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Controversies in nephrology: Response to ‘renal albumin handling, facts, and artifacts’

2007; Elsevier BV; Volume: 72; Issue: 10 Linguagem: Inglês

10.1038/sj.ki.5002528

1523-1755

Leileata M. Russo, Ruben M. Sandoval, Dennis Brown, Bruce A. Molitoris, Wayne D. Comper,

Dialysis and Renal Disease Management

For 40 years indirect measurements of the glomerular sieving coefficient of albumin yielded very low values. The first direct measurement by 2-photon microscopy by Russo et al (Kidney Int (2007) 71, 504–513) gives values 50-times higher. This demonstrated that relatively large quantities of albumin are normally filtered based on size selectivity alone. Most of this albumin is retrieved and returned to the blood supply. These new discoveries represent a paradigm shift in our understanding of albumin processing by the kidney. They also serve to explain several anomalous aspects of previous studies on glomerular filtration and mechanism of albuminuria and support the fact that glomerular charge selectivity is not a major factor controlling glomerular permselectivity. For 40 years indirect measurements of the glomerular sieving coefficient of albumin yielded very low values. The first direct measurement by 2-photon microscopy by Russo et al (Kidney Int (2007) 71, 504–513) gives values 50-times higher. This demonstrated that relatively large quantities of albumin are normally filtered based on size selectivity alone. Most of this albumin is retrieved and returned to the blood supply. These new discoveries represent a paradigm shift in our understanding of albumin processing by the kidney. They also serve to explain several anomalous aspects of previous studies on glomerular filtration and mechanism of albuminuria and support the fact that glomerular charge selectivity is not a major factor controlling glomerular permselectivity. We thank Professor Christensen and his colleagues for taking their time to comment on our paper. They cite a select number of published papers using different techniques, as well as electron microscopical data, that they claim do not support the concept of nephrotic leakage of normal glomeruli to albumin as proposed by Russo et al.1.Russo L.M. Sandoval R.M. McKee M. et al.The normal kidney filters nephrotic levels of albumin retrieved by proximal tubule cells: retrieval is disrupted in nephrotic states.Kidney Int. 2007; 71: 504-513Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar However, it is important to examine each one of these cited studies in detail as the retrieval pathway and its receptor may confound the original author's interpretation. The very low albumin glomerular sieving coefficients (GSCs) of 10−4–10−5 cited by Christensen et al., suggesting glomerular impermeability, have never been biophysically justified: None can explain why they are so low in terms of glomerular permselectivity. They are also inconsistent with the original observations of Ryan and Karnovsky2.Ryan G.B. Karnovsky M.J. Distribution of endogenous albumin in the rat glomerulus: role of hemodynamic factors in glomerular barrier function.Kidney Int. 1976; 9: 36-45Abstract Full Text PDF PubMed Scopus (182) Google Scholar of the massive amounts of albumin that rapidly appear within minutes in the urinary space when the renal artery and vein are ligated. Of course, it was originally thought that these GSCs were so low because of albumin electrostatic repulsion from the fixed negative charges of glomerular capillary wall (glomerular charge selectivity). However, charge selectivity has now been shown to be essentially nonexistent. This has come from the discoveries that the low renal clearance of dextran sulfate, used to support the charge selectivity concept, is due to cell-mediated3.Comper W.D. Tay M. Wells X. et al.Desulphation of dextran sulphate during kidney ultrafiltration.Biochem J. 1994; 297: 31-34Crossref PubMed Scopus (43) Google Scholar,4.Vyas S.V. Burne M.J. Pratt L.M. et al.Glomerular processing of dextran sulphate.Arch Biochem Biophys. 1996; 332: 205-212Crossref PubMed Scopus (39) Google Scholar processing of dextran sulfate, and that use of stable negatively charged transport probes have shown that they do not have lower renal clearance compared to their uncharged counterparts.4.Vyas S.V. Burne M.J. Pratt L.M. et al.Glomerular processing of dextran sulphate.Arch Biochem Biophys. 1996; 332: 205-212Crossref PubMed Scopus (39) Google Scholar, 5.Greive K.A. Nikolic-Paterson D.J. Guimarães M.A.M. et al.Glomerular permselectivity factors are not responsible for the increase in fractional clearance of albumin in rat glomerulonephritis.Am J Pathol. 2001; 159: 1159-1170Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 6.Schaeffer R.C. Gratrix M.L. Mucha D.R. et al.The rat glomerular filtration barrier does not show negative charge selectivity.Microcirculation. 2002; 9: 329-342Crossref PubMed Scopus (51) Google Scholar, 7.Guimarães M.A.M. Nikolovski J. Pratt L.M. et al.Anomalous fractional clearance of negatively charged Ficoll relative to uncharged Ficoll.Am J Physiol. 2003; 285: F1118-F1124Crossref PubMed Scopus (48) Google Scholar, 8.Yu W. Sandoval R.M. Molitoris B.A. Quantitative intravital microscopy using generalized polarity concept for kidney studies.Am J Physiol. 2005; 289: C1197-C1208Crossref PubMed Scopus (47) Google Scholar, 9.Asgeirsson D. Rippe B. Venturoli D. Rippe C. Increased glomerular permeability to negatively charged Ficoll relative to neutral Ficoll in rats.Am J Physiol. 2006; 291: F1083-F1089Crossref PubMed Scopus (59) Google Scholar In addition, many investigations outside the renal area have shown the electrostatic interaction of albumin with negatively charged glycosaminoglycans, both in the test tube and in extracellular matrices of various connective tissues, is essentially nonexistent.10.Zamparo O. Comper W.D. Model anionic polysaccharide matrices exhibit lower charge selectivity than is normally associated with kidney ultrafiltration.Biophys Chem. 1990; 38: 167-178Crossref PubMed Scopus (19) Google Scholar, 11.Russo L.M. Bakris G. Comper W.D. Renal handling of albumin. A critical review of basic concepts and perspective.Am J Kidney Dis. 2002; 39: 899-919Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 12.Comper W.D. Osicka T.M. Russo L.M. Renal filtration, transport, and metabolism of albumin and albuminuria.in: Alpern R. Hebert S. The Kidney: Physiology and Pathophysiology. 2007Google Scholar Therefore, the only restrictive force that has been identified in the transport of albumin across the glomerular capillary wall is one of the size selectivity, which agrees with the conclusions of our two-photon study.1.Russo L.M. Sandoval R.M. McKee M. et al.The normal kidney filters nephrotic levels of albumin retrieved by proximal tubule cells: retrieval is disrupted in nephrotic states.Kidney Int. 2007; 71: 504-513Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar Christensen et al.13.Norden A.G. Lapsley M. Lee P.J. et al.Glomerular protein sieving and implications for renal failure in Fanconi syndrome.Kidney Int. 2001; 60: 1885-1892Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar cite GSC studies from Fanconi syndrome (that leads to non-nephrotic albuminuria) and tissue uptake14.Lund U. Rippe A. Venturoli D. et al.Glomerular filtration rate dependence of sieving of albumin and some neutral proteins in rat kidneys.Am J Physiol. 2003; 284: F1226-F1234Crossref PubMed Scopus (120) Google Scholar as evidence of low GSCs. However, these studies will be confounded by the existence of the retrieval pathway. There has been no evidence presented to suggest that tubular uptake is completely inhibited in Fanconi syndrome, particularly the retrieval pathway. Tissue uptake techniques through glomerular extraction and urinary excretion of albumin in the first 8–12 min after intravenous injection will not measure the amount of albumin that is retrieved and returned to the blood supply (a process likely to occur in 20–60 s15.Eppel G.A. Osicka T.M. Pratt L.M. et al.The return of glomerular filtered albumin to the rat renal vein.Kidney Int. 1999; 55: 1861-1870Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar or faster) during that time. The original and elegant observations by Park and Maack16.Park C.H. Maack T. Albumin absorption and catabolism by isolated perfused proximal convoluted tubules of the rabbit.J Clin Invest. 1984; 73: 767-777Crossref PubMed Scopus (185) Google Scholar are important as they discovered two albumin-binding sites in the perfused isolated rabbit proximal tubules: one with a Michaelis constant (Km) value of 0.031 mg ml−1 and a maximum binding range of 0.1–0.2 mg ml−1, and a second with a Km value of 1.2 mg ml−1 with a maximum of ∼10 mg ml−1. These binding sites closely correspond to the degradation and retrieval pathways, respectively.1.Russo L.M. Sandoval R.M. McKee M. et al.The normal kidney filters nephrotic levels of albumin retrieved by proximal tubule cells: retrieval is disrupted in nephrotic states.Kidney Int. 2007; 71: 504-513Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 5.Greive K.A. Nikolic-Paterson D.J. Guimarães M.A.M. et al.Glomerular permselectivity factors are not responsible for the increase in fractional clearance of albumin in rat glomerulonephritis.Am J Pathol. 2001; 159: 1159-1170Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 11.Russo L.M. Bakris G. Comper W.D. Renal handling of albumin. A critical review of basic concepts and perspective.Am J Kidney Dis. 2002; 39: 899-919Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 12.Comper W.D. Osicka T.M. Russo L.M. Renal filtration, transport, and metabolism of albumin and albuminuria.in: Alpern R. Hebert S. The Kidney: Physiology and Pathophysiology. 2007Google Scholar When Park and Maack16.Park C.H. Maack T. Albumin absorption and catabolism by isolated perfused proximal convoluted tubules of the rabbit.J Clin Invest. 1984; 73: 767-777Crossref PubMed Scopus (185) Google Scholar studied transcellular transport they only used low albumin concentrations in the tubular perfusate, where albumin would be processed predominantly by the low-capacity/high-affinity pathway. To examine transcytosis, they should have studied much higher albumin perfusate concentrations near 1 mg ml−1 so as to activate the low-affinity/high-capacity retrieval/transcytosis pathway which they did not do. Therefore, Christensen et al. are incorrect to suggest that Park and Maack16.Park C.H. Maack T. Albumin absorption and catabolism by isolated perfused proximal convoluted tubules of the rabbit.J Clin Invest. 1984; 73: 767-777Crossref PubMed Scopus (185) Google Scholar did not observe transcytosis of intact albumin; they simply did not set up the correct conditions for transcytosis to occur. Evidence points to the fact that the high-affinity/low-capacity receptor is the megalin/cubilin complex. In this case, we agree with Christensen et al. that megalin-deficient mice would only lead to relatively low levels of albuminuria. We reiterate Russo et al.1.Russo L.M. Sandoval R.M. McKee M. et al.The normal kidney filters nephrotic levels of albumin retrieved by proximal tubule cells: retrieval is disrupted in nephrotic states.Kidney Int. 2007; 71: 504-513Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar that the nature of the low-affinity/high-capacity receptor remains to be determined. Christensen et al. cite the low GSCs measured in cold-perfused kidneys by Haraldsson et al.,17.Ohlson M. Sörensson J. Lindström K. et al.Effects of filtration rate on the glomerular barrier and clearance of four differently shaped molecules.Am J Physiol. 2001; 281: F103-F113PubMed Google Scholar where cellular activity has been eliminated by low temperatures; Haraldsson et al. measured a fractional clearance of albumin as ∼0.001 (assumed to be same as GSC as tubular reabsorption was inhibited), whereas the GSC for 36-Å radius Ficoll was normal near ∼0.04.17.Ohlson M. Sörensson J. Lindström K. et al.Effects of filtration rate on the glomerular barrier and clearance of four differently shaped molecules.Am J Physiol. 2001; 281: F103-F113PubMed Google Scholar They cite this as evidence of glomerular charge selectivity to albumin. The high-capacity albumin receptors16.Park C.H. Maack T. Albumin absorption and catabolism by isolated perfused proximal convoluted tubules of the rabbit.J Clin Invest. 1984; 73: 767-777Crossref PubMed Scopus (185) Google Scholar appear to play a role in clearance measurements in cold-perfused kidney technique as the albumin sieving appears to be heavily influenced by the experimental set-up. The perfusate used by Haraldsson et al. was hypoalbuminemic with an albumin concentration at 18 mg ml−1. The glomerular filtration rate was also very low, because of the low temperatures, being 1–10% of normal. This means that albumin flux across the glomerular capillary wall was 0.5–5% of normal and therefore represents hypofiltration of albumin. Urinary excretion of albumin in cold-perfused kidneys will be the net result of albumin binding to albumin receptors of the proximal tubular cells and possibly other low temperature-sensitive binding sites. Albumin that is not bound is excreted. If the amount of albumin filtered is very low due to hypoalbuminemia and hypofiltration, the binding to albumin receptors will dominate, and it appears that this occurs in the cold-perfused kidneys. When albumin flux is increased in the cold-perfused kidney through increases in glomerular filtration rate (GFR),17.Ohlson M. Sörensson J. Lindström K. et al.Effects of filtration rate on the glomerular barrier and clearance of four differently shaped molecules.Am J Physiol. 2001; 281: F103-F113PubMed Google Scholar without alteration in the sieving of 36-Å radius Ficoll, the albumin-sieving coefficient increases exponentially to very high values17.Ohlson M. Sörensson J. Lindström K. et al.Effects of filtration rate on the glomerular barrier and clearance of four differently shaped molecules.Am J Physiol. 2001; 281: F103-F113PubMed Google Scholar that mimic the GSC for 36-Å radius Ficoll and the two-photon GSC estimate for albumin. Therefore, the albumin fractional clearances in cold-perfused kidneys are markedly GFR dependent but, critically, mimic those GSCs controlled by size selectivity alone when low temperature-induced hypofiltration and albumin binding is overcome. Contrary to the claim by Christensen et al. of low GSC with tubular inhibition, nephrotic states are characterized by albumin fractional clearances in the range of 0.03–0.06 (as determined in rats), which is consistent with inhibition of the retrieval pathway without any change in glomerular permeability of 36-Å molecules.5.Greive K.A. Nikolic-Paterson D.J. Guimarães M.A.M. et al.Glomerular permselectivity factors are not responsible for the increase in fractional clearance of albumin in rat glomerulonephritis.Am J Pathol. 2001; 159: 1159-1170Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 18.Tencer J. Frick I.M. Oqvist B.W. et al.Size-selectivity of the glomerular barrier to high molecular weight proteins: upper size limitations of shunt pathways.Kidney Int. 1998; 53: 709-715Abstract Full Text PDF PubMed Scopus (123) Google Scholar, 19.Osicka T.M. Hankin A.R. Comper W.D. Puromycin aminonucleoside nephrosis results in a marked increase in fractional clearance of albumin.Am J Physiol. 1999; 277: F139-F145PubMed Google Scholar, 20.Koltun M. Comper W.D. Retention of albumin in the circulation is governed by saturable renal cell-mediated processes.Microcirculation. 2004; 11: 351-360Crossref PubMed Scopus (22) Google Scholar, 21.Koltun M. Nikolovski J. Strong K. et al.Mechanism of hypoalbuminemia in rodents.Am J Physiol. 2005; 288: H1604-H1610Crossref PubMed Scopus (28) Google Scholar Our approach to the electron microscopical analysis was different to conventional analysis; here, we were confronted with the detection of a unique, very rapid, high-capacity transport process where tissue preparation and fixation conditions were often slow compared to the dynamic phenomena that we were dealing with. When we employed techniques to address this problem, we then began to see many large albumin-laden vesicles located along the apical/basolateral length of the cell and in association with the basolateral membrane. We do not deny that some of these vesicles may merge with lysosomes but, as in all types of TEM, it is very difficult to quantify dynamic phenomena and the examples provided by Christensen et al. are consistent with that. The TEM results certainly support all the other data demonstrating transcytosis/retrieval, but we agree that more work will be necessary to determine the extent to which this process is involved in the albumin-processing/retrieval pathway. We have already discussed the issues raised about the micropuncture and two-photon data in other responses by us published in this issue. However, it is pertinent to point out that retrieval of 230 g albumin day−1 in humans corresponds to ∼80 ng nephron−1 min. In summary, we firmly believe the Russo et al.1.Russo L.M. Sandoval R.M. McKee M. et al.The normal kidney filters nephrotic levels of albumin retrieved by proximal tubule cells: retrieval is disrupted in nephrotic states.Kidney Int. 2007; 71: 504-513Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar study when taken together with the extensive experimental evidence that overturns charge selectivity, the universal lack of change of the glomerular permselectivity of 36-Å molecules, like albumin, in nephrotic syndrome, and other studies support the concept that heavy albuminuria and nephrotic syndrome are primarily a tubular defect rather than a primary glomerular defect.