Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...

Indiana University School of Medicine

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 Î¼m 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 0.035Â±0.005 to 0.016Â±0.004 (P<0.01). FrÃ¶mter 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.

Multiple factors influence glomerular albumin permeability in rats.

Diff&eacute;rents laboratoires a r&eacute;cemment rapport&eacute; des r&eacute;sultats incongrus d&eacute;crivant la quantification de la filtration de l&#39;albumine par microscopie biphotonique. Nous avons &eacute;tudi&eacute; les facteurs qui influencent le coefficient de tamisage glom&eacute;rulaire de l&#39;albumine (GSC(A)) dans le but d&#39;expliquer ces rapports discordants et de d&eacute;velopper des modes op&eacute;ratoires normalis&eacute;s pour la d&eacute;termination de GSC(A). Plusieurs facteurs ont influenc&eacute; GSC(A), y compris la profondeur de rein d&#39;acquisition d&#39;images (10-20 &mu;m convenait), la s&eacute;lection du fluorophore (sondes &eacute;mettant des longueurs d&#39;onde sont sup&eacute;rieurs), la s&eacute;lection des diff&eacute;rentes r&eacute;gions de plasma pour mesures de fluorescence, la taille et caract&eacute;ristiques de la dispersion mol&eacute;culaire de polym&egrave;res de dextran si utilis&eacute;, statut alimentaire et la souche g&eacute;n&eacute;tique du rat. Le je&ucirc;ne r&eacute;duit la GSC(A) chez des rats Wistar Munich Simonsen de 0.035&plusmn;0.005 &agrave; 0.016&plusmn;0.004 (P &lt; 0,01). Des rats Wistar Munich Fr&ouml;mter avaient un GSC(A) beaucoup plus faible dans la fed et les &Eacute;tats &agrave; jeun. Enfin, nous avons document&eacute; transcytose vaste albumine avec livraison v&eacute;siculaire et tubulaire &agrave; et fusion avec la membrane baso-lat&eacute;rale dans les cellules du tubule proximal S1. En r&eacute;sum&eacute;, ces r&eacute;sultats mieux expliquer la microscopie pr&eacute;c&eacute;demment en conflit et la microponction donn&eacute;es d&eacute;crivant la filtration de l&#39;albumine et de mettre en &eacute;vidence la nature dynamique de la perm&eacute;abilit&eacute; glom&eacute;rulaire albumine.

Plusieurs facteurs influencent la perm&eacute;abilit&eacute; glom&eacute;rulaire albumine chez le rat.

Diversi laboratori recentemente segnalato risultati incongrui che descrive la quantificazione di filtrazione di albumina utilizzando la microscopia del due-fotone. Abbiamo studiato i fattori che influenzano il coefficiente di setacciatura glomerulare per l&#39;albumina (GSC(A)) nel tentativo di spiegare queste relazioni discordanti e sviluppare procedure operative standard per la determinazione GSC(A). Molteplici fattori influenzato GSC(A), tra cui la profondit&agrave; di rene di acquisizione immagine (10-20 &mu;m era appropriato), la selezione del fluoroforo (sonde che emettono lunghezze d&#39;onda pi&ugrave; lunghe erano superiori), la selezione delle regioni al plasma per misure in fluorescenza, le dimensioni e caratteristiche di dispersione molecolare dei polimeri Destrano se usato, stato dietetico e il ceppo genetico del ratto. Digiuno ridotta la GSC(A) in ratti Wistar Monaco Simonsen da 0.035&plusmn;0.005 a 0.016&plusmn;0.004 (P &lt; 0,01). Ratti Wistar Monaco Fr&ouml;mter avevano un GSC(A) molto bassa in entrambi la fed e gli Stati di digiuno. Infine, abbiamo documentato albumina ampio transcytosis con consegna vescicolare dei suini e tubolare a e fusione con la membrana basolaterale nelle cellule del tubulo prossimale S1. In sintesi, questi risultati aiutano a spiegare la microscopia precedentemente contrastanti e microponfi dati che descrivono la filtrazione di albumina ed evidenziare la natura dinamica della permeabilit&agrave; glomerulare albumina.

Diversi fattori influenzano la permeabilit&agrave; glomerulare albumina nei ratti.

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.

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

Nous avons constat&eacute; pr&eacute;c&eacute;demment que chronique l&#39;endoth&eacute;line-1 (ET-1) infusion chez le rat Sprague-Dawley augmente la perm&eacute;abilit&eacute; glom&eacute;rulaire &agrave; l&#39;albumine (P(alb)) quotes ind&eacute;pendant in vitro de la tension art&eacute;rielle avec aucun albuminurie observ&eacute;e. Dans cette &eacute;tude, nous avons suppos&eacute; que, lorsque accompagn&eacute;s absorption d&#39;albumine par l&#39;interm&eacute;diaire du tube proximal, qui masque l&#39;accroissement pr&eacute;vu de l&#39;excr&eacute;tion urinaire de l&#39;albumine, ET-1 augmente filtration glom&eacute;rulaire de l&#39;albumine.

Chronique l&#39;endoth&eacute;line-1 infusion &eacute;l&egrave;ve glom&eacute;rulaire coefficient de tamisage et de r&eacute;absorption tubulaire proximale de l&#39;albumine chez le rat.

In precedenza abbiamo trovato che cronica endotelina-1 (ET-1) infusione in ratti Sprague-Dawley aumenta la permeabilit&agrave; glomerulare all&#39;albumina (P(alb)) come valutato in vitro indipendente della pressione sanguigna con nessun osservata albuminuria. In questo studio, abbiamo ipotizzato che ET-1 aumenta la filtrazione glomerulare albumina con accompagnato aumento di assorbimento di albumina attraverso il tubulo prossimale, che maschera il previsto aumento dell&#39;escrezione urinaria di albumina.

Cronica endotelina-1 infusione eleva glomerulare coefficiente di setacciatura e albumina tubulare prossimale reuptake nel ratto.

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. 

Shear stress is normalized in glomerular capillaries following ⅚ nephrectomy.

Evidence from multiple studies supports the concept that both glomerular filtration and proximal tubule (PT) reclamation affect urinary albumin excretion rate. To better understand these roles of glomerular filtration and PT uptake, we investigated these processes in two distinct animal models. In a rat model of acute exogenous albumin overload, we quantified glomerular sieving coefficients (GSC) and PT uptake of Texas Red-labeled rat serum albumin using two-photon intravital microscopy. No change in GSC was observed, but a significant decrease in PT albumin uptake was quantified. In a second model, loss of endogenous albumin was induced in rats by podocyte-specific transgenic expression of diphtheria toxin receptor. In these albumin-deficient rats, exposure to diphtheria toxin induced an increase in albumin GSC and albumin filtration, resulting in increased exposure of the PTs to endogenous albumin. In this case, PT albumin reabsorption was markedly increased. Analysis of known albumin receptors and assessment of cortical protein expression in the albumin overload model, conducted to identify potential proteins and pathways affected by acute protein overload, revealed changes in the expression levels of calreticulin, disabled homolog 2, NRF2, angiopoietin-2, and proteins involved in ATP synthesis. Taken together, these results suggest that a regulated PT cell albumin uptake system can respond rapidly to different physiologic conditions to minimize alterations in serum albumin level. 

Proximal Tubules Have the Capacity to Regulate Uptake of Albumin.

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.

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

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 v5 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 v5 in a rat model of renal IRI. Pretreatment with this anti-v5 antibody significantly reduced serum creatinine levels, diminished renal damage detected by histopathologic evaluation, and decreased levels of injury biomarkers. Notably, therapeutic treatment with the v5 antibody 8 hours after IRI also provided protection from injury. Global gene expression profiling of post-ischemic kidneys showed that v5 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 v5 antibody treatment. Immunostaining for v5 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 v5 in modulating vascular leak. Additional studies showed v5 functions in the adhesion and migration of kidney pericytes  Initial studies monitoring renal blood flow after IRI did not find significant effects with v5 inhibition; however, future studies should explore the contribution of vasomotor effects. These studies identify a role for v5 in modulating injury-induced renal vascular leak, possibly through effects on pericyte adhesion and migration, and reveal v5 inhibition as a promising therapeutic strategy for AKI.

Inhibition of v5 Integrin Attenuates Vascular Permeability and Protects against Renal Ischemia-Reperfusion Injury.

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  peripheral infusion (<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.

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

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 (+∆16%) and impaired volume hemodynamics (-∆14%). Serum albumin, but not serum globulins or creatinine, concentration was decreased (-0.54g/dL), which was concomitant with increased filtration of albumin (3.7 vs 0.8 g per 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 (+∆2.20score/image), progressive augmentation of red blood cell velocity (+∆269µm/s) and micro vessel diameter (+∆4.3µm), and increased vascular injury (+∆0.61leakage/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.

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

Chronic kidney disease results in high serum urea concentrations leading to excessive protein carbamylation, primarily albumin. This is associated with increased cardiovascular disease and mortality. Multiple methods were used to address whether carbamylation alters albumin metabolism. Intravital two-photon imaging of the Munich Wistar Frömter (MWF) rat kidney and liver allowed us to characterize filtration and proximal tubule uptake and liver uptake. Microscale thermophoresis enabled quantification of cubilin (CUB7,8 domain) and FcRn binding. Finally, multiple biophysical methods including dynamic light scattering, small-angle X-ray scattering, LC-MS/MS and in silico analyses were used to identify the critical structural alterations and amino acid modifications of rat albumin. Carbamylation of albumin reduced binding to CUB7,8 and FcRn in a dose-dependent fashion. Carbamylation markedly increased vascular clearance of carbamylated rat serum albumin (cRSA) and altered distribution of cRSA in both the kidney and liver at 16 h post intravenous injection. By evaluating the time course of carbamylation and associated charge, size, shape, and binding parameters in combination with in silico analysis and mass spectrometry, the critical binding interaction impacting carbamylated albumin's reduced FcRn binding was identified as K524. Carbamylation of RSA had no effect on glomerular filtration or proximal tubule uptake. These data indicate urea-mediated time-dependent carbamylation of albumin lysine K524 resulted in reduced binding to CUB7,8 and FcRn that contribute to altered albumin transport, leading to increased vascular clearance and increased liver and endothelial tissue accumulation.

Silvia B. Campos-Bilderback

Department of Medicine,
Division of Nephrology,
Department of Medicine, Division of Nephrology

Indiana University School of Medicine

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Silvia B. Campos-Bilderback

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Medicine,Division of Nephrology,Department of Medicine, Division of Nephrology

Indiana University School of Medicine

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Department of Medicine,
Division of Nephrology,
Department of Medicine, Division of Nephrology