Diabetic Nephropathy Diabetic Kidney Disease: the Problem Pathology, Pathogenesis, Potential for Reversal Charles E. Alpers, MD Incidence of end stage renal disease (thousands of cases per year) 50 40 30 20 10 0 80 84 88 92 96 00 04 08 Calendar Year Diabetes Hypertension Glomerulonephritis Cystic kidney 2010 USRDS Data Report DISCLOSURES FOR CHARLES ALPERS PRESENTATION Diabetic nephropathy (ies) is/are a glomerular, tubulointerstitial, and vascular disease Nothing to disclose 4 1
Traditional Natural History: Diabetic Nephropathy Diabetic Nephropathy(ies): Challenges to the Paradigm Diabetic Nephropathy is a progressive disorder - But how to account for reversibility of albuminuria in some patients? - How to account for structural reversibility? Curr Opin Nephrol Hypertens 2011; 20:246-57 7 Traditional Natural History: Diabetic Nephropathy DN is a Glomerular Disorder, and it Functional Manifestation is Albuminuria Curr Opin Nephrol Hypertens 2011; 20:246-57 MacIsaac and Jerums Curr. Opin Nephrol Hypertens. 2011; 20:246 8 2
DN is a Tubulointerstitial Disorder, and its functional manifestation is a decline in GFR MacIsaac and Jerums Curr. Opin Nephrol Hypertens. 2011; 20:246 9 11 DN is a Glomerular Disorder, and it Functional Manifestation is Albuminuria MacIsaac and Jerums Curr. Opin Nephrol Hypertens. 2011; 20:246 10 12 3
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Mesangiolysis in Diabetic Nephropathy Fraying of the mesangium Lamination of mesangial matrix Implicated in the formation of mesangial nodules Capillary microaneurysms 17 Podocyte (Glomerular Epithelial Cell) Characteristics/Functions Terminally differentiated (has many similar charactersistics to neurons). Synthesis of GBM. Glomerular barrier function (prevents proteinuria). Counteracts capillary wall distension/hydrostatic pressure. Makes survival factors for endothelial cells (e.g., VEGF). 19 Podocytes Type 1 Diabetics have Fewer Podocytes DO THEY HAVE A ROLE IN DIABETIC NEPHROPATHY? 18 Steffes, Kidney Int; 2001, 59: 2104 20 5
Podocyte Number Predicts Albuminuria in Pima Indians Podocyte Loss and Mesangiolysis WT-1 staining p = 0.05 r = -0.49 p = 0.02 r = -0.57 Meyer et al.: Diabetologica 1999; 42: 1341 21 Control Matsusaka T et al JASN 2005; 16:1013 Podo knockout tissue blocks courtesy of: Agnes Fogo 23 Podocyte Loss in Diabetic Nephropathy- What we Know Podocyte Loss and Mesangiolysis Podocyte loss is a feature of diabetic glomerulosclerosis Podocytes have a limited capacity for replication Podocyte loss or loss of function can lead to mesangiolysis-studies of Ichikawa and Quaggin and Alpers Mesangiolysis CAN lead to nodular sclerosis Silver methenamine Matsusaka T et al JASN 2005; 16:1013 PAS tissue blocks courtesy of: Agnes Fogo 24 6
VEGF and Podocyte/Endothelial Interactions Diabetic Nephropathy Is it Reversible? Eremina, et al 25 J. Clin. Invest. 2003; 111:707 27 Anti-VEGF Antibody and TMA A Renal Biopsy Study Glomerular Sclerosis After Pancreas Transplantation V Eremina, JA Jefferson, et al. NEJM 2008 26 N Engl J Med 339:69-75, 1998 28 7
Diabetic Nephropathy Is it Reversible? But How? 29 Podocyte Loss in Diabetic Nephropathy Yes! IF If Podocyte Loss is a feature of diabetic glomerulosclerosis And, podocytes have a limited capacity for replication How can we account for the possibility of regression of diabetic glomerulosclerosis? 30 8
BTBR control 15 weeks: The BTBR mouse with the ob/ob leptin deficiency mutation is a good model of diabetic nephropathy BTBRob/ob: 14 weeks BTBR littermates Glucose: 135 mg/dl Chol: 53 mg/dl TGs: 120 mg/dl Uprot: Normal BTBR ob/ob Glu: 3-4x Chol: 3x TGs: 2x Proteinuria + Hudkins KL et.al. J Am Soc Nephrol. 2010; 21: 1533-42. 36 9
BTBR ob/ob: 21 weeks The BTBR ob/ob Mouse Model is a Good Model for Studying Reversibility of Diabetic Nephropathy Female BTBR ob/ob 24 week BTBR ob/ob mouse with leptin pump 10
Serum Glucose in BTBR ob/ob Mice 600 Podocyte specific WT-1 positive cells in BTBRob/ob mice glucose (mg/dl) 500 400 300 200 100 BTBRob/ob BTBRob/ob Leptin Rx BTBR WT 0 20 21 22 23 24 25 26 27 28 age (wks) BTBR WT 20 wk Pichaiwong et al. JASN 2013 BTBR ob/ob 20 wk BTBR ob/ob 28wk treated 8 weeks with leptin 43 Progressive accumulation of mesangial matrix in diabetic BTBR ob/ob mice is reversed by leptin. WT-1 positive cells in BTBRob/ob mice PODOCYTE NUMBER 250 P< 0.01 P< 0.01 P< 0.01 # OF PODOCYTE/GLOM 200 150 100 BTBR WT 20 wks Pichaiwong et al. JASN 2013 BTBR ob/ob 20 wks BTBR ob/ob 28wks BTBR ob/ob 28wks treated 8 weeks with leptin 50 WT 8wk BTBRob/ob 8wk WT 20wk BTBRob/ob 20wk BTBRob/ob leptin 28 wk Modified counting method. Reference: Sanden SK et al. J Am Soc Nephrol 14: 2484-2493, 2003. 11
p57 Positive Podocytes Counted Using Fractionator/Dissector Method On Serial Sections STUDY DESIGN Leptin replace Route: subcutaneous Dose: 0.15 µl/hr. 0 18 N=7 24 0 Enalapril Route: Drinking w ater Dose: 30 mg/kg BW/day N=9 18 24 Losartan Route: Drinking w ater Dose: 25 mg/kg BW/day 0 18 N=7 24 0 Hydralazine Route: Drinking w ater Dose: 50 mg/kg BW/day 18 N=7 24 0 18 Control Untreated mice N=7 24 Pichaiwong, et al JASN 2013 24: 1088-1102 Where do Podocytes come from? Efficacy of Treatment on Glomerular Matrix BTBR WT BTBRob/ob BTBR WT BTBRob/ob BTBRob/ob leptin WT-1 staining Pichaiwong et al. JASN 2013 *** p<0.001, **p<0.01, *p<0.05 vs BTBR WT Pichaiwong, et al JASN 2013 24(7): 1088-1102 12
It s All About the Podocyte (and PECs) Detection of renin lineage cell transdifferentiation to podocytes in the kidney glomerulus with dual lineage tracing Diana G. Eng, Natalya V. Kaverina, Remington R.S. Schneider, Benjamin S. Freedman, Kenneth W. Gross, Jeffrey H. Miner, Jeffrey W. Pippin, Stuart J. Shankland Kidney International Volume 93, Issue 5, Pages 1240-1246 (May 2018) Pichaiwong et al. JASN 2013 Copyright 2018 International Society of Nephrology Podocyte progenitor Mature podocyte Regeneration of the glomerulus Renal progenitor Mitotic progenitor Dead tubular cell Mitotic tubular cell The BTBR ob/ob Mouse is a Good Model of Diabetic Nephropathy For Testing Therapeutic Interventions 1. Inhibition of Renin-Angiotensin- Aldosterone System (RAAS) 2. Endothelin Receptor Blockade 3. Reduction of ROS Lazzeri, Elena; Mazzinghi, Benedetta; Romagnani, Paola Current Opinion in Nephrology & Hypertension. 19(3):248-253, May 2010. 13
ROS and Podocyte Injury: Dihydroethidium (DHE) stain BTBR WT DHE BTBR ob/ob DHE BTBR ob/ob leptin DHE Podocyte Density (p57) BTBR ob/ob: DHE WT-1 merge DHE/WT-1 BTBR ob/ob + Saline BTBR ob/ob BTBR ob/ob + SS31 BTBR WT BTBR WT + SS31 Podocyte Density (p57) BTBR ob/ob + Saline BTBR ob/ob BTBR ob/ob + SS31 Birk et al. J Am Soc Nephrol. 2013. 24: 1250-61 BTBR WT BTBR WT + SS31 14
Podocyte Density (p57) Conclusions: Nephropathy 1) Diabetic nephropathy is a dynamic process involving acute and chronic injury and repair. BTBR ob/ob + Saline BTBR ob/ob BTBR ob/ob + SS31 2) WE CAN CURE DIABETIC NEPHROPATHY - IF you are a Type I diabetic with a functioning pancreas transplant for 10 years - OR, IF you are a BTBR ob/ob mouse. 3) Podocytes can regenerate! The mechanisms of podocyte regeneration from parietal epithelial cell progenitors are unknown but currently being tested. BTBR WT BTBR WT + SS31 4) Limitations of current therapies for DN may be their insufficiency for enabling adequate podocyte replacement. Summary Podocyte density is partially restored in diabetic mice after SS-31treatment, but an effect on proteinuria is not identified Mesangial matrix accumulation is significantly reduced in SS-31 treated diabetic mice Parietal Epithelial Cells and Podocyte Regeneration in Diabetic Nephropathy Nicole K Andeen, TQ Nguyen, Floor Steegh, Kelly L Hudkins, Behzad Najafian, CE Alpers Kidney Int 2015; 88: 1099-1107 15
Control p57 p57 in Advanced Diabetic Nephropathy Early DN Advanced DN p57 in Early Diabetic Nephropathy Results 1. The number of WT-1 and/or p57 positive podocytes per glomerular profile was progressively reduced in early and advanced diabetic nephropathy. Average # cells per glomerular profile 25 20 15 10 5 0 p57 positive podocytes Control Early DN * * Advanced DN con con earl Average # Number cells per glomerular of positive profile cells 40 30 20 10 0 WT-1 positive podocytes **** **** Control Early DN Advanced DN co co ea *p<0.01 16
Results 4. p57 positive cells (marker of podos) on Bowman s capsule were significantly increased in histologically early DN. Average Number # cells per of glomerular positive cells profile 0.4 0.2 0.0 Control p57 positive PECs 0.8 * control vs early: p <.01 control vs advanced: ns early vs advanced: ns 0.6 Early DN Advanced DN *p<0.01 Acknowledgements Alpers Lab Kelly Hudkins Anja Mühlfeld Stephan Segerer Jolanta Kowalewska Miriam Banas Tomasz Wietecha Warangkana Pichaiwong Floor Steegh Tri Nguyen Minseob Eom Noppanit Pittanchaiwit Anna Batorsky Collaborators John Brunzell Renee LeBoeuf Stuart Shankland Jeff Pippin Bardia Askari Kevin O Brien Nicole Andeen Alan Attie Mark Keller (U. Wisconsin) Funding from: Kidney and Urology Foundation of America The American People (N.I.H.) Potential impact These findings support suggest that podocytes are progressively lost in DN but may regenerate from PEC progenitors in human DN, particularly early DN, and that progression of disease represents a balance between podocyte loss and regeneration. This potentially could be modified by new therapeutics for diabetic nephropathy. 17