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Increased albuminuria is associated with obesity and diabetes and is a risk factor for cardiovascular and renal disease. However, the link between early albuminuria and adiposity remains unclear. To determine whether adiponectin, an adipocyte-derived hormone, is a communication signal between adipocytes and the kidney, we performed studies in a cohort of patients at high risk for diabetes and kidney disease as well as in adiponectin-knockout (Ad-/-) mice. Albuminuria had a negative correlation with plasma adiponectin in obese patients, and Ad-/- mice exhibited increased albuminuria and fusion of podocyte foot processes. In cultured podocytes, adiponectin administration was associated with increased activity of AMPK, and both adiponectin and AMPK activation reduced podocyte permeability to albumin and podocyte dysfunction, as evidenced by zona occludens-1 translocation to the membrane. These effects seemed to be caused by reduction of oxidative stress, as adiponectin and AMPK activation both reduced protein levels of the NADPH oxidase Nox4 in podocytes. Ad-/- mice treated with adiponectin exhibited normalization of albuminuria, improvement of podocyte foot process effacement, increased glomerular AMPK activation, and reduced urinary and glomerular markers of oxidant stress. These results suggest that adiponectin is a key regulator of albuminuria, likely acting through the AMPK pathway to modulate oxidant stress in podocytes., Introduction Chronic kidney disease and albuminuria have recently been recognized as among the most significant clinical risk factors for cardiovascular disease (CVD), hospitalization, and all-cause mortality (1-5). Microalbuminuria (albumin/creatinine ratio [...]

We used the mouse nephrin promoter to express a constitutively active G[alpha]q [G[alpha]q(Q>L)] transgene in mice. As previously reported, the transgene was expressed in kidney, pancreas, and brain, and the kidney phenotype was characterized by albuminuria and reduced nephron mass. Additional studies revealed a second phenotype characterized by polyuria and polydipsia. The polyuric phenotype was not caused by abnormal glucose metabolism or hypercalcemia but was accompanied by reduced urinary concentrating ability. Additional studies found that 1) water restriction was associated with an appropriate increase in serum vasopressin levels in transgenic (TG) mice; 2) the urinary concentrating defect was not corrected by administration of desamino-D-arginine vasopressin (DDAVP); and 3) papillary length was similar in TG and non-TG mice. To examine the renal response to DDAVP at the molecular level, we monitored aquaporin 2 (AQP2) and vasopressin V2 receptor (V2R) mRNA levels in mouse kidney. Consistent with the known effects of vasopressin, administration of DDAVP caused a decrease in V2R mRNA levels and an increase in AQP2 mRNA levels in both TG and non-TG animals, suggesting an appropriate renal response to DDAVP in the TG mice. To determine whether the urine concentrating abnormality was the result of primary polydipsia, water intake by TG mice was restricted to the amount ingested by non-TG animals. After 5 days, urinary concentrating ability was similar in TG mice and non-TG littermate controls. These data are consistent with the notion that expression of the G[alpha]q(Q>L) transgene in the brain induced primary polydipsia in the TG mice. polyuria; vasopressin; aquaporin

A simplified method for HPLC determination of creatinine in mouse serum. Am J Physiol Renal Physiol 286:F1116-F1119, 2004. First published February 17, 2004; 10.1152/ajprenal.00366.2003.--Mouse models are frequently used to study renal function. However, mouse serum contains chromagens that interfere with standard picric acid-based assays for serum creatinine. Several alternative methods exist for serum creatinine measurements, including assay by high-performance liquid chromatography (HPLC), but only one has been adapted to mouse serum. Creatinine was measured in serum by acetonitrile deproteinization, followed by isocratic, cation exchange HPLC. The HPLC method was compared with a standard alkaline picrate colorimetric assay, using serum from animals with low-to-moderate renal injury. Acidification of acetonitrile with HCI in the deproteinization step produced variable results, including an extra peak that interfered with integration of the creatinine peak or loss of the creatinine peak. Deproteinizing with acetonitrile alone resulted in a more reliable measurement of serum creatinine, which was validated by a series of known additions of creatinine standard. The HPLC assay was reproducible with coefficients of variation from 1.6 to 5.1%. The picric acid assay overestimated serum creatinine, when directly compared with the HPLC assay. The extent of overestimation, up to sixfold, was greatest at normal (0.1 to 0.2 mg/dl) to moderately elevated (0.5 mg/dl) serum creatinine levels. Mouse serum contains substances that interfere with standard picric acid assays for creatinine. Our new HPLC assay can accurately detect creatinine from 5 [micro]l of mouse serum. These results support the widespread adoption of HPLC to accurately measure serum creatinine in mouse models of renal injury. Jaffe assay; mouse models; renal failure

Diabetic nephropathy is increasing in incidence and is now the number one cause of end-stage renal disease in the industrialized world. To gain insight into the genetic susceptibility and pathophysiology of diabetic nephropathy, an appropriate mouse model of diabetic nephropathy would be critical. A large number of mouse models of diabetes have been identified and their kidney disease characterized to various degrees. Perhaps the best characterized and most intensively investigated model is the db/db mouse. Because this model appears to exhibit the most consistent and robust increase in albuminuria and mesangial matrix expansion, it has been used as a model of progressive diabetic renal disease. In this review, we present the findings from various studies on the renal pathology of the db/db mouse model of diabetes in the context of human diabetic nephropathy. Furthermore, we discuss shortfalls of assessing functional renal disease in mouse models of diabetic kidney disease. diabetic nephropathy; creatinine; albuminuria; mesangial matrix

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We used the mouse nephrin promoter to express a constitutively active Gαq [Gαq(Q>L)] transgene in mice. As previously reported, the transgene was expressed in kidney, pancreas, and brain, and the kidney phenotype was characterized by albuminuria and reduced nephron mass. Additional studies revealed a second phenotype characterized by polyuria and polydipsia. The polyuric phenotype was not caused by abnormal glucose metabolism or hypercalcemia but was accompanied by reduced urinary concentrating ability. Additional studies found that 1) water restriction was associated with an appropriate increase in serum vasopressin levels in transgenic (TG) mice; 2) the urinary concentrating defect was not corrected by administration of desaminoᴅ-arginine vasopressin (DDAVP); and 3) papillary length was similar in TG and non-TG mice. To examine the renal response to DDAVP at the molecular level, we monitored aquaporin 2 (AQP2) and vasopressin V2 receptor (V2R) mRNA levels in mouse kidney. Consistent with the known effects of vasopressin, administration of DDAVP caused a decrease in V2R mRNA levels and an increase in AQP2 mRNA levels in both TG and non-TG animals, suggesting an appropriate renal response to DDAVP in the TG mice. To determine whether the urine concentrating abnormality was the result of primary polydipsia, water intake by TG mice was restricted to the amount ingested by non-TG animals. After 5 days, urinary concentrating ability was similar in TG mice and non-TG littermate controls. These data are consistent with the notion that expression of the Gαq(Q>L) transgene in the brain induced primary polydipsia in the TG mice. [ABSTRACT FROM AUTHOR]

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.

TGF-beta and oxidant stress have been considered to play key roles in the pathogenesis of diabetic vascular complications; however, the stimulus for these factors in humans is not clear. The purpose of this in vivo study was to determine whether transient hyperglycemia in humans is sufficient to increase renal production of TGF-beta1 and urinary isoprostanes in normal humans. A hyperglycemic clamp procedure was performed on 13 healthy volunteers. An infusion of glucose was delivered to maintain the plasma glucose between 200 and 250 mg/dl for 120 min. Timed urine samples, collected on an overnight period before the study, at each void on completion of the procedure, and the following overnight, were assayed for TGF-beta1, F2-isoprostanes, and creatinine. Plasma samples were assayed for TGF-beta1 before and at timed intervals throughout hyperglycemia. Mean baseline TGF-beta1 in plasma was 4.57 +/- 0.22 ng/ml, and no change in plasma TGF-beta1 levels was detected throughout the hyperglycemia period. Baseline urine TGF-beta1 was 4.14 +/- 1.16 pg/mg creatinine. The fractional urine samples showed a sharp increase in TGF-beta1 excretion in the 12-h period after exposure to hyperglycemia, with a mean peak TGF-beta1 of 30.43 +/- 8.05 pg/mg (P = 0.002). TGF-beta1 excretion in the subsequent overnight urine sample was not different from baseline (4.62 +/- 1.21 pg/mg). Urinary isoprostanes increased from a baseline of 4.92 +/- 0.74 to 13.8 +/- 3.37 ng/mg creatinine. It is concluded that 120 min of hyperglycemia in normal humans is sufficient to induce an increase in renal TGF-beta1 and isoprostane production.

The present study was to test the hypothesis that, selected bacteria instilled into the gastrointestinal tract could help in converting nitrogenous wastes accumulated due to renal insufficiency into non-toxic compounds; thereby, ameliorating the biochemical imbalance. Herein we describe a prospective, blinded, placebo controlled pilot-study, using 5/6th nephrectomized Sprague Dawley rat, as a chronic renal failure model. The study group consisted of 36 nephrectomized and 7 non-nephrectomized (control) rats. After two-week nephrectomy stabilization, cohorts of six nephrectomized rats were fed casein-based diet plus one of the following regimens: (A) Control, (B) Placebo (casein-based diet without probiotics), (C) Bacillus pasteurii, (D) Sporolac(R), (E) Kibow cocktail, (F) CHR Hansen Cocktail, and (G) ECONORM. Subsequently, blood (retro-orbital) and urine (collected for measurements of blood urea-nitrogen and creatinine respectively), body weight and bacterial counts (feces) were obtained at regular intervals. The study end-points were to determine if any of the probiotic dietary supplements facilitated, (1) decreased blood concentrations of uremic toxins, (2) altered renal function, and (3) prolonged survival. After 16 weeks of treatment, regimens C and D significantly prolonged the life span of uremic rats, in addition to showing a reduction in blood urea-nitrogen levels, concluding that supplementation of probiotic formulation to uremic rats slows the progression of azotemia, which may correlate with prolonged life span of uremic rats. Derivative trials of probiotic treatment of larger animals and humans will further assess the potential role of probiotic formulations in delaying the onset and clinical severity of clinical illness at different stages of renal failure.

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.