Pericyte-mediated constriction of renal capillaries evokes no-reflow and kidney injury following ischaemia
- Department of Neuroscience, Physiology and Pharmacology, University College London, United Kingdom
Figures
Figure 1 with 1 supplement
Ischaemia and reperfusion lead to cortical and medullary no-reflow.
(a, b) Ischaemia and reperfusion (I/R) evoked changes of blood flow (measured by laser Doppler) in the rat renal (a) medulla (n = 4 animals) and (b) cortex (n = 10 animals). CONT indicates blood flow on the contralateral (non-ischaemic) side. Traces labelled +HF show the effect on recovery of perfusion of administering the Rho kinase inhibitor hydroxyfasudil (HF) immediately on reperfusion (I/R + HF) (n = 4 animals). (c–e) Top: low power views of kidney slices after perfusion in vivo with FITC-albumin gelatin, from (c) control (contralateral) kidney, (d) a kidney after ischaemia and 30 min reperfusion, and (e) a kidney 30 min after treatment with HF on reperfusion Bottom: regions of interest (ROIs) are shown in red and blue for the cortex and medulla. (f) Medullary perfusion (assessed in slices of fixed kidney as the total intensity of FITC-albumin summed over the ROIs) was reduced after 30 min of post-ischaemic reperfusion (51 stacks, 6 animals) by ~50% compared with control kidneys (52 stacks, 7 animals). Treatment with HF increased medullary perfusion 2.3-fold at this time compared with non-treated ischaemic kidneys (20 stacks, 4 animals). (g) Cortex perfusion (assessed as in c-e) after 30 min of reperfusion after ischaemia was reduced by ~23.5% compared with control kidneys. Treatment with HF (I/R + HF) increased cortex perfusion by 25% at this time compared with non-treated ischaemic kidneys (I/R). Data are mean ± s.e.m. P values are corrected for multiple comparisons. Statistical tests used the number of animals as the N value (not the stack number).
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Figure 1—source data 1
Ischaemia and reperfusion lead to cortical and medullary no-reflow.
- https://cdn.elifesciences.org/articles/74211/elife-74211-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Ischaemia (I/R) evoked changes of blood flow measured by laser Doppler in the rat renal medulla and cortex.
(a, b) Ischaemia (I/R) evoked changes of blood flow (measured by laser Doppler) in the rat renal (a) medulla (n = 3 animals) and (b) cortex (n = 3 animals). CONT indicates blood flow on the contralateral (non-ischaemic) side. At 60 min following reperfusion, medullary perfusion remained compromised at 40% of its control value (P = 0.017), but cortical perfusion was fully recovered (to ~20% above the control value, although this did not reach significance, P = 0.092). (c) Hydroxyfasudil (3 mg/kg; i.v.) (n = 4 animals) treatment immediately after reperfusion (I/R + HF) induced a faster recovery to the pre-ischaemic value of of medullary blood flow than did BQ123 (0.5 mg/kg, i.v., given on reperfusion: I/R + BQ123) (n = 3 animals), a selective endothelin-A receptor antagonist. After 30 min reperfusion both agents resulted in blood flow that was not significantly different from control (P = 0.8 and P = 0.38, respectively) but was significantly different from ischaemia (P = 0.01 for both drugs). Valsartan (1 mg/kg i.v., given on reperfusion: I/R + VAL) (n = 2 animals), an angiotensin II type 1 (AT1) receptor antagonist, increased medullary perfusion by 52% after 30 min reperfusion compared with non-treated ischaemic kidneys, although this did not reach significance (P = 0.11 vs. I/R) and valsartan had not reversed medullary blood flow to the baseline level after 30 mins (P = 0.19 vs. CONT). (d) Recovery of cortical blood flow to its control level on reperfusion was faster in the presence of hydroxyfasudil (I/R + HF) (n = 4 animals). BQ123 (n = 4 animals) (P = 0.05 vs. I/R) and valsartan (n = 3 animals) (P = 0.04 vs. I/R) also promoted recovery of cortical blood flow at 30 min reperfusion compared with non-treated ischaemic kidneys (I/R). Statistical tests used the number of animals as the N value.
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Figure 1—figure supplement 1—source data 1
Ischaemia (I/R) evoked changes of blood flow measured by laser Doppler in the rat renal medulla and cortex.
- https://cdn.elifesciences.org/articles/74211/elife-74211-fig1-figsupp1-data1-v2.xlsx
Figure 2
Ischaemia and reperfusion reduce medullary microvascular perfusion.
(a) Representative images of slices after perfusion with FITC-albumin gelatin, showing the rat kidney microcirculation in 100 µm deep confocal z-stacks. Images depict renal cortical arterioles, the glomeruli and peritubular capillaries, as well as the vasa recta capillaries (VR) that supply blood to the renal medulla. (b–d) Representative images of the medullary microcirculation: (b) in control conditions (CONT), (c) after ischaemia and 30 min reperfusion (I/R), and (d) after ischaemia and reperfusion for 30 min with hydroxyfasudil (HF) applied during reperfusion (I/R + HF). Images show NG2-labelling of pericytes (red), FITC-albumin labeling (green) of vessels that are perfused, a merge of the NG2 and FITC-albumin images, and the analysed skeleton (yellow) of the perfused microvessels. (e–g) After ischaemia and reperfusion (12 stacks, 4 animals), the total perfused capillary length (e), the number of perfused capillary segments (f) and the overall volume fraction of vessels perfused (g) in 100 µm deep confocal z-stacks were reduced compared with control kidneys (14 stacks, 6–7 animals), and treatment with hydroxyfasudil immediately after reperfusion (10 stacks, 4 animals) increased all of these parameters.Data are mean ± s.e.m. P values are corrected for multiple comparisons. Statistical tests used the number of animals as the N value (not the stack number).
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Figure 2—source data 1
Ischaemia and reperfusion reduce medullary microvascular perfusion.
- https://cdn.elifesciences.org/articles/74211/elife-74211-fig2-data1-v2.xlsx
Figure 3
Ischaemia and reperfusion of renal cortex evoke no-reflow in capillaries but not arterioles.
(a–c) Representative images of rat renal cortex slices containing arterioles, glomeruli and peritubular capillaries, after perfusion with FITC-albumin gelatin: (a) for control kidneys (CONT), (b) after ischaemia and reperfusion (I/R), and (c) after ischaemia with hydroxyfasudil (I/R + HF). NG2-labelling (red) is seen of arterioles (blue arrowheads) and pericytes (yellow arrowheads), while FITC-albumin labelling (green) shows vessels that are perfused. (d-f) After ischaemia and reperfusion (I/R) (12 stacks, 6 animals), the total perfused capillary length (d), the number of perfused segments (e), and the overall perfused microvascular volume fraction (f) were reduced compared with control kidneys (CONT) (14 stacks, 7 animals), and treatment with hydroxyfasudil immediately after reperfusion (I/R + HF) (10 stacks, 4 animals) increased cortical microvascular perfusion compared with non-treated ischaemic kidneys. (g) Percentage of afferent and efferent arterioles (blue arrowheads in a-c), and of glomeruli (white arrowheads), perfused after ischaemia, compared with control conditions. (h–i) Diameters of perfused (h) afferent and (i) efferent arterioles in the renal cortex for the three experimental conditions (15 arterioles, 4 animals for each group). Data are mean ± s.e.m. P values are corrected for multiple comparisons.Statistical tests used the number of animals as the N value (not the stack number).
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Figure 3—source data 1
Ischaemia and reperfusion of renal cortex evoke no-reflow in capillaries but not arterioles.
- https://cdn.elifesciences.org/articles/74211/elife-74211-fig3-data1-v2.xlsx
Figure 4 with 2 supplements
Descending vasa recta are constricted by pericytes after ischaemia.
(a) Descending vasa recta (DVR) in slices of rat renal medulla after perfusion with FITC-albumin gelatin (re-coloured red), and labelled for pericytes with antibody to the proteoglycan NG2 (green); FITC-albumin labelling shows perfused and blocked vessels. White arrow indicates flow direction; white lines indicate blocked vessels. (b–c) Representative images showing DVR capillaries blocked near pericyte somata. NG2-labelling of pericytes shows pericyte processes presumed to be constricting vessels at block site. (d) Percentage of DVR capillaries blocked in the renal medulla in control conditions (CONT) (127 capillaries, 12 stacks, 9 animals), after ischaemia and reperfusion (I/R) (77 capillaries, 10 stacks, 6 animals), and after ischaemia with hydroxyfasudil present in the reperfusion period (IR + HF) (60 capillaries, 8 stacks, 4 animals). Statistical tests used number of animals as the N value. (e) Diameter at block sites. (f) Probability distribution per 2.5 μm bin of distance from blockage to nearest pericyte soma after ischaemia and reperfusion (for 27 block sites), and of the distance between adjacent pericytes on DVR capillaries (for 118 pericyte pairs). (g) DVR diameter versus distance from pericyte somata (10 µm is approximately half the separation between pericytes) in the same three conditions as d (number of pericytes was 31, 20, and 17 respectively). P values by each point are from t-tests. Slope of the best-fit ISCH regression line is significantly greater than zero (P = 0.039) while that of the CONT line is not (P = 0.084). Data are mean ± s.e.m.
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Figure 4—source data 1
Descending vasa recta are constricted by pericytes after ischaemia.
- https://cdn.elifesciences.org/articles/74211/elife-74211-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
The effect of renal ischaemia and reperfusion on red blood cell trapping and endothelial glycocalyx integrity in the descending vasa recta.
(a) Red blood cells (RBCs, indicated by yellow arrowheads, labelled for glycophorin A) were associated with a small percentage of blockage sites (indicated by blue arrowheads) in ischaemic rat kidneys (5.8% of 85 blockages from 137 vessels analysed from two animals), and even where red blood cells were near the capillary blockages it did not always lead to a block of blood flow (as shown by FITC-albumin, re-coloured white, passing the red blood cells [purple arrow]). Note that the vasculature was perfused with PBS to remove loose RBCs before perfusing PFA and FITC-albumin, so the only RBCs remaining should be those bound to the vessel walls. (b) Percentage of DVR that were blocked, and percentage of blocked DVR that had an associated RBC. (c) Endothelial glycocalyx (eGCX) was labelled in vivo using wheat germ agglutinin-Alexa Fluor 647 (WGA, re-coloured green). White boxes show ROIs for measuring eGCX mean fluorescence intensities at different distances from the pericyte soma. (d) Plots of WGA signal across capillary at different distances from arrowed pericyte in (c). (e) eGCX is fairly evenly distributed along the vessel wall in normal kidneys (CONT), and also after ischaemia and reperfusion (I/R). Blockages (indicated by blue arrowheads) are highly associated with pericyte location (indicated by white arrowheads) in ischaemic kidneys (I/R). (f) Mean level of eGCX averaged across vessel at different distances from the pericyte soma in control kidney and after ischaemia with 30 min reperfusion. For the control condition, black P values compare the value at each position with that at the soma. Red P values compare the ischaemic and control groups for each position. (g) eGCX mean fluorescence averaged over all positions measured. (h) eGCX intensity and diameter have no correlation in control or ischaemic conditions. Data are mean ± s.e.m, 30 pericytes from two animals for each experimental condition. Statistical tests used the number of pericytes as the N value.
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Figure 4—figure supplement 1—source data 1
The effect of renal ischaemia and reperfusion on red blood cell trapping and endothelial glycocalyx integrity in the descending vasa recta.
- https://cdn.elifesciences.org/articles/74211/elife-74211-fig4-figsupp1-data1-v2.xlsx
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Figure 4—figure supplement 1—source data 2
The effect of renal ischaemia and reperfusion on red blood cell trapping and endothelial glycocalyx integrity in the descending vasa recta.
- https://cdn.elifesciences.org/articles/74211/elife-74211-fig4-figsupp1-data2-v2.xlsx
Figure 4—figure supplement 2
Morphology of renal pericytes.
(a) Cortical pericyte showing longitudinal processes and a limited number of circumferential processes. (b) Medullary pericytes showing a large number of circumferential processes.
Figure 5
Pericytes constrict capillaries after renal ischaemia in vivo.
(a) Overview two-photon in vivo imaging stack of the mouse renal cortex microcirculation, showing pericytes expressing NG2-DsRed (red), intraluminal FITC-albumin given intravenously (green), and Hoechst 33,342 labelling nuclei (blue, 1 mg/kg in 0.5 ml of sterile, isotonic saline was administered intravenously: Dunn et al., 2018). Images were acquired in a plane parallel to the cortical surface. (b, c) Higher magnification images showing apericyte on a cortical peritubular capillary in control conditions, and post-ischaemic capillary block (dashed lines show path of blocked vessel). (d) Capillary diameter versus distance from pericyte somata after ischaemia and reperfusion (I/R), and for control kidneys (CONT) (number of pericytes was 15 and 10 respectively from 10 stacks from three animals from each group). Slope of the best-fit ISCH regression line is significantly greater than zero (P = 0.046) while that of the CONT line is negative but not significantly different from zero (P = 0.10). Data are mean ± s.e.m. P values comparing data at each distance are corrected for multiple comparisons. Statistical tests used number of stacks as the N value.
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Figure 5—source data 1
Pericytes constrict capillaries after renal ischaemia in vivo.
- https://cdn.elifesciences.org/articles/74211/elife-74211-fig5-data1-v2.xlsx
Figure 6
Pericyte contraction is mediated by α-SMA and regulated by Rho kinase.
Representative images of the rat renal medulla containing descending vasa recta (DVR) pericytes (a–d) and cortical peritubular capillary pericytes (f–i), labelled with antibody to phosphorylated myosin light chain (p-MLC, green), Alexa Fluor 647-isolectin B4 which labels kidney tubules and pericytes (red), and DAPI which labels nuclei (blue). Labelling is shown for kidneys in control conditions (CONT) (a, f), after ischaemia and reperfusion (I/R) (b, d, g, i), and after ischaemia with hydroxyfasudil present during reperfusion (I/R + HF) (c, h). (e, j) Cortical (e) and medullary (j) p-MLC levels in pericytes for the three experimental conditions (10 stacks, 4 animals for each group). (k–m) DVR pericytes labelled for NG2 (purple), α-SMA (green), Alexa647-isolectin B4 (red) and DAPI (blue). (n) DVR blockage-associated pericyte labelled for α-SMA. Statistical tests used the numbers of animals for N values(not the stack number). Data are mean ± s.e.m. P values are corrected for multiple comparisons.
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Figure 6—source data 1
Pericyte contraction is mediated by α-SMA and regulated by Rho kinase.
- https://cdn.elifesciences.org/articles/74211/elife-74211-fig6-data1-v2.xlsx
Figure 7
Rho kinase inhibition reduces kidney injury induced by ischaemia and reperfusion.
(a–c) Images of the rat renal cortex containing proximal tubules, showing isolectin B4 labelling kidney tubules (red), DAPI labelling nuclei (blue), and kidney injury molecule-1 (Kim-1) labelling as an injury marker (white lines indicate examples of injured tubules labelled in green), for control conditions (CONT) (a), after ischaemia and reperfusion (I/R) (b), and after ischaemia with hydroxyfasudil present during reperfusion (I/R + HF) (c).(d) Kim-1 levels for the three experimental conditions (six stacks, 3 animals for each group). Data are mean ± s.e.m. P values are corrected for multiple comparisons. Statistical tests used the number of animals as the N value (not the stack number).
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Figure 7—source data 1
Rho kinase inhibition reduces kidney injury induced by ischaemia and reperfusion.
- https://cdn.elifesciences.org/articles/74211/elife-74211-fig7-data1-v2.xlsx
Figure 8
Schematic diagram of loci of blood flow reductions after renal ischaemia and reperfusion.
The afferent arteriole feeding the glomerulus (top arrow) and the efferent arteriole leaving the glomerulus are little affected by ischaemia and reperfusion. In contrast, pericytes on peritubular capillaries and the descending vasa recta (upper descending arrow) constrict the capillaries, reducing blood flow and causing blockages as schematised at the lower right, and indicated by the crossed lower descending arrow signifying impaired DVR flow. The resulting ischaemia leads to kidney damage detectable by Kim-1 labelling. Hydroxyfasudil - a Rho kinase inhibitor - reduces these effects. Created with https://biorender.com/.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain backgroundRattusnorvegicus (Sprague Dawley, male) | Rat | UCL Biological Services | ||
| Genetic reagent (Mus musculus/spretus, male) | NG2-DsRed mice | https://doi.org/10.1242/dev.004895 | JAX 008241 | |
| Antibody | anti-NG2 (mouse monoclonal) | AbCam | ab50009 | (1:200) |
| Antibody | Anti-Myosin light chain (phospho S20) (rabbit polyclonal) | AbCam | ab5694 | (1:100) |
| Antibody | kidney injury molecule-1 (Kim-1) (rabbit polyclonal) | NovusBiologicals | NBP1-76701 | (1:100) |
| Antibody | anti-alpha smooth muscle actin (rabbit polyclonal) | AbCam | ab5694 | (1:100) |
| Antibody | anti-glycophorin A (mouse monoclonal) | AbCam | ab9520 | (1:2000) |
| Antibody | Alexa Fluor 405 goat anti-rabbit (polyclonal) | ThermoFisher | A31556 | (1:500) |
| Antibody | Alexa Fluor 555 donkey anti-rabbit (polyclonal) | ThermoFisher | A31572 | (1:500) |
| Antibody | Alexa Fluor 555 donkey anti-mouse (polyclonal) | ThermoFisher | A31570 | (1:500) |
| Chemical compound, drug | isolectin B4 - AlexaFluor 647 | ThermoFisher | I32450 | (1:200) |
| Chemical compound, drug | wheat germ agglutinin Alexa Fluor 647 conjugate | ThermoFisher | W32466 | 200 μl (1 mg/ml) |
| Chemical compound, drug | Hoechst 33,342 | ThermoFisher | H21492 | 1 mg/kg in 0.5 ml saline |
| Chemical compound, drug | gelatin | Sigma-Aldrich | G2625 | 5% in PBS |
| Chemical compound, drug | FITC-albumin | Sigma-Aldrich | A9771 | 1:200 in 5% gelatin |
| Chemical compound, drug | FITC-albumin | Sigma-Aldrich | A9771 | (1 mg in 100 μl; i.v.) |
| Chemical compound, drug | Hydroxyfasudilhydrochloride | Santa Cruz Biotechnology | sc-202176 | (3 mg/kg; i.v.) |
| Software, algorithm | MATLAB R2015a | MathWorks, Inc. | in vivo data acquisition | |
| Software, algorithm | ImageJ | https://imagej.nih.gov/ij/ | imageanalysis | |
| Software, algorithm | GraphPadPrism 6 | GraphPad Software, Inc | statisticalanalysis | |
| Other | DAPI stain | Molecular Probes | D1306 | 200 μl (5 μg/ml) |
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Pericyte-mediated constriction of renal capillaries evokes no-reflow and kidney injury following ischaemia
eLife 11:e74211.
https://doi.org/10.7554/eLife.74211
