Multiphoton Imaging of the Glomerular Permeability of Angiotensinogen

Abstract

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

Albuminuria is controlled primarily by proximal tubules

“…7 The two photon microscopy method has been associated with technical issues, with some reports of albumin GSC values <0.0006. 8,9 These have been resolved by testing various background settings on the microscope. 10 Another technical issue raised has been 'out of focus' fluor escence, 11 but this has no physical basis in two photon microscopy.…”

Urinary Angiotensinogen: A Promising Biomarker of AKI Progression in Acute Decompensated Heart Failure: What Does It Mean?

“…Moreover, efficient tubular reabsorption or degradation of any filtered AGT could prevent it from appearing in the urine. The fact that AGT is detectable in urines from humans and animals without kidney disease supports the view that uAGT originates primarily from local kidney sources (17,20). Moreover, kidney angiotensin II levels are much higher than those detected in the circulation.…”

Podocytes are key—although albumin never reaches the slit diaphragm

“…This, and other factors, explains their high value of 0.034 for albumin GSC. 4 Recently, three two-photon microscopy studies [11][12][13] reported very low albumin GSCs (approximately 0.0006). Sandoval et al 14 criticized these studies because large negative voltage offsets were applied to external photodetector amplifiers, which can result in inaccurate, low values.…”