Patients with albuminuria and CKD frequently have vascular dysfunction but the underlying mechanisms remain unclear. Because the endothelial surface layer, a meshwork of surface-bound and loosely adherent glycosaminoglycans and proteoglycans, modulates vascular function, its loss could contribute to both renal and systemic vascular dysfunction in proteinuric CKD. Using Munich-Wistar-Fromter (MWF) rats as a model of spontaneous albuminuric CKD, multiphoton fluorescence imaging and single-vessel physiology measurements revealed that old MWF rats exhibited widespread loss of the endothelial surface layer in parallel with defects in microvascular permeability to both water and albumin, in both continuous mesenteric microvessels and fenestrated glomerular microvessels. In contrast to young MWF rats, enzymatic disruption of the endothelial surface layer in old MWF rats resulted in neither additional loss of the layer nor additional changes in permeability. Intravenous injection of wheat germ agglutinin lectin and its adsorption onto the endothelial surface layer significantly improved glomerular albumin permeability. Taken together, these results suggest that widespread loss of the endothelial surface layer links albuminuric kidney disease with systemic vascular dysfunction, providing a potential therapeutic target for proteinuric kidney disease.

Podocytes are critical in the maintenance of a healthy glomerular filter, however they have been difficult to study in the intact kidney due to technical limitations. Here we report the development of serial multiphoton microscopy (MPM) of the same glomeruli over several days to visualize the motility of podocytes and parietal epithelial cells (PEC) in vivo. In Podocin-GFP mice podocytes formed sporadic multi-cellular clusters after unilateral ureteral ligation (UUO) and migrated into the parietal Bowman’s capsule. The tracking of single cells in Podocin-confetti mice featuring cell-specific expression of CFP, GFP, YFP, or RFP revealed the simultaneous migration of multiple podocytes. In PEPCK-GFP mice serial MPM found PEC-to-podocyte migration and nanotubule connections. Our data support the highly dynamic rather than static nature of the glomerular environment and cellular composition. Future application of this new approach promises to advance our understanding of the mechanisms of glomerular injury and regeneration.

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

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signaling is a robust and key pathogenic mechanism in podocyte injury. This in vivo imaging approach will allow future detailed investigation of the molecular and cellular mechanisms of glomerular disease in the intact living kidney.

Peti-Peterdi J, Burford JL, Hackl MJ. The first decade of using multiphoton microscopy for high-power kidney imaging. Am J Physiol Renal Physiol 302: F227-F233, 2012. First published October 26, 2011 doi:10.1152/ajprenal.00561.2011In this review, we highlight the major scientific breakthroughs in kidney research achieved using multiphoton microscopy (MPM) and summarize the milestones in the technological development of kidney MPM during the past 10 years. Since more and more renal laboratories invest in MPM worldwide, we discuss future directions and provide practical, useful tips and examples for the application of this still-emerging optical sectioning technology. Advantages of using MPM in various kidney preparations that range from freshly dissected individual glomeruli or the whole kidney in vitro to MPM of the intact mouse and rat kidney in vivo are reviewed. Potential combinations of MPM with micromanipulation techniques including microperfusion and micropuncture are also included. However, we emphasize the most advanced and complex, quantitative in vivo imaging applications as the ultimate use of MPM since the true mandate of this technology is to look inside intact organs in live animals and humans. intravital imaging; juxtaglomerular apparatus; podocyte; two-photon microscopy IT HAS BEEN MORE THAN 10 YEARS since two-or three-photon excitation, collectively called multiphoton microscopy (MPM), was first applied to the study of the living kidney tissue by the Peti-Peterdi-Bell (37, 38) and Dunn-Molitoris groups (6, 42) that pioneered several applications of this powerful new imaging technology. Over the past decade, MPM has provided stunning images and real-time movies of the structure and function of the intact kidney in unparalleled spatial and temporal resolution. Because of the ability of this noninvasive imaging approach to directly visualize dynamic intrarenal processes in vivo and in near real-time without causing tissue damage, MPM has revolutionized renal physiology research and served as the perfect complement to more traditional biological and histological techniques. MPM also opened the door to studying otherwise inaccessible cell types and complex tissue structures like the glomerular podocyte (30, 39) and the juxtaglomerular apparatus (JGA) (37) in their intact environment. Applications of MPM helped to change several paradigms in renal (patho)physiology. Imaging data have been incorporated into a number of textbooks on kidney function (3,8,41), and MPM videos (30,37,39,40) have been used as visual aid and teaching material in graduate and medical student classrooms worldwide.MPM offers a state-of-the-art imaging technique superior for deep optical sectioning of living tissues. The higher resolution and minimal phototoxicity of this method permit longer time periods of continuous tissue scanning with uses in near realtime imaging of intact organs in vivo. MPM has applications far beyond the generation of superior images: dynamic intracellular processes as well as more complex physiological ...

Our study identified a molecular mechanism for laminar flow-activated LEC proliferation.

Diabetes is a major epidemic, and diabetic nephropathy is the most common cause of end-stage renal disease. Two critical components of diabetic nephropathy are persistent inflammation and chronic renal ischemia from widespread vasculopathy. Moreover, acute ischemic renal injury is common in diabetes, potentially causing chronic kidney disease or end-stage renal disease. Accordingly, we tested the hypothesis that acute renal ischemia accelerates nephropathy in diabetes by activating proinflammatory pathways. Lean and obese-diabetic ZS rats (F(1) hybrids of spontaneously hypertensive heart failure and Zucker fatty diabetic rats) were subjected to bilateral renal ischemia or sham surgery before the onset of proteinuria. The postischemic state in rats with obesity-diabetes was characterized by progressive chronic renal failure, increased proteinuria, and renal expression of proinflammatory mediators. Leukocyte number in obese-diabetic rat kidney was markedly increased for months after ischemia. Intrarenal blood flow velocity was decreased after ischemia in lean control and obese-diabetic rats, although it recovered in lean rats. At 2 mo after ischemia, blood flow velocity decreased further in sham-surgery and postischemia obese-diabetic rats, so that RBC flow velocity was only 39% of control in the obese-diabetic rats after ischemia. In addition, microvascular density remained depressed at 2 mo in kidneys of obese-diabetic rats after ischemia. Abnormal microvascular permeability and increases in interstitial fibrosis and apoptotic renal cell death were also more pronounced after ischemia in obese-diabetic rats. These data support the hypothesis that acute renal ischemia in obesity-diabetes severely aggravates chronic inflammation and vasculopathy, creating a self-perpetuating postischemia inflammatory syndrome, which accelerates renal failure.