Multiphoton Imaging of the Glomerular Permeability of Angiotensinogen

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 cell-specific 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 Intravenously injected human recombinant AGT (hAGT), which has little affinity for enzymatic cleavage by rodent renin because of high species specificity,^12,13 was undetectable in the urine of angiotensin II-induced hypertensive rats,⁸ suggesting that the increase in urinary AGT is likely of tubular rather than glomerular (plasma) origin. However, the plasma level of AGT is much greater than the plasma level of urinary AGT, and therefore, the increase in urinary AGT in a kidney injury model could result from the damage of the glomerular filtration barrier (GFB) and AGT leakage into the urine.Therefore, the present study was conducted to measure exactly how much AGT is filtered through the GFB and ultimately, excreted through the urine in normal circumstances and the glomerulosclerosis model of Munich-Wistar-Frömter (MWF) rats that shows significant GFB damage. We compared the glomerular permeability of AGT with the glomerular permeability of albumin, because albumin (66 kD) has a molecular mass similar to AGT (∼60 kD); also, the increase in its urinary excretion closely reflects the decline of renal function. Indirectly, these studies also addressed the currently highly debated issue of the glomerular filtration of albumin.^14–19 To compare the glomerular processing of these two proteins, we employed intravital multiphoton fluorescence microscopy, which has been used by our laboratory^15,20,21 and the laboratories of others,^16–19 to directly and quantitatively visualize the glomerular permeability of macromolecules.