Metabolic processing of proteins during renal passage

The underlying mechanism of albuminuria has generally been attributed to a loss of glomerular permselectivity, involving a loss of charge selectivity and/or the formation of large pores in the glomerular capillary wall (GCW). Contrary to this, studies have shown that the GCW is not significantly charge or conformation selective, and that the mechanisms of albuminuria are centred at the post-filtration processing events. Despite this body of evidence highlighting the importance of post- filtration processing of albumin, there are ongoing controversies in the literature regarding the mechanisms of albuminuria. This thesis aims to address these controversies and re-examines the renal processing of proteins, in particular albumin, and low molecular weight proteins (LMWP). Although studies have shown the contrary, albuminuria, or increased fractional clearance (FC) of albumin, in nephrotic states is still being attributed to glomerular permeability dysfunction. A portion of this thesis re-examines this issue and presents studies confirming that charge selectivity does not govern the FC of albumin, and that large pores in the GCW do not account for albuminuria. In the same manner, hypoalbuminemia, or increased albumin loss from the plasma, is thought to be controlled by mechanisms centred at the GCW. A large portion of this thesis focuses on the plasma elimination of albumin and re-evaluates the issue of glomerular permselectivity. The studies presented demonstrate that increased plasma clearance of albumin, with accompanying increase in urinary excretion, is not due to increased glomerular permeability, but rather, is associated with post-filtration processing events. The majority of studies on renal function and proteinuria have been performed on albumin. There are no similar studies on LMWP. The LMWP have not been examined since the very early studies performed more than 30 years ago using ex- vivo techniques. A section of this thesis examines the renal processing of LMWP in- vivo, in comparison studies with albumin. Although it has generally been assumed that all proteins are processed by the same mechanism in the kidney, this thesis demonstrates differential renal processing of albumin as compared to LMWP. This finding has great impact on the interpretation of proteinuria and its underlying mechanisms, and provides plausible explanation why low molecular weight proteinuria and albuminuria are entirely separate processes. Finally, this thesis discusses the findings that highlight the important and under- estimated role of post-filtration processing events in the mechanisms of albuminuria and hypoalbuminemia. This has great clinical implication, as it is important for clinicians to distinguish albuminuria that gives rise to clinically significant hypoalbuminemia, from albuminuria with no associated hypoalbuminemia

Urinary excretion of [³H]lysozyme and [¹⁴C]RSA in control and PAN rats, shown as Bq and total excretion in 24 h (as percentage of the injected dose of radioactivity).

<p>^† = <i>P</i> < 0.0001 versus control.</p><p>Urinary excretion of [³H]lysozyme and [¹⁴C]RSA in control and PAN rats, shown as Bq and total excretion in 24 h (as percentage of the injected dose of radioactivity).</p

Specific activities of radiolabeled preparations, expressed as Becquerels per milligram of sample (Bq/mg).

<p>Specific activities of radiolabeled preparations, expressed as Becquerels per milligram of sample (Bq/mg).</p

Inhibition of the Metabolic Degradation of Filtered Albumin Is a Major Determinant of Albuminuria

<div><p>Inhibition of the degradation of filtered albumin has been proposed as a widespread, benign form of albuminuria. There have however been recent reports that radiolabeled albumin fragments in urine are not exclusively generated by the kidney and that in albuminuric states albumin fragment excretion is not inhibited. In order to resolve this controversy we have examined the fate of various radiolabeled low molecular weight protein degradation products (LMWDPs) introduced into the circulation in rats. The influence of puromycin aminonucleoside nephrosis on the processing and excretion of LMWDPs is also examined. The status and destinies of radiolabeled LMWDPs in the circulation are complex. A major finding is that LMWDPs are rapidly eliminated from the circulation (>97% in 2 h) but only small quantities (<4%) are excreted in urine. Small (<4%) but significant amounts of LMWDPs may have prolonged elimination (>24 h) due to binding to high molecular weight components in the circulation. If LMWDPs of albumin seen in the urine are produced by extra renal degradation it would require the degradation to far exceed the known catabolic rate of albumin. Alternatively, if an estimate of the role of extra renal degradation is made from the limit of detection of LMWDPs in plasma, then extra renal degradation would only contribute <1% of the total excretion of LMWDPs of albumin. We confirm that the degradation process for albumin is specifically associated with filtered albumin and this is inhibited in albuminuric states. This inhibition is also the primary determinant of the massive change in intact albuminuria in nephrotic states.</p></div

Representative size exclusion profiles of [³H]lysozyme in plasma taken at 0.25 h (A), 2 h (B), and 24 h (C), after intravenous administration in PAN (<i>closed circles</i>) and control (<i>open circles</i>) rats.

<p>Representative size exclusion profiles of [³H]lysozyme in urine collected at 0.25 h (<b>D</b>), 2 h (<b>E</b>), and 24 h (<b>F</b>). Native [³H]lysozyme elutes at Kav 0.46. Chromatography was performed on Sephadex G-50. For both plasma and urine profiles, <i>n</i> = 3 for each time point.</p

Representative size exclusion profiles of [¹⁴C]RSA in urine collected after intravenous administration in PAN (<i>closed circles</i>) and control (<i>open circles</i>) rats.

<p>The same levels of radioactivity in the urine of PAN and controls was loaded onto the column. Identical profiles were obtained at 2 h and 24 h (from <i>n</i> = 5 for each time point). Native [¹⁴C]RSA elutes at Kav 0.18. Chromatography was performed on Sephadex G-100.</p

The percentage of administered radioactivity recovered in the plasma, urine, kidney and extra-renal (ER) tissues, at 2 h after injecting [³H]lysozyme, [¹⁴C]cytochrome c, [¹⁴C]RSA or [³H]RSA tryptic peptides.

<p>The extra-renal tissues analyzed were muscle, spleen and liver. Values are mean (± SD).</p><p>The percentage of administered radioactivity recovered in the plasma, urine, kidney and extra-renal (ER) tissues, at 2 h after injecting [³H]lysozyme, [¹⁴C]cytochrome c, [¹⁴C]RSA or [³H]RSA tryptic peptides.</p

Representative size exclusion profiles of [³H]lysozyme in plasma (A) and in urine (B) taken at various times after intravenous administration in normal rats.

<p>The elution profile of native [³H]lysozyme (<i>open circles</i>) is shown for comparison. Chromatography was performed on Sephadex G-50. For both plasma and urine samples <i>n</i> = 3 for each time point.</p

Testing the educational potential of 3D visualization software in oral radiographic interpretation

There is heightened optimism about the potential of 3D visualization software as an alternative learning resource in radiology education. The purpose of this study was to investigate the effect of 3D visualization software on students’ learning of oral radiographic interpretation from 2D radiographic images. Fourth-year dental students underwent a learning intervention phase of radiographic interpretation of oral pathoses using 3D visualization software. The success of the educational intervention was assessed by quantitative means, using a radiographic interpretation test, and by qualitative means, using a structured Likert-scale survey, asking students to evaluate their own learning outcomes. It was anticipated that training with the rotational mode of 3D visualization software would provide additional depth cues, enabling students to create spatial-mental models of anatomy that they can apply to 2D radiographic interpretation of oral pathoses. Although quantitative assessment did not support this, questionnaire evaluations demonstrated a positive effect of the 3D visualization software by enhancing students’ learning about radiographic interpretation. Despite much optimism about the educational potential of 3D visualization software, it is important to understand the interactions between learners and such new technologies in order to identify potential advantages and limitations prior to embracing them as learning resources