TGF-β 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-β receptor expression in the glomeruli of normotensive WKY rats.ConclusionThese studies support the hypothesis that hemodynamic factors and metabolic factors influence glomerular TGF-β receptor in vivo in the SHRs

Activation of mesangial cell MAPK in responseto homocysteine

Activation of mesangial cell MAPK in response to homocysteine.BackgroundAlteration in mesangial cell function is central to the progression of glomerular disease in numerous models of chronic renal failure (CRF). Animal models of chronic glomerular disease are characterized by mesangial cell proliferation and elaboration of extracellular matrix protein (ECM), resulting in glomerulosclerosis. Elevated plasma levels of homocysteine (Hcy) are seen in both animal models and humans with CRF, and have been proposed to contribute to the high prevalence of vascular disease in this group. Some of the pathogenetic effects of Hcy are thought to be mediated via the induction of endoplasmic reticulum stress. Thus, Hcy effects on mesangial cells could contribute to the progression of CRF. Previous work has shown Hcy- mediated induction of Erk mitogen-activated protein kinase (MAPK) in vascular smooth muscle cells (VSMCs). Erk induces increases in activator protein-1 (AP-1) transcription factor activity which may augment mesangial cell proliferation and ECM protein production. Consequently, we studied the effect of Hcy on mesangial cell Erk signaling.MethodsMesangial cells were exposed to Hcy after 24 hours of serum starvation and Erk activity assessed. Nuclear translocation of phospho-Erk was visualized by confocal microscopy. AP-1 nuclear protein binding was measured in response to Hcy by mobility shift assay. Hcy-induced mesangial cell calcium flux was measured in Fura-2 loaded cells. Mesangial cell DNA synthesis in response to Hcy was assessed by [3H]-thymidine incorporation and proliferation by Western blotting for proliferating cell nuclear antigen (PCNA). Expression of endoplasmic reticulum stress response genes were determined by Northern and Western analysis.ResultsHcy led to an increase in Erk activity that was maximal at 50 μmol/L and 20 minutes of treatment. Subsequent experiments used this concentration and time point. Erk activity in response to Hcy was insensitive to n-acetylcysteine and catalase, indicating oxidative stress did not play a role. However, Hcy50 μmol/L induced a brief increase in intracellular mesangial cell calcium within 5 minutes, and the calcium ionophores A23187 and ionomycin increased Erk activity while chelation of intracellular calcium with BAPTA-AM abrogated the Erk response to Hcy. Confocal microscopy of activated Erk nuclear translocation mirrored these results as did mesangial cell nuclear protein binding to AP-1 consensus sequences. Hcy- induced increases in thymidine incorporation and PCNA expression at 24 hours were Erk dependent. The expression of endoplasmic reticulum stress response genes was significantly elevated by Hcy in an Erk-dependent manner.ConclusionHcy increases Erk activity in mesangial cells via a calcium-dependent mechanism, resulting in increased AP-1 nuclear protein binding, cell DNA synthesis and proliferation and induction of endoplasmic reticulum stress. These observations suggest potential mechanisms by which Hcy may contribute to progressive glomerular injury

Social Determinants of Health Are Associated with Markers of Renal Injury in Adolescents with Type 1 Diabetes.

OBJECTIVE: To examine the relationship between the social determinants of health and markers of early renal injury in adolescent patients with type 1 diabetes (T1D). STUDY DESIGN: Renal outcomes included estimated glomerular filtration rate (eGFR) and albumin-creatinine excretion ratio (ACR). Differences in urinary and serum inflammatory markers also were assessed in relation to social determinants of health. Regression analysis was used to evaluate the association between the Ontario Marginalization Index (ON-Marg) as a measure of the social determinants of health, patient characteristics, ACR, eGFR, and renal filtration status (hyperfiltration vs normofiltration). RESULTS: Participants with T1D (n = 199) with a mean age of 14.4 ± 1.7 years and diabetes duration of 7.2 ± 3.1 years were studied. Mean eGFR was 122.0 ± 19.4 mL/min/1.73 m2. Increasing marginalization was positively associated with eGFR (P < .0001) but not with ACR (P = .605). Greater marginalization was associated with greater median levels of urinary interleukin (IL)-2, IL-12 (p40), macrophage-derived chemokine, monocyte chemoattractant protein-3, and tumor necrosis factor-β and serum IL-2. ON-Marg was significantly associated with eGFR after we controlled for age, sex, body mass index z score, ethnicity, serum glucose, and hemoglobin A1c in linear regression. A similar association between hyperfiltration and ON-Marg score was observed in multivariable logistic regression. CONCLUSION: Increasing marginalization is significantly associated with both eGFR and hyperfiltration in adolescents with T1D and is associated with significant changes in urinary inflammatory biomarkers. These findings highlight a potentially important interaction between social and biological determinants of health in adolescents with T1D

Early changes in cardiovascular structure and function in adolescents with type 1 diabetes.

BACKGROUND: Children with type 1 diabetes (T1D) are at higher risk of early adult-onset cardiovascular disease. We assessed cardiovascular structure and function in adolescents with T1D compared with healthy controls and the relationships between peripheral vascular function and myocardial parameters. METHODS AND RESULTS: 199 T1D [14.4 ± 1.6 years, diabetes duration 6.2 (2.0-12.8) years] and 178 controls (14.4 ± 2.1 years) completed endothelial function by flow mediated vasodilatation (FMD), arterial stiffness using pulse wave velocity (PWV) along with M-mode, pulse wave and tissue Doppler, and myocardial deformation echocardiographic imaging. Systolic (113 ± 10 vs. 110 ± 9 mmHg; p = 0.0005) and diastolic (62 ± 7 vs. 58 ± 7 mmHg; p < 0.0001) blood pressures, carotid femoral PWV and endothelial dysfunction measurements were increased in T1D compared with controls. Systolic and diastolic left ventricular dimensions and function by M-mode and pulse wave Doppler assessment were not significantly different. Mitral valve lateral e' (17.6 ± 2.6 vs. 18.6 ± 2.6 cm/s; p < 0.001) and a' (5.4 ± 1.1 vs. 5.9 ± 1.1 cm/s; p < 0.001) myocardial velocities were decreased and E/e' (7.3 ± 1.2 vs. 6.7 ± 1.3; p = 0.0003) increased in T1D. Left ventricular mid circumferential strain (-20.4 ± 2.3 vs. -19.5 ± 1.7 %; p < 0.001) was higher, whereas global longitudinal strain was lower (-19.0 ± 1.9 vs. -19.8 ± 1.5 % p < 0.001) in T1D. CONCLUSIONS: Adolescents with T1D exhibit early changes in blood pressure, peripheral vascular function and left ventricular myocardial deformation indices with a shift from longitudinal to circumferential shortening. Longitudinal follow-up of these changes in ongoing prospective trials may allow detection of those most at risk for cardiovascular abnormalities including hypertension that could preferentially benefit from early therapeutic interventions.Funding was provided by the Juvenile Diabetes Research Foundation- Canadian Clinical Trial Network (JDRF-CCTN), the Canadian Diabetes Association, the Heart and Stroke Foundation of Canada and the Sick Kids Labatt Family Heart Center Innovation fund. Funding was also provided by the British Heart Foundation, Diabetes UK and the Juvenile Diabetes Research Foundation

The urinary cytokine/chemokine signature of renal hyperfiltration in adolescents with type 1 diabetes.

OBJECTIVE: Urinary cytokine/chemokine levels are elevated in adults with type 1 diabetes (T1D) exhibiting renal hyperfiltration. Whether this observation extends to adolescents with T1D remains unknown. Our first objective was to determine the relationship between hyperfiltration and urinary cytokines/chemokines in normotensive, normoalbuminuric adolescents with T1D using GFR(cystatin). Our second aim was to determine the relationship between urine and plasma levels of inflammatory biomarkers, to clarify the origin of these factors. METHODS: Urine and serum cytokines/chemokines (Luminex platform) and GFR(cystatin) were measured in normofiltering (n = 111, T1D-N, GFR<135 ml/min/1.73 m(2)) and hyperfiltering (n = 31, T1D-H, GFR ≥ 135 ml/min/1.73 m(2)) adolescents with T1D (ages 10-16), and in age and sex matched healthy control subjects (HC, n = 59). RESULTS: We noted significant step-wise increases in urinary cytokine/chemokine excretion according to filtration status with highest levels in T1D-H, with parallel trends in serum analyte concentrations. After adjusting for serum glucose at the time of sampling, differences in urinary cytokine excretion were not statistically significant. Only serum IL-2 significantly differed between HC and T1D (p = 0.0076). CONCLUSIONS: Hyperfiltration is associated with increased urinary cytokine/chemokine excretion in T1D adolescents, and parallel trends in serum cytokine concentration. The GFR-associated trends in cytokine excretion may be driven by the effects of ambient hyperglycemia. The relationship between hyperfiltration, glycemia, and variations in serum and urine cytokine expression and their impact on future renal and systemic vascular complications requires further study

IFT Proteins Accumulate during Cell Division and Localize to the Cleavage Furrow in Chlamydomonas

Intraflagellar transport (IFT) proteins are well established as conserved mediators of flagellum/cilium assembly and disassembly. However, data has begun to accumulate in support of IFT protein involvement in other processes elsewhere in the cell. Here, we used synchronous cultures of Chlamydomonas to investigate the temporal patterns of accumulation and localization of IFT proteins during the cell cycle. Their mRNAs showed periodic expression that peaked during S and M phase (S/M). Unlike most proteins that are synthesized continuously during G1 phase, IFT27 and IFT46 levels were found to increase only during S/M phase. During cell division, IFT27, IFT46, IFT72, and IFT139 re-localized from the flagella and basal bodies to the cleavage furrow. IFT27 was further shown to be associated with membrane vesicles in this region. This localization pattern suggests a role for IFT in cell division

A Molecular Signature of Proteinuria in Glomerulonephritis

Proteinuria is the most important predictor of outcome in glomerulonephritis and experimental data suggest that the tubular cell response to proteinuria is an important determinant of progressive fibrosis in the kidney. However, it is unclear whether proteinuria is a marker of disease severity or has a direct effect on tubular cells in the kidneys of patients with glomerulonephritis. Accordingly we studied an in vitro model of proteinuria, and identified 231 “albumin-regulated genes” differentially expressed by primary human kidney tubular epithelial cells exposed to albumin. We translated these findings to human disease by studying mRNA levels of these genes in the tubulo-interstitial compartment of kidney biopsies from patients with IgA nephropathy using microarrays. Biopsies from patients with IgAN (n = 25) could be distinguished from those of control subjects (n = 6) based solely upon the expression of these 231 “albumin-regulated genes.” The expression of an 11-transcript subset related to the degree of proteinuria, and this 11-mRNA subset was also sufficient to distinguish biopsies of subjects with IgAN from control biopsies. We tested if these findings could be extrapolated to other proteinuric diseases beyond IgAN and found that all forms of primary glomerulonephritis (n = 33) can be distinguished from controls (n = 21) based solely on the expression levels of these 11 genes derived from our in vitro proteinuria model. Pathway analysis suggests common regulatory elements shared by these 11 transcripts. In conclusion, we have identified an albumin-regulated 11-gene signature shared between all forms of primary glomerulonephritis. Our findings support the hypothesis that albuminuria may directly promote injury in the tubulo-interstitial compartment of the kidney in patients with glomerulonephritis