Quantitative imaging of basic functions in renal (patho)physiology

Abstract

Multiphoton fluorescence microscopy offers the advantages of deep optical sectioning of living tissue with minimal phototoxicity and high optical resolution. More importantly, dynamic processes and multiple functions of an intact organ can be visualized in real time using noninvasive methods, and quantified. These studies aimed to extend existing methods of multiphoton fluorescence imaging to directly observe and quantify basic physiological parameters of the kidney including glomerular filtration rate (GFR) and permeability, blood flow, urinary concentration/dilution, renin content and release, as well as more integrated and complex functions like the tubuloglomerular feedback (TGF)-mediated oscillations in glomerular filtration and tubular flow. Streptozotocin-induced diabetes significantly increased single-nephron GFR (SNGFR) from 32.4 +/- 0.4 to 59.5 +/- 2.5 nl/min and glomerular permeability to a 70-kDa fluorophore approximately eightfold. The loop diuretic furosemide 2-fold diluted and increased approximately 10-fold the volume of distal tubular fluid, while also causing the release of 20% of juxtaglomerular renin content. Significantly higher speeds of individual red blood cells were measured in intraglomerular capillaries (16.7 +/- 0.4 mm/s) compared with peritubular vessels (4.7 +/- 0.2 mm/s). Regular periods of glomerular contraction-relaxation were observed, resulting in oscillations of filtration and tubular flow rate. Oscillations in proximal and distal tubular flow showed similar cycle times ( approximately 45 s) to glomerular filtration, with a delay of approximately 5-10 and 25-30 s, respectively. These innovative technologies provide the most complex, immediate, and dynamic portrayal of renal function, clearly depicting the components and mechanisms involved in normal physiology and pathophysiology.

Visualization of Calcium Dynamics in Kidney Proximal Tubules

“…This material is preferentially retained in the renal blood circulation, but slow filtration and tubular passage can also be followed. 1,23 For two-photon imaging of cortical areas containing mostly PTs (Supplemental Figure Figure 3A shows the renal PT baseline intracellular calcium levels visualized by GCaMP2 fluorescence, in relatively large kidney cortex areas. Of note, despite the relatively homogeneous GCaMP2 expression in the PTs (Figure 1), different PTareas showed different GCaMP2 fluorescence.…”

Two‐photon microscopy of deep intravital tissues and its merits in clinical research

“…Multiphoton excitation imaging was employed to quantitatively reveal microvascular function (Dunn et al, 2003;Molitoris & Sandoval, 2005) as well as glomerular filtration and permeability within kidney with living animals (Kang et al, 2006;Sipos et al, 2007). In a further study, they showed how this nonlinear optical imaging can be used to observe and quantify drug delivery in an intact, living and functioning kidney.…”

Multiphoton Imaging of the Glomerular Permeability of Angiotensinogen

“…Indirectly, these studies also addressed the currently highly debated issue of the glomerular filtration of albumin. [14][15][16][17][18][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][17][18][19] to directly and quantitatively visualize the glomerular permeability of macromolecules.…”