Generation of Urinary Albumin Fragments Does Not Require Proximal Tubular Uptake

109

118

Patients and animals with renal injury exhibit increased urinary excretion of angiotensinogen. Although increased tubular synthesis of angiotensinogen contributes to the increased excretion, we do not know to what degree glomerular filtration of systemic angiotensinogen, especially through an abnormal glomerular filtration barrier, contributes to the increase in urinary levels. Here, we used multiphoton microscopy to visualize and quantify the glomerular permeability of angiotensinogen in the intact mouse and rat kidney. In healthy mice and Munich-Wistar-Frömter rats at the early stage of glomerulosclerosis, the glomerular sieving coefficient of systemically infused Atto565-labeled human angiotensinogen (Atto565-hAGT), which rodent renin cannot cleave, was only 25% of the glomerular sieving coefficient of albumin, and its urinary excretion was undetectable. In a more advanced phase of kidney disease, the glomerular permeability of Atto565-hAGT was slightly higher but still very low. Furthermore, unlike urinary albumin, the significantly higher urinary excretion of endogenous rat angiotensinogen did not correlate with either the Atto565-hAGT or Atto565-albumin glomerular sieving coefficients. These results strongly suggest that the vast majority of urinary angiotensinogen originates from the tubules rather than glomerular filtration. The renin-angiotensin system (RAS) is one of the most important regulatory mechanisms of body fluid, electrolyte homeostasis, and BP. 1-4 RAS in the kidney is independently regulated from RAS in the systemic circulation, and it has been implicated in the development of hypertension and kidney diseases. For example, renal epithelial cellspecific overexpression of human angiotensinogen (AGT) in human renin transgenic mice resulted in increased renal angiotensin II, hypertension, and renal fibrosis without any changes in circulating angiotensin II. 5 Also, Dahl salt-sensitive rats, which show low plasma renin activity under high salt feeding, had higher renal angiotensin II content in the kidney compared with normal salt-fed control animals. 6 Although all components that are necessary for angiotensin II production are expressed in the kidney, 3 AGT is, currently, the only component that is noninvasively measurable in the urine of patients. The level of urinary AGT reflects the activity of the intrarenal RAS, and it is associated with pathologic states in several experimental 7,8 and clinical studies. 9,10 Although the major source of the circulating AGT is the liver, we and others have previously shown that AGT is produced in the proximal tubules through a de novo pathway. 3,11

Section: Discussionmentioning

confidence: 79%

“…A very recent report by Weyer et al 29 found small amounts of low-molecular weight fragments of injected labeled albumin in the plasma of mice. Because these small fragments can be readily filtered in the kidney, they may help explain the higher albumin GSC results.…”

Section: Discussionmentioning

confidence: 94%

Multiphoton Imaging of the Glomerular Permeability of Angiotensinogen

Nakano

Kobori

Burford

et al. 2012

109

118

“…35 More recently, it could be shown that albumin degradation products typically found in the urine 36 are not formed within proximal tubular cells of the kidney but rather, are formed in other tissues. 37 By two-photon microscopy, fluorescently labeled albumin was observed within endocytotic vesicles of proximal tubules in vivo. 38 Some of the vesicles fused with the basolateral membrane during the observation period, indicating a transcytosis mechanism.…”

Section: Discussionmentioning

confidence: 99%

Albumin Is Recycled from the Primary Urine by Tubular Transcytosis

Tenten¹,

Menzel²,

Kunter³

et al. 2013

117

103

Under physiologic conditions, significant amounts of plasma protein pass the renal filter and are reabsorbed by proximal tubular cells, but it is not clear whether the endocytosed protein, particularly albumin, is degraded in lysosomes or returned to the circulatory system intact. To resolve this question, a transgenic mouse with podocyte-specific expression of doxycycline-inducible tagged murine albumin was developed. To assess potential glomerular backfiltration, two types of albumin with different charges were expressed. On administration of doxycycline, podocytes expressed either of the two types of transgenic albumin, which were secreted into the primary filtrate and reabsorbed by proximal tubular cells, resulting in serum accumulation. Renal transplantation experiments confirmed that extrarenal transcription of transgenic albumin was unlikely to account for these results. Genetic deletion of the neonatal Fc receptor (FcRn), which rescues albumin and IgG from lysosomal degradation, abolished transcytosis of both types of transgenic albumin and IgG in proximal tubular cells. In summary, we provide evidence of a transcytosis within the kidney tubular system that protects albumin and IgG from lysosomal degradation, allowing these proteins to be recycled intact. 24: 196624: -198024: , 201324: . doi: 10.1681 The main function of the kidney is to filter plasma, while at the same time, retain the majority of plasma proteins. However, a certain fraction of plasma proteins inevitably passes the glomerular filtration barrier. The most abundant and most studied plasma protein, albumin, is produced at a rate of about 15 g per day in humans. 1 In the renal glomerulus, the albumin sieving coefficient (i.e., the transport rate of albumin across the glomerular filter in relation to water) has been estimated to be below 0.001. [2][3][4] In more recent studies using intravital microscopy, the albumin sieving coefficient has been estimated to be significantly higher, although this result is still controversial. 5-7 Thus, even when assuming a tight renal filtration barrier, significant amounts of albumin pass the glomerular filterroughly in the range of 1 g per day in healthy humans, J Am Soc Nephrol

“…21 PTCs are specialized to capture ultrafiltrated proteins by apical receptor-mediated endocytosis (ARME) through abundantly expressed, rapidly recycling multiligand tandem receptors, megalin, and cubilin. 22 Megalin-and cubilin-knockout mice show defective PTC endocytosis and urinary loss of ultrafitrated plasma proteins [23][24][25][26] and lysosomal enzymes. 27,28 Although defective ARME caused by loss of megalin/cubilin was a possible explanation for proteinuria of cystinotic patients, normal immunolabeling of megalin/cubilin was reported in an end stage cystinotic kidney despite massive proteinuria, calling attention to glomerular leakage.…”

mentioning

confidence: 99%

Time Course of Pathogenic and Adaptation Mechanisms in Cystinotic Mouse Kidneys

Chevronnay

Janssens

Smissen

et al. 2014

126

Cystinosis, a main cause of Fanconi syndrome, is reproduced in congenic C57BL/6 cystinosin knockout (KO) mice. To identify the sequence of pathogenic and adaptation mechanisms of nephropathic cystinosis, we defined the onset of Fanconi syndrome in KO mice between 3 and 6 months of age and analyzed the correlation with structural and functional changes in proximal tubular cells (PTCs), with focus on endocytosis of ultrafiltrated disulfide-rich proteins as a key source of cystine. Despite considerable variation between mice at the same age, typical event sequences were delineated. At the cellular level, amorphous lysosomal inclusions preceded cystine crystals and eventual atrophy without crystals. At the nephron level, lesions started at the glomerulotubular junction and then extended distally. In situ hybridization and immunofluorescence revealed progressive loss of expression of megalin, cubilin, sodiumglucose cotransporter 2, and type IIa sodium-dependent phosphate cotransporter, suggesting apical dedifferentiation accounting for Fanconi syndrome before atrophy. Injection of labeled proteins revealed that defective endocytosis in S1 PTCs led to partial compensatory uptake by S3 PTCs, suggesting displacement of endocytic load and injury by disulfide-rich cargo. Increased PTC apoptosis allowed luminal shedding of cystine crystals and was partially compensated for by tubular proliferation. We conclude that lysosomal storage triggered by soluble cystine accumulation induces apical PTC dedifferentiation, which causes transfer of the harmful load of disulfide-rich proteins to more distal cells, possibly explaining longitudinal progression of swan-neck lesions. Furthermore, our results suggest that subsequent adaptation mechanisms include lysosomal clearance of free and crystalline cystine into urine and ongoing tissue repair.