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Utility of endogenous creatinine clearance as a measure of renal function in mice. Background. The use of endogenous plasma creatinine levels and creatinine clearance as a tool to evaluate renal function in mice has come under scrutiny as prior studies have reported that the Jaffé alkaline picrate method grossly overestimates true plasma creatinine in mice. As members of the NIDDK Animal Models of Diabetic Complications Consortium (AMDCC), we evaluated the performance and feasibility of an alternative high-performance liquid chromatography (HPLC)-based method for standard determination of plasma creatinine and creatinine clearance in mice. Our purpose was to develop a simple method that provides a reliable, reproducible, and sensitive assay for small volumes (<25 μL) of mouse plasma and sera. Methods. We compared creatinine clearance measured by HPLC with the Jaffé method and HPLC creatinine clearance with inulin clearance [fluoroscein isothiocyanate (FITC) inulin in an osmotic pump implanted in mouse] in C57BL/6J mice. Different groups of mice underwent either one of two protocols. Protocol A included dietary intervention with normal, low salt plus enalapril, or high salt. Protocol B induced diabetes using streptozotocin. Results. First, mean plasma creatinine levels were significantly lower ( P < 0.0001) by HPLC (0.128 ± 0.026 mg/dL) vs. Jaffé (0.4 ± 0.12 mg/dL) for mice on a normal diet. Urine creatinine concentrations measured by HPLC were 10% lower than by Jaffé ( P < 0.01). Second, mean creatinine clearance by HPLC for mice on a normal diet was 255 ± 68 μL/min. Mice on low salt diet plus enalapril had reduced creatinine clearance (72.8 ± 24.2 μL/min) while mice on high salt diet had an elevated creatinine clearance (355 ± 105 μL/min). Third, diabetic mice (19 to 24 weeks of diabetes) exhibited hyperfiltration as creatinine clearance was 524 ± 214 μL/min whereas nondiabetic age/gender-matched mice showed a mean creatinine clearance of 206 ± 41 μL/min. Finally, significant correlation was demonstrated for creatinine clearance by HPLC vs. inulin clearance ( R= 0.643; P < 0.001). Conclusion. HPLC is highly accurate, much more sensitive and specific than the Jaffé method for plasma creatinine measurements in mice. Creatinine clearance in mice measured by HPLC reflects changes in renal function induced by diet and diabetes. [ABSTRACT FROM AUTHOR]

Insulin resistance has been associated with kidney disease even in the absence of diabetes; however, pathways linking insulin resistance to kidney disease are unclear. The purpose of this study was to determine if transforming growth factor (TGF)-β1, a key cytokine associated with kidney disease, responds to circulating levels of glucose and/or insulin. Urinary TGF-β1 levels were measured in 249 young adult African Americans (mean age 40) at baseline, after an oral glucose tolerance test and after a euglycemic hyperinsulinemic clamp procedure. Baseline urinary geometric mean TGF-β1 levels were somewhat lower in those with normal compared with the impaired glucose tolerance. The urinary TGF-β1 level increased by 56% followed by a 23% decrease in the normal glucose tolerance group, changes that were significant and corresponded to the changes in the plasma glucose and insulin concentrations. The impaired tolerance group showed little change in the urinary TGF-β1 level following glucose ingestion. All participants had a significant increase in urinary TGF-β1 level after steady-state hyperinsulinemia, with sustained euglycemia during the clamp procedure in both of the groups. At baseline, there was a significant correlation between the urinary TGF-β1 level and urinary albumin excretion. Thus our results suggest that insulin contributes to increased TGF-β1 production and possible early renal injury in prediabetic young African Americans. [ABSTRACT FROM AUTHOR]

Insulin resistance has been associated with kidney disease even in the absence of diabetes; however, pathways linking insulin resistance to kidney disease are unclear. The purpose of this study was to determine if transforming growth factor (TGF)-beta1, a key cytokine associated with kidney disease, responds to circulating levels of glucose and/or insulin. Urinary TGF-beta1 levels were measured in 249 young adult African Americans (mean age 40) at baseline, after an oral glucose tolerance test and after a euglycemic hyperinsulinemic clamp procedure. Baseline urinary geometric mean TGF-beta1 levels were somewhat lower in those with normal compared with the impaired glucose tolerance. The urinary TGF-beta1 level increased by 56% followed by a 23% decrease in the normal glucose tolerance group, changes that were significant and corresponded to the changes in the plasma glucose and insulin concentrations. The impaired tolerance group showed little change in the urinary TGF-beta1 level following glucose ingestion. All participants had a significant increase in urinary TGF-beta1 level after steady-state hyperinsulinemia, with sustained euglycemia during the clamp procedure in both of the groups. At baseline, there was a significant correlation between the urinary TGF-beta1 level and urinary albumin excretion. Thus our results suggest that insulin contributes to increased TGF-beta1 production and possible early renal injury in prediabetic young African Americans.

Background: The best characterized signaling pathway employed by transforming growth factor-beta (TGF-beta) is the Smad pathway; however, its role in matrix production in mesangial cells is unclear. We focused on Smad4, as Smad4 is essential for the activation of Smad-dependent target genes., Methods: To investigate the function of Smad4 in extracellular matrix (ECM) production, we generated several stably transfected mesangial cell lines (MMC) that have a deletion in the linker region (Smad4 Delta M4: Delta 275-322) or have a deletion in MH1 of Smad4 (Smad4N4: Delta 1-136). The ECM genes, alpha1 type I collagen (COL1A1), plasminogen activator inhibitor-1 (PAI-1) and fibronectin (FN) were assessed in wild-type mesangial cells and stably transfected Smad4-DN cell lines in the absence and presence of TGF-beta., Results: As compared to wild-type MMC that had a 10.8-fold stimulation of TGF-beta-induced p3TP-Lux activity, MMC stably transfected with Smad4 Delta M4 and Smad4N4 had only a 2.0-fold and 1.3-fold stimulation, respectively, indicating that they had dominant-negative effects on TGF-beta signaling. Basal and TGF-beta-induced COL1A1 expression in Smad4 dominant-negative cells were dramatically reduced to very low levels. The early (2 hours) TGF-beta-induced PAI-1 mRNA expression was inhibited; however, the sustained (24 to 48 hours) TGF-beta-induced expression was not affected in Smad4 dominant-negative cells. For FN, TGF-beta-induced expression was maintained in Smad4-dominant negative cells., Conclusion: These results indicate that Smad4 is essential for basal and TGF-beta-induced COL1A1 expression, and contributes to the early, but not sustained TGF-beta-induced PAI-1 expression in mesangial cells. However, TGF-beta-induced FN expression is independent of Smad4. In conclusion, Smad4 has a discriminate effect in mediating specific ECM molecules stimulated by TGF-beta in mesangial cells.