Research ArticleEndocrinologyNephrology Open Access | 10.1172/jci.insight.154164

Michael Crompton,1 Joanne K. Ferguson,1 Raina D. Ramnath,1 Karen L. Onions,1 Anna S. Ogier,1 Monica Gamez,1 Colin J. Down,1 Laura Skinner,1 Kitty H. Wong,1 Lauren K. Dixon,1 Judit Sutak,2 Steven J. Harper,3 Paola Pontrelli,4 Loreto Gesualdo,4 Hiddo L. Heerspink,5 Robert D. Toto,6 Gavin I. Welsh,1 Rebecca R. Foster,1 Simon C. Satchell,1 and Matthew J. Butler1

1Bristol Renal, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.

2Pathology Department, Southmead Hospital, Bristol, United Kingdom.

3School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.

4Division of Nephrology, Dialysis and Transplantation, Department of Emergency and Organ Transplantation, Aldo Moro University of Bari, Bari, Italy.

5Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.

6Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Address correspondence to: Matthew J. Butler, Bristol Renal, Dorothy Hodgkin Building, Whitson St., Bristol, BS1 3NY, United Kingdom. Phone: 44.0.117.331.3086; Email: matthew.butler@bristol.ac.uk.

Authorship note: MJB and SCS are co–senior authors.

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1Bristol Renal, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.

2Pathology Department, Southmead Hospital, Bristol, United Kingdom.

3School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.

4Division of Nephrology, Dialysis and Transplantation, Department of Emergency and Organ Transplantation, Aldo Moro University of Bari, Bari, Italy.

5Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.

6Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Address correspondence to: Matthew J. Butler, Bristol Renal, Dorothy Hodgkin Building, Whitson St., Bristol, BS1 3NY, United Kingdom. Phone: 44.0.117.331.3086; Email: matthew.butler@bristol.ac.uk.

Authorship note: MJB and SCS are co–senior authors.

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1Bristol Renal, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.

2Pathology Department, Southmead Hospital, Bristol, United Kingdom.

3School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.

4Division of Nephrology, Dialysis and Transplantation, Department of Emergency and Organ Transplantation, Aldo Moro University of Bari, Bari, Italy.

5Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.

6Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Address correspondence to: Matthew J. Butler, Bristol Renal, Dorothy Hodgkin Building, Whitson St., Bristol, BS1 3NY, United Kingdom. Phone: 44.0.117.331.3086; Email: matthew.butler@bristol.ac.uk.

Authorship note: MJB and SCS are co–senior authors.

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1Bristol Renal, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.

2Pathology Department, Southmead Hospital, Bristol, United Kingdom.

3School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.

4Division of Nephrology, Dialysis and Transplantation, Department of Emergency and Organ Transplantation, Aldo Moro University of Bari, Bari, Italy.

5Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.

6Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Address correspondence to: Matthew J. Butler, Bristol Renal, Dorothy Hodgkin Building, Whitson St., Bristol, BS1 3NY, United Kingdom. Phone: 44.0.117.331.3086; Email: matthew.butler@bristol.ac.uk.

Authorship note: MJB and SCS are co–senior authors.

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1Bristol Renal, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.

2Pathology Department, Southmead Hospital, Bristol, United Kingdom.

3School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.

4Division of Nephrology, Dialysis and Transplantation, Department of Emergency and Organ Transplantation, Aldo Moro University of Bari, Bari, Italy.

5Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.

6Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Address correspondence to: Matthew J. Butler, Bristol Renal, Dorothy Hodgkin Building, Whitson St., Bristol, BS1 3NY, United Kingdom. Phone: 44.0.117.331.3086; Email: matthew.butler@bristol.ac.uk.

Authorship note: MJB and SCS are co–senior authors.

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1Bristol Renal, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.

2Pathology Department, Southmead Hospital, Bristol, United Kingdom.

3School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.

4Division of Nephrology, Dialysis and Transplantation, Department of Emergency and Organ Transplantation, Aldo Moro University of Bari, Bari, Italy.

5Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.

6Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Address correspondence to: Matthew J. Butler, Bristol Renal, Dorothy Hodgkin Building, Whitson St., Bristol, BS1 3NY, United Kingdom. Phone: 44.0.117.331.3086; Email: matthew.butler@bristol.ac.uk.

Authorship note: MJB and SCS are co–senior authors.

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1Bristol Renal, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.

2Pathology Department, Southmead Hospital, Bristol, United Kingdom.

3School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.

4Division of Nephrology, Dialysis and Transplantation, Department of Emergency and Organ Transplantation, Aldo Moro University of Bari, Bari, Italy.

5Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.

6Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Address correspondence to: Matthew J. Butler, Bristol Renal, Dorothy Hodgkin Building, Whitson St., Bristol, BS1 3NY, United Kingdom. Phone: 44.0.117.331.3086; Email: matthew.butler@bristol.ac.uk.

Authorship note: MJB and SCS are co–senior authors.

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1Bristol Renal, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.

2Pathology Department, Southmead Hospital, Bristol, United Kingdom.

3School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.

4Division of Nephrology, Dialysis and Transplantation, Department of Emergency and Organ Transplantation, Aldo Moro University of Bari, Bari, Italy.

5Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.

6Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Address correspondence to: Matthew J. Butler, Bristol Renal, Dorothy Hodgkin Building, Whitson St., Bristol, BS1 3NY, United Kingdom. Phone: 44.0.117.331.3086; Email: matthew.butler@bristol.ac.uk.

Authorship note: MJB and SCS are co–senior authors.

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1Bristol Renal, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.

2Pathology Department, Southmead Hospital, Bristol, United Kingdom.

3School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.

4Division of Nephrology, Dialysis and Transplantation, Department of Emergency and Organ Transplantation, Aldo Moro University of Bari, Bari, Italy.

5Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.

6Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Address correspondence to: Matthew J. Butler, Bristol Renal, Dorothy Hodgkin Building, Whitson St., Bristol, BS1 3NY, United Kingdom. Phone: 44.0.117.331.3086; Email: matthew.butler@bristol.ac.uk.

Authorship note: MJB and SCS are co–senior authors.

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1Bristol Renal, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.

2Pathology Department, Southmead Hospital, Bristol, United Kingdom.

3School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.

4Division of Nephrology, Dialysis and Transplantation, Department of Emergency and Organ Transplantation, Aldo Moro University of Bari, Bari, Italy.

5Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.

6Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Address correspondence to: Matthew J. Butler, Bristol Renal, Dorothy Hodgkin Building, Whitson St., Bristol, BS1 3NY, United Kingdom. Phone: 44.0.117.331.3086; Email: matthew.butler@bristol.ac.uk.

Authorship note: MJB and SCS are co–senior authors.

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1Bristol Renal, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.

2Pathology Department, Southmead Hospital, Bristol, United Kingdom.

3School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.

4Division of Nephrology, Dialysis and Transplantation, Department of Emergency and Organ Transplantation, Aldo Moro University of Bari, Bari, Italy.

5Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.

6Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Address correspondence to: Matthew J. Butler, Bristol Renal, Dorothy Hodgkin Building, Whitson St., Bristol, BS1 3NY, United Kingdom. Phone: 44.0.117.331.3086; Email: matthew.butler@bristol.ac.uk.

Authorship note: MJB and SCS are co–senior authors.

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1Bristol Renal, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.

2Pathology Department, Southmead Hospital, Bristol, United Kingdom.

3School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.

4Division of Nephrology, Dialysis and Transplantation, Department of Emergency and Organ Transplantation, Aldo Moro University of Bari, Bari, Italy.

5Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.

6Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Address correspondence to: Matthew J. Butler, Bristol Renal, Dorothy Hodgkin Building, Whitson St., Bristol, BS1 3NY, United Kingdom. Phone: 44.0.117.331.3086; Email: matthew.butler@bristol.ac.uk.

Authorship note: MJB and SCS are co–senior authors.

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1Bristol Renal, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.

2Pathology Department, Southmead Hospital, Bristol, United Kingdom.

3School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.

4Division of Nephrology, Dialysis and Transplantation, Department of Emergency and Organ Transplantation, Aldo Moro University of Bari, Bari, Italy.

5Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.

6Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Address correspondence to: Matthew J. Butler, Bristol Renal, Dorothy Hodgkin Building, Whitson St., Bristol, BS1 3NY, United Kingdom. Phone: 44.0.117.331.3086; Email: matthew.butler@bristol.ac.uk.

Authorship note: MJB and SCS are co–senior authors.

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1Bristol Renal, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.

2Pathology Department, Southmead Hospital, Bristol, United Kingdom.

3School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.

4Division of Nephrology, Dialysis and Transplantation, Department of Emergency and Organ Transplantation, Aldo Moro University of Bari, Bari, Italy.

5Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.

6Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Address correspondence to: Matthew J. Butler, Bristol Renal, Dorothy Hodgkin Building, Whitson St., Bristol, BS1 3NY, United Kingdom. Phone: 44.0.117.331.3086; Email: matthew.butler@bristol.ac.uk.

Authorship note: MJB and SCS are co–senior authors.

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1Bristol Renal, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.

2Pathology Department, Southmead Hospital, Bristol, United Kingdom.

3School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.

4Division of Nephrology, Dialysis and Transplantation, Department of Emergency and Organ Transplantation, Aldo Moro University of Bari, Bari, Italy.

5Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.

6Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Address correspondence to: Matthew J. Butler, Bristol Renal, Dorothy Hodgkin Building, Whitson St., Bristol, BS1 3NY, United Kingdom. Phone: 44.0.117.331.3086; Email: matthew.butler@bristol.ac.uk.

Authorship note: MJB and SCS are co–senior authors.

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1Bristol Renal, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.

2Pathology Department, Southmead Hospital, Bristol, United Kingdom.

3School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.

4Division of Nephrology, Dialysis and Transplantation, Department of Emergency and Organ Transplantation, Aldo Moro University of Bari, Bari, Italy.

5Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.

6Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Address correspondence to: Matthew J. Butler, Bristol Renal, Dorothy Hodgkin Building, Whitson St., Bristol, BS1 3NY, United Kingdom. Phone: 44.0.117.331.3086; Email: matthew.butler@bristol.ac.uk.

Authorship note: MJB and SCS are co–senior authors.

Find articles by Toto, R. in: JCI | PubMed | Google Scholar

1Bristol Renal, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.

2Pathology Department, Southmead Hospital, Bristol, United Kingdom.

3School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.

4Division of Nephrology, Dialysis and Transplantation, Department of Emergency and Organ Transplantation, Aldo Moro University of Bari, Bari, Italy.

5Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.

6Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Address correspondence to: Matthew J. Butler, Bristol Renal, Dorothy Hodgkin Building, Whitson St., Bristol, BS1 3NY, United Kingdom. Phone: 44.0.117.331.3086; Email: matthew.butler@bristol.ac.uk.

Authorship note: MJB and SCS are co–senior authors.

Find articles by Welsh, G. in: JCI | PubMed | Google Scholar

1Bristol Renal, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.

2Pathology Department, Southmead Hospital, Bristol, United Kingdom.

3School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.

4Division of Nephrology, Dialysis and Transplantation, Department of Emergency and Organ Transplantation, Aldo Moro University of Bari, Bari, Italy.

5Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.

6Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Address correspondence to: Matthew J. Butler, Bristol Renal, Dorothy Hodgkin Building, Whitson St., Bristol, BS1 3NY, United Kingdom. Phone: 44.0.117.331.3086; Email: matthew.butler@bristol.ac.uk.

Authorship note: MJB and SCS are co–senior authors.

Find articles by Foster, R. in: JCI | PubMed | Google Scholar |

1Bristol Renal, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.

2Pathology Department, Southmead Hospital, Bristol, United Kingdom.

3School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.

4Division of Nephrology, Dialysis and Transplantation, Department of Emergency and Organ Transplantation, Aldo Moro University of Bari, Bari, Italy.

5Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.

6Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Address correspondence to: Matthew J. Butler, Bristol Renal, Dorothy Hodgkin Building, Whitson St., Bristol, BS1 3NY, United Kingdom. Phone: 44.0.117.331.3086; Email: matthew.butler@bristol.ac.uk.

Authorship note: MJB and SCS are co–senior authors.

Find articles by Satchell, S. in: JCI | PubMed | Google Scholar

1Bristol Renal, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.

2Pathology Department, Southmead Hospital, Bristol, United Kingdom.

3School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.

4Division of Nephrology, Dialysis and Transplantation, Department of Emergency and Organ Transplantation, Aldo Moro University of Bari, Bari, Italy.

5Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.

6Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Address correspondence to: Matthew J. Butler, Bristol Renal, Dorothy Hodgkin Building, Whitson St., Bristol, BS1 3NY, United Kingdom. Phone: 44.0.117.331.3086; Email: matthew.butler@bristol.ac.uk.

Authorship note: MJB and SCS are co–senior authors.

Find articles by Butler, M. in: JCI | PubMed | Google Scholar |

Authorship note: MJB and SCS are co–senior authors.

Published February 7, 2023 - More info

Published in Volume 8, Issue 5 on March 8, 2023
JCI Insight. 2023;8(5):e154164. https://doi.org/10.1172/jci.insight.154164.
© 2023 Crompton et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published February 7, 2023 - Version history
Received: August 16, 2021; Accepted: January 23, 2023 View PDF Abstract

The glomerular endothelial glycocalyx (GEnGlx) forms the first part of the glomerular filtration barrier. Previously, we showed that mineralocorticoid receptor (MR) activation caused GEnGlx damage and albuminuria. In this study, we investigated whether MR antagonism could limit albuminuria in diabetes and studied the site of action. Streptozotocin-induced diabetic Wistar rats developed albuminuria, increased glomerular albumin permeability (Ps’alb), and increased glomerular matrix metalloproteinase (MMP) activity with corresponding GEnGlx loss. MR antagonism prevented albuminuria progression, restored Ps’alb, preserved GEnGlx, and reduced MMP activity. Enzymatic degradation of the GEnGlx negated the benefits of MR antagonism, confirming their dependence on GEnGlx integrity. Exposing human glomerular endothelial cells (GEnC) to diabetic conditions in vitro increased MMPs and caused glycocalyx damage. Amelioration of these effects confirmed a direct effect of MR antagonism on GEnC. To confirm relevance to human disease, we used a potentially novel confocal imaging method to show loss of GEnGlx in renal biopsy specimens from patients with diabetic nephropathy (DN). In addition, patients with DN randomized to receive an MR antagonist had reduced urinary MMP2 activity and albuminuria compared with placebo and baseline levels. Taken together, our work suggests that MR antagonists reduce MMP activity and thereby preserve GEnGlx, resulting in reduced glomerular permeability and albuminuria in diabetes.

Graphical Abstract Introduction

Glomerular diseases, including diabetic nephropathy (DN), are the most common cause of end-stage renal failure (1). Approximately 1 in 5 people with diabetes need treatment for DN during their lifetime (2). In 2018, more than 700,000 people in the United States were being treated for end-stage renal disease (ESRD), and diabetes accounted for 47% of all new ESRD cases (3). Renin-angiotensin-aldosterone system (RAAS) blockade with angiotensin-converting enzyme inhibitors (ACEi) or angiotensin-receptor blockers (ARB) reduce albuminuria and the risk of ESRD (4). However, because of aldosterone escape, use of an ACEi or ARB may not reduce aldosterone-mediated mineralocorticoid receptor (MR) stimulation (5, 6). In DN, the addition of an MR antagonist to ACEi or ARB therapy further reduces albuminuria, suggesting MR activation directly contributes to albuminuria (7–12). Most recently, the large Phase III FIDELIO-DKD trial reported that, in patients with chronic kidney disease (CKD) and type 2 diabetes, MR antagonism reduced the risk of CKD progression, albuminuria, and cardiovascular events (13). However, side effects, including hyperkalemia, limit the clinical use of MR antagonists (10, 12, 14–16). Thus, a better definition of the mechanisms of glomerular protection mediated by MR antagonists is needed to identify novel tissue-specific therapeutic targets.

MR is expressed in the vascular endothelium (17, 18) and is also expressed in glomerular endothelial cells (GEnC) (19) — highly specialized fenestrated vascular endothelial cells. Changes in the glomerular endothelium are increasingly recognized in DN and other glomerular diseases (20). The glomerulus is the filtering unit of the kidney. Its function is dependent on the multilayer structure of the glomerular filtration barrier (GFB) consisting of GEnC, glomerular basement membrane (GBM), and podocytes (21). The glomerular endothelial glycocalyx (GEnGlx) covers the luminal surface of the GEnC, filling the fenestrations and contributing to GFB function (22, 23). Our group and others have shown that GEnGlx specifically limits albumin permeability in vitro (24–26) and in vivo (27–30). The EnGlx is a hydrated poly-anionic gel composed principally of proteoglycan core proteins, glycosaminoglycan chains, and sialoglycoproteins (31). In healthy vascular physiology, the EnGlx has multiple roles, including regulating vascular permeability (32, 33), mediating shear stress mechanotransduction (34, 35), and attenuating immune cell–endothelium interactions (36, 37), with further roles under investigation (31, 38, 39). Disruption of the EnGlx occurs in multiple clinical conditions, including diabetes, sepsis, preeclampsia, and atherosclerosis (22, 31, 38, 40).

In humans, DN is characterized by albuminuria (22, 41). In early DN, no macroscopic GBM or podocyte changes are detectable, but systemic endothelial and Glx dysfunction in both type 1 (42) and type 2 (43) diabetes have been shown to occur. Others have previously shown that glycosaminoglycans are lost from the GFB in diabetes (44, 45), and we have confirmed GEnGlx loss in diabetic mice (46, 47) and rats (30). Together, these findings strongly implicate GEnGlx damage as a key initiator of albuminuria in DN (22, 23, 41).

EnGlx components are cleaved from the cell surface by sheddases, including matrix metalloproteinases (MMPs) (48, 49). We have recently defined a pathway whereby excess MR activation results in increased MMP2 and MMP9 activity and consequent GEnGlx dysfunction and albuminuria (19). Here, we sought to determine whether this pathological pathway contributes to GEnGlx damage in diabetes, hypothesizing that MR antagonism reduces MMP activity in diabetes, preserving the GEnGlx and limiting the development of albuminuria. Hence, we sought to determine whether MR antagonism, with spironolactone, could prevent the development of albuminuria in a DN rat model by preserving the GEnGlx to maintain the GFB. Furthermore, we examined GEnGlx damage and MR-mediated MMP inhibition in human DN.

Results

Development of albuminuria and increased glomerular permeability in early DN is ameliorated by MR antagonism. After receiving streptozotocin (STZ), rats were hyperglycemic at week 3 (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.154164DS1). STZ-induced diabetic rats gained significantly less weight than controls. Spironolactone treatment had no significant impact on body weight (Supplemental Figure 1B). Diabetic rats developed albuminuria by week 4 (Supplemental Figure 1C). Spironolactone significantly reduced albuminuria in diabetic rats compared with vehicle. The fold change in urinary albumin/creatinine ratio (uACR) in vehicle-treated diabetic rats significantly increased compared with controls and spironolactone-treated diabetic rats (Figure 1B). There was no significant difference in uACR fold change between spironolactone-treated diabetic rats and controls. Similarly, diabetic rats had a significant increase in fold change of total protein/creatinine ratio (Supplemental Figure 1D). Our glomerular permeability assay was used to directly measure the albumin permeability (Ps’alb) of individually trapped glomeruli (30). In contrast to urine-based measurements, this ex vivo assay directly measures the GFB permeability to albumin in isolation, independently of hemodynamic factors and tubular albumin handling (Figure 1C). Increased glomerular capillary wall protein permeability was confirmed by an increase in Ps’alb (Figure 1D). Spironolactone restored Ps’alb to control values, significantly reducing Ps’alb compared with vehicle-treated diabetic rats.

Figure 1

Development of albuminuria and increased glomerular permeability in early diabetic nephropathy is ameliorated by MR antagonism. (A) Schematic overview of STZ-induced diabetic model and spironolactone (spiro) treatment protocol for male Wistar rats. An injection of STZ was given at week 0. Four weeks after STZ injection, spiro (an MR inhibitor) was given for 28 days, and rats were culled at week 8 after STZ injection. Rats were randomized to receive STZ and spiro. (B) Treatment with spiro for 28 days reduced the fold change in urinary albumin/creatinine ratio (uACR) from initiation of treatment, week 4 to week 8 (control, n = 10; diabetes, n = 12; diabetes-spiro, n = 13). Data were log transformed and presented as log2 (fold change). (C) Representative images of an isolated glomerulus stained with R18 and Alexa Fluor 488–BSA (AF488-BSA). Magnification, 20×. (D) Glomerular albumin permeability (Ps’alb) was measured at week 8 (control, n = 7 rats [32 glomeruli]; diabetes, n = 8 [36 glomeruli]; diabetes-spiro, n = 5 [21 glomeruli]). In B and D, 1-way ANOVA was used for statistical analysis, followed by Tukey’s multiple comparisons. Each dot, triangle, and square on the graph represents a rat. Data are expressed as mean ± SEM. **P < 0.01; ***P < 0.001.

Diabetes-induced GEnGlx damage in early DN is prevented by MR antagonism. Marasmium oreades agglutinin (MOA) and wheat germ agglutinin (WGA) lectins both bound to the EnGlx on the luminal surface of the labeled GEnC membrane (Figure 2A). We analyzed the images generated using a fluorescence profile peak-to-peak measurement technique (19, 47, 50) to provide an index of GEnGlx thickness (Figure 2B). Manual peak-to-peak measurement of MOA labeling demonstrated a reduction in GEnGlx thickness in diabetic rats (Figure 2C). Spironolactone treatment in diabetic rats restored the GEnGlx thickness, with no significant differences compared with controls. Ps’alb correlated inversely with the GEnGlx thickness measured by peak-to-peak analysis of MOA/R18 labeling (Figure 2D). To confirm the validity of our findings with MOA labeling, we applied a second lectin, WGA, for peak-to-peak measurements, and we developed an automated methodology. As with MOA, we found significant Glx damage in diabetic rats, with a decrease in GEnGlx thickness (Figure 2E). Spironolactone treatment significantly restored the GEnGlx thickness, with no significant differences compared with controls. Again, Ps’alb changes correlated inversely, and strongly, with GEnGlx thickness measured from peak to peak of WGA/octadecyl rhodamine B chloride (WGA/R18) labeling (Figure 2F). Perfusion-fixed, Alcian blue–labeled, kidneys were used for transmission electron microscopy (TEM) of the glomerular capillary wall to validate peak-to-peak assessment of the Glx (Supplemental Figure 2A) and study GFB changes (Supplemental Figure 3A) (30, 46). Diabetic rats had decreased GEnGlx coverage and thickness, which were both restored by spironolactone treatment (Supplemental Figure 2, B and C). Ps’alb changes were weakly associated with GEnGlx thickness measured by electron microscopy (EM) (Supplemental Figure 2D), suggesting that peak-to-peak assessment provides a superior measure of Glx structural and functional integrity. Mesangial matrix expansion was assessed using periodic acid–Schiff staining, with no significant changes in glomerular fibrosis in diabetic rats (Supplemental Figure 1, E and F). In addition, comprehensive TEM analysis confirmed that no other significant ultrastructural changes were visible (Supplemental Figure 3, B–H), further confirming that this model represents early DN.

Figure 2

Fluorescence profile peak-to-peak measurements confirm that glomerular endothelial glycocalyx damage is prevented by MR antagonism and correlates strongly with glomerular albumin permeability. Rats were perfused with Ringer solution, and the left kidney was removed for lectin staining. (A) Representative images show glomerular capillaries labeled red (R18) and the luminal glomerular endothelial glycocalyx (GEnGlx) labeled green with Marasmium oreades agglutinin (MOA) or wheat germ agglutinin (WGA). Scale bars: 20 μm and 5 μm. ROI, re