Cell. Isolated Perfused Kidney. Isolated Perfused Tubule. Intact Kidney. Isolated Cells

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1 Bruce A. Molitoris i Departmentt of Medicine Indiana Center for Biological Microscopy Indiana University i Sh School of Medicine Mdii

2 Functional and Therapeutic Studies in Vivo Cell Intact Kidney Isolated Perfused Kidney Isolated Perfused Tubule Isolated Cells Molitoris and Sandoval AJP, 2005

3 mv CL mv DT DT bs PT S1 S1 PT Femoral Artery & Venous access

4 500K Mw FITC-Dextran (Green) 10K Mw Rhodamine Dextran (Red Hoechst (Blue)

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6 Glomerular Permeability and Vascular Clearance

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9 Question: What are the Underlying Mechanisms of Proteinuria? Hypothesis: Glomerular Filtration as well as Proxmal Tubule Reabsorption are Citi Critical ldt Determinants t of fproteinuria ti i

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11 Renal albumin handling: A look at the dark side of the filter. Gekle, KI (2007) 2

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17 5 seconds 19 seconds ~3 minutes

18 69 kda FITC Dextran ALEXA 586 Albumin Russo et.al. JASN 2009

19 Russo et.al. JASN 2009

20 Challenges 1. Dogma, Assumptions, Biology, Reagents, Sensitivity 2. Quantitative Analysis wit thout Gold Standards 3. You See What you are Looking For 4. Correcting for Depth of ff Field 5. Going Deeper 6. Out of Focus Fluorescence 7. Physiologic state of the ra at

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22 24 Hr CLP Glomerular Flow Heterogeneity Large 150 kda dextran Small 3 kda dextran

23 DT bs CL mv DT PT S1 S1 PT

24 GFR as a Marker of Kidney Function in AKI. Historical Marker of Global Kidney Function. Multiple Techniques but either Lack Accuracy or Speed of Determination. No Clinically Usable Technique for AKI. In AKI would have Diagnostic and Severity of Injury Capabilities. An Accurate GFR would allow for Earlier Initiation and Termination of RRT. May have a Role in Surveillance Technology

25 Ideal Characteristicss of GFR Technique 1. Safe, Inexpensive, Repeatable and Accurate 2. Rapid at Bedside Readout 3. Display Data for Interpretation and Evaluation of Test 4. Minimally Invasive or Noninvasive 5. Administered by Nursing Personnel 6. Independent of Vascular Permeability

26 Why is GFR not Determined in AKI Now? 1. Multiple Gold Standard Techniques have been developed 2. Sampling methods such as inulin, i ohexol, or iothalimate clearance: All require 6+ hours to admini ister test - multiple blood draws Require samples to be sent for outside lab analysis, requiring days Possible radiation exposure from injected marker Require moving the patient Too expensive, time consuming and cumbersome to be practical

27 SCr and egfr R: Inadequate Measures in AKI By the time SCr rises above normal, 50% of kidney function has been lost

28 DT bs CL mv DT PT S1 S1 PT

29 GFR, ml/m min/100g Time, hr Data Collection Only Required minutes per GFR Determination Post Injection. Advantages of Dextrans Solubility None Immunogenic High labeling effeciency Wang. Am J Physiol Renal Physiol, 2010

30 Converting from a Microsco ope to Portable Technique 1. Fluorescent Markers, No Change form Microscopic Technique A. Large Dextran for Vascular Volume Measurement B. Small Free Filterable Dextran for Rate Determination 2. Excitation Generation and Em mission Detection Device Needed 3. Optical Probe for Delivery of Emission Signal Excitation Pulse and Recovery of 4. Data Storage and Software An nalysis 5. Raising MONEY

31 Advancing to the Dark Side Academia Industry

32 Commercializa ation: Bench to Bedside Discovery using expensive 2-photon laser requiring surgery to visualize exteriorized kidney Inexpensive, portable LED-based device using fiber optic introduced through standard 18g catheter to read markers

33 Excitation Emission Processing

34 Measuring Fluoreescence Through 18g Caatheter Self Centering Fiber Optic Inserted Through 18g Peripheral Catheter

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36 GFR Determination in Dogs Via A Peripheral Vein

37 New Technology In Development for Rapid GFR 1. Repeatable, accurate, rapid GFR determination at bedside 2. Minimally invasive optical device 3. Rich data for interpretation and evaluation of test 4. Able to be administered by nursing gpersonnel 5. Independent of vascular permeability

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39 IUSM Cellular & Integrative Physiology Mouhamad Alloosh Michael S Sturek

40 NIH O Brien Center for Advanced Renal Microscopic Analysis Develop new optical methodologies for renal investigators. Assist investigators in implementing these new techniques in their laboratories, or in the facilities of the Center. Investigators interested in using the facilities of the Center can find details and application information at:

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