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

Abstract

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-β1 (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.

Effect of Cytochrome P450 Metabolites of Arachidonic Acid in Nephrology

“…135,136 TGF-b inhibits 20-HETE production and increases Palb in isolated glomeruli. 133,134 Chronic blockade of 20-HETE increased Palb and proteinuria in normotensive rats. 1 3 4 , 1 3 5 More recently, McCarthy et al 137 reported that increased 20-HETE levels in CYP4A14 knockout mice protected podocytes from the detrimental effects of ethanol by blocking superoxide production.…”

“…The decrease in 20-HETE also impairs the myogenic and tubuloglomerular feedback responses of the Af-art, elevates glomerular capillary pressure, increases the permeability of the glomerulus to albumin (Palb), and upregulates the expression of TGF-b leading to the development of proteinuria and CKD. 11,117,133 Normalization of the 20-HETE levels with fibrates or transfer of wild-type CYP4A genes in congenic Dahl S strains 11,[115][116][117]134 prevented the development of proteinuria and renal injury. We hypothesize that the deficiency in renal 20-HETE may play a similar role in the development of hypertension and kidney injury in patients with inactivating mutations in CYP4A11 and CYP4F2.…”

Free radical scavenging decreases endothelin‐1 excretion and glomerular albumin permeability during type 1 diabetes

“…; Fan et al. ) and O 2 •− production (Stanton ). Results of this study demonstrate that chronic treatment with the free radical scavenger tempol not only blunts glomerular O 2 •− production and decreases urinary TBARS excretion in rats with T1D, but also reduces glomerular P alb as well as renal ET‐1 synthesis and excretion.…”

“…; Fan et al. )that can be prevented or reversed by treatment with an ET A receptor antagonist (Saleh et al. ).…”

“…We previously reported that chronic ET-1 infusion in normal rats increases glomerular P alb (Saleh et al 2010) through an ET A -dependent mechanism. Moreover, rats with STZ-induced T1D display not only an increase in renal ET-1 production (Hocher et al 1998;Chen et al 2005;Sasser et al 2007;Chiu et al 2008) but also a substantial increase in glomerular P alb (Saleh et al 2011;Fan et al 2015)that can be prevented or reversed by treatment with an ET A receptor antagonist (Saleh et al 2011). Although these observations indicate that activation of the ET system increases glomerular P alb in T1D, the interaction between these events and the oxidative stress accompanying T1D remains unexplored.…”