Fluorescence dilution technique for measurement of albumin reflection coefficient in isolated glomeruli.

This study describes a high-throughput fluorescence dilution technique to measure the albumin reflection coefficient (σAlb) of isolated glomeruli. Rats were injected with FITC-dextran 250 (75 mg/kg), and the glomeruli were isolated in a 6% BSA solution. Changes in the fluorescence of the glomerulus due to water influx in response to an imposed oncotic gradient was used to determine σAlb. Adjustment of the albumin concentration of the bath from 6 to 5, 4, 3, and 2% produced a 10, 25, 35, and 50% decrease in the fluorescence of the glomeruli. Pretreatment of glomeruli with protamine sulfate (2 mg/ml) or TGF-ß1 (10 ng/ml) decreased σAlb from 1 to 0.54 and 0.48, respectively. Water and solute movement were modeled using Kedem-Katchalsky equations, and the measured responses closely fit the predicted behavior, indicating that loss of albumin by solvent drag or diffusion is negligible compared with the movement of water. We also found that σAlb was reduced by 17% in fawn hooded hypertensive rats, 33% in hypertensive Dahl salt-sensitive (SS) rats, 26% in streptozotocin-treated diabetic Dahl SS rats, and 21% in 6-mo old type II diabetic nephropathy rats relative to control Sprague-Dawley rats. The changes in glomerular permeability to albumin were correlated with the degree of proteinuria in these strains. These findings indicate that the fluorescence dilution technique can be used to measure σAlb in populations of isolated glomeruli and provides a means to assess the development of glomerular injury in hypertensive and diabetic models.

Heterozygous knockout of transforming growth factor-ß1 protects Dahl S rats against high salt-induced renal injury.

The present study employed a zinc-finger nuclease strategy to create heterozygous knockout (KO) rats for the transforming growth factor-ß1 (Tgfb1) gene on the Dahl SS/Jr genetic background (TGF-ß1(+/-) Dahl S). Intercrossing TGF-ß1(+/-) rats did not produce any homozygous KO rats (66.4% +/-, 33.6% +/+), indicating that the mutation is embryonic lethal. Six-week-old wild-type (WT) littermates and TGF-ß1(+/-) Dahl S rats were fed a 0.4% (low salt, LS) or 8% NaCl (high salt, HS) diet for 5 wk. Renal cortical expression of TGF-ß1, urinary TGF-ß1 excretion, proteinuria, glomerular injury and tubulointerstitial fibrosis, and systolic blood pressure were similar in WT and TGF-ß1(+/-) Dahl S rats maintained on the LS diet. The expression and urinary excretion of TGF-ß1 increased to a greater extent in WT than in TGF-ß1(+/-)Dahl S rats fed an HS diet for 1 wk. Systolic blood pressure rose by the same extent to 235 ± 2 mmHg in WT and 239 ± 4 mmHg in TGF-ß1(+/-) Dahl S rats fed a HS diet for 5 wk. However, urinary protein excretion was significantly lower in TGF-ß1(+/-) Dahl S than in the WT animals. The degree of glomerular injury and renal cortical and outer medullary fibrosis was markedly less in TGF-ß1(+/-) than in WT rats. These findings suggest that the loss of one copy of the TGF-ß1 gene blunts the increase in renal TGF-ß1 protein expression and slows the progression of proteinuria, glomerulosclerosis, and renal interstitial fibrosis in Dahl S rats fed an HS diet independently of changes in blood pressure.

Renoprotective effects of anti-TGF-ß antibody and antihypertensive therapies in Dahl S rats.

This study examined the effects of anti-TGF-ß antibody (1D11) therapy in Dahl S (S) rats fed a 4% NaCl diet. Baseline renal expression of TGF-ß1 and the degree of injury were lower in female than male S rats maintained on a 0.4% NaCl diet. 4% NaCl diet increased mean arterial pressure (MAP), proteinuria, and renal injury to the same extent in both male and female S rats. Chronic treatment with 1D11 had renoprotective effects in both sexes. The ability of 1D11 to oppose the development of proteinuria when given alone or in combination with antihypertensive agents was further studied in 6-wk-old female S rats, since baseline renal injury was less than that seen in male rats. 1D11, diltiazem, and hydrochlorothiazide (HCT) attenuated the development of hypertension, proteinuria, and glomerular injury. 1D11 had no additional effect when given in combination with these antihypertensive agents. We also explored whether 1D11 could reverse renal injury in 9-wk-old male S rats with preexisting renal injury. MAP increased to 197 ± 4 mmHg and proteinuria rose to >300 mg/day after 3 wk on a 4% NaCl diet. Proteinuria was reduced by 30-40% in rats treated with 1D11, HCT, or captopril + 1D11, but the protective effect was lost in rats fed the 4% NaCl diet for 6 wk. Nevertheless, 1D11, HCT, and captopril + 1D11 still reduced renomedullary and cardiac fibrosis. These results indicate that anti-TGF-ß antibody therapy reduces renal and cardiac fibrosis and affords additional renoprotection when given in combination with various antihypertensive agents in Dahl S rats.

TNFR1-deficient mice display altered blood pressure and renal responses to ANG II infusion.

The hypothesis that TNF receptor 1-deficient (TNFR1(-/-)) mice display blood pressure (BP) and renal functional responses that differ from wild-type (WT) mice was tested in an angiotensin II (ANG II)-dependent model of hypertension. Basal systolic BP (SBP), mean arterial pressure, diastolic BP, heart rate (HR), and pulse pressure were similar in WT and TNFR1(-/-) mice. Infusion of ANG II for 7 days elevated SBP to a greater extent in TNFR1(-/-) compared with WT mice; pulse pressure was also elevated in TNFR1(-/-). HR decreased in TNFR1(-/-) mice infused with ANG II, an effect prominent on day 1. Basal urinary albumin excretion was similar in WT and TNFR1(-/-) mice but was higher in TNFR1(-/-) in response to ANG II infusion. Water intake and urine volume were increased by ANG II infusion; this increase was higher in TNFR1(-/-) vs. WT mice, whereas body weight and food intake were unaffected. Baseline creatinine clearance (Ccr), urinary sodium excretion, and fractional excretion of sodium (FE(Na)%) were similar in vehicle-treated WT and TNFR1(-/-) mice. ANG II infusion for 7 days increased Ccr and filtered load of sodium in TNFR1(-/-) but not WT mice, whereas it elicited an increase in FE(Na)% and urinary sodium excretion in WT but not TNFR1(-/-) mice. ANG II also inhibited renal TNFR1 mRNA accumulation while increasing that of TNFR2. These findings indicate deletion of TNFR1 is associated with an exacerbated SBP response, decrease in HR, and altered renal function in ANG II-dependent hypertension.