Silvia B. Campos-Bilderback

Multiple Factors Influence Glomerular Albumin Permeability in Rats

Different laboratories recently reported incongruous results describing the quantification of albumin filtration using two-photon microscopy. We investigated the factors that influence the glomerular sieving coefficient for albumin (GSC(A)) in an effort to explain these discordant reports and to develop standard operating procedures for determining GSC(A). Multiple factors influenced GSC(A), including the kidney depth of image acquisition (10-20 μm was appropriate), the selection of fluorophore (probes emitting longer wavelengths were superior), the selection of plasma regions for fluorescence measurements, the size and molecular dispersion characteristics of dextran polymers if used, dietary status, and the genetic strain of rat. Fasting reduced the GSC(A) in Simonsen Munich Wistar rats from 0.035±0.005 to 0.016±0.004 (P<0.01). Frömter Munich Wistar rats had a much lower GSC(A) in both the fed and the fasted states. Finally, we documented extensive albumin transcytosis with vesicular and tubular delivery to and fusion with the basolateral membrane in S1 proximal tubule cells. In summary, these results help explain the previously conflicting microscopy and micropuncture data describing albumin filtration and highlight the dynamic nature of glomerular albumin permeability.

Chronic endothelin-1 infusion elevates glomerular sieving coefficient and proximal tubular albumin reuptake in the rat

AIM We have previously found that chronic endothelin-1 (ET-1) infusion in Sprague-Dawley rats increases glomerular permeability to albumin (P(alb)) as assessed in vitro independent of blood pressure with no observed albuminuria. In this study, we hypothesized that ET-1 increases glomerular albumin filtration with accompanied increase in albumin uptake via the proximal tubule, which masks the expected increase in urinary albumin excretion. MAIN METHODS Nonfasting Munich-Wistar Fromter rats were surgically prepared for in vivo imaging (n=6). Rats were placed on the microscope stage with the exposed kidney placed in a cover slip-bottomed dish bathed in warm isotonic saline. Rats were then injected i.v. with rat serum albumin conjugated to Texas Red that was observed to enter capillary loops of superficial glomeruli, move into Bowmans space, bind to the proximal tubular cell brush border and reabsorbed across the apical membrane. Glomerular sieving coefficient (GSC) was calculated as the ratio of conjugated albumin within the glomerular capillary versus that in Bowmans space. Rats were again studied after 2 weeks of chronic ET-1 (2 pmol/kg/min; i.v. osmotic minipump). KEY FINDINGS Glomerular sieving coefficient was significantly increased in rats following chronic ET-1 infusion (0.025 ± 0.005 vs. 0.017 ± 0.003, p<0.05). Mean fluorescence intensity for conjugated albumin within proximal tubules was increased by ET-1 infusion: 118.40 ± 6.34 vs. 74.27 ± 4.45 pixel intensity (p<0.01). SIGNIFICANCE These data provide in vivo evidence that ET-1 directly increases glomerular permeability to albumin and that albuminuria is prevented by increased PT albumin uptake in the rat.

Shear stress is normalized in glomerular capillaries following ⅚ nephrectomy

Loss of significant functional renal mass results in compensatory structural and hemodynamic adaptations in the nephron. While these changes have been characterized in several injury models, how they affect hemodynamic forces at the glomerular capillary wall has not been adequately characterized, despite their potential physiological significance. Therefore, we used intravital multiphoton microscopy to measure the velocity of red blood cells in individual glomerular capillaries of normal rats and rats subjected to ⅚ nephrectomy. Glomerular capillary blood flow rate and wall shear stress were then estimated using previously established experimental and mathematical models to account for changes in hematocrit and blood rheology in small vessels. We found little change in the hemodynamic parameters in glomerular capillaries immediately following injury. At 2 wk postnephrectomy, significant changes in individual capillary blood flow velocity and volume flow rate were present. Despite these changes, estimated capillary wall shear stress was unchanged. This was a result of an increase in capillary diameter and changes in capillary blood rheology in nephrectomized rats.

Mechanism of increased clearance of glycated albumin by proximal tubule cells.

Serum albumin is the most abundant plasma protein and has a long half-life due to neonatal Fc receptor (FcRn)-mediated transcytosis by many cell types, including proximal tubule cells of the kidney. Albumin also interacts with, and is modified by, many small and large molecules. Therefore, the focus of the present study was to address the impact of specific known biological albumin modifications on albumin-FcRn binding and cellular handling. Binding at pH 6.0 and 7.4 was performed since FcRn binds albumin strongly at acidic pH and releases it after transcytosis at physiological pH. Equilibrium dissociation constants were measured using microscale thermophoresis. Since studies have shown that glycated albumin is excreted in the urine at a higher rate than unmodified albumin, we studied glucose and methylgloxal modified albumins (21 days). All had reduced affinity to FcRn at pH 6.0, suggesting these albumins would not be returned to the circulation via the transcytotic pathway. To address why modified albumin has reduced affinity, we analyzed the structure of the modified albumins using small-angle X-ray scattering. This analysis showed significant structural changes occurring to albumin with glycation, particularly in the FcRn-binding region, which could explain the reduced affinity to FcRn. These results offer an explanation for enhanced proximal tubule-mediated sorting and clearance of abnormal albumins.

Hydrodynamic Isotonic Fluid Delivery Ameliorates Moderate-to-Severe Ischemia-Reperfusion Injury in Rat Kidneys

Highly aerobic organs like the kidney are innately susceptible to ischemia-reperfusion (I/R) injury, which can originate from sources including myocardial infarction, renal trauma, and transplant. Therapy is mainly supportive and depends on the cause(s) of damage. In the absence of hypervolemia, intravenous fluid delivery is frequently the first course of treatment but does not reverse established AKI. Evidence suggests that disrupting leukocyte adhesion may prevent the impairment of renal microvascular perfusion and the heightened inflammatory response that exacerbate ischemic renal injury. We investigated the therapeutic potential of hydrodynamic isotonic fluid delivery (HIFD) to the left renal vein 24 hours after inducing moderate-to-severe unilateral IRI in rats. HIFD significantly increased hydrostatic pressure within the renal vein. When conducted after established AKI, 24 hours after I/R injury, HIFD produced substantial and statistically significant decreases in serum creatinine levels compared with levels in animals given an equivalent volume of saline via peripheral infusion (P<0.05). Intravital confocal microscopy performed immediately after HIFD showed improved microvascular perfusion. Notably, HIFD also resulted in immediate enhancement of parenchymal labeling with the fluorescent dye Hoechst 33342. HIFD also associated with a significant reduction in the accumulation of renal leukocytes, including proinflammatory T cells. Additionally, HIFD significantly reduced peritubular capillary erythrocyte congestion and improved histologic scores of tubular injury 4 days after IRI. Taken together, these results indicate that HIFD performed after establishment of AKI rapidly restores microvascular perfusion and small molecule accessibility, with improvement in overall renal function.

Intravital Imaging of the Kidney in a Rat Model of Salt-Sensitive Hypertension

Hypertension is one of the most prevalent diseases worldwide and a major risk factor for renal failure and cardiovascular disease. The role of albuminuria, a common feature of hypertension and robust predictor of cardiorenal disorders, remains incompletely understood. The goal of this study was to investigate the mechanisms leading to albuminuria in the kidney of a rat model of hypertension, the Dahl salt-sensitive (SS) rat. To determine the relative contributions of the glomerulus and proximal tubule (PT) to albuminuria, we applied intravital two-photon-based imaging to investigate the complex renal physiological changes that occur during salt-induced hypertension. Following a high-salt diet, SS rats exhibited elevated blood pressure, increased glomerular sieving of albumin (GSCalb = 0.0686), relative permeability to albumin (+Δ16%), and impaired volume hemodynamics (-Δ14%). Serum albumin but not serum globulins or creatinine concentration was decreased (-0.54 g/dl), which was concomitant with increased filtration of albumin (3.7 vs. 0.8 g/day normal diet). Pathologically, hypertensive animals had significant tubular damage, as indicated by increased prevalence of granular casts, expansion and necrosis of PT epithelial cells (+Δ2.20 score/image), progressive augmentation of red blood cell velocity (+Δ269 µm/s) and micro vessel diameter (+Δ4.3 µm), and increased vascular injury (+Δ0.61 leakage/image). Therefore, development of salt-induced hypertension can be triggered by fast and progressive pathogenic remodeling of PT epithelia, which can be associated with changes in albumin handling. Collectively, these results indicate that both the glomerulus and the PT contribute to albuminuria, and dual treatment of glomerular filtration and albumin reabsorption may represent an effective treatment of salt-sensitive hypertension.

Inhibition of αvβ5 Integrin Attenuates Vascular Permeability and Protects against Renal Ischemia-Reperfusion Injury

Ischemia-reperfusion injury (IRI) is a leading cause of AKI. This common clinical complication lacks effective therapies and can lead to the development of CKD. The αvβ5 integrin may have an important role in acute injury, including septic shock and acute lung injury. To examine its function in AKI, we utilized a specific function-blocking antibody to inhibit αvβ5 in a rat model of renal IRI. Pretreatment with this anti-αvβ5 antibody significantly reduced serum creatinine levels, diminished renal damage detected by histopathologic evaluation, and decreased levels of injury biomarkers. Notably, therapeutic treatment with the αvβ5 antibody 8 hours after IRI also provided protection from injury. Global gene expression profiling of post-ischemic kidneys showed that αvβ5 inhibition affected established injury markers and induced pathway alterations previously shown to be protective. Intravital imaging of post-ischemic kidneys revealed reduced vascular leak with αvβ5 antibody treatment. Immunostaining for αvβ5 in the kidney detected evident expression in perivascular cells, with negligible expression in the endothelium. Studies in a three-dimensional microfluidics system identified a pericyte-dependent role for αvβ5 in modulating vascular leak. Additional studies showed αvβ5 functions in the adhesion and migration of kidney pericytes in vitro Initial studies monitoring renal blood flow after IRI did not find significant effects with αvβ5 inhibition; however, future studies should explore the contribution of vasomotor effects. These studies identify a role for αvβ5 in modulating injury-induced renal vascular leak, possibly through effects on pericyte adhesion and migration, and reveal αvβ5 inhibition as a promising therapeutic strategy for AKI.