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. 2016 Dec 1;311(6):F1230-F1242.
doi: 10.1152/ajprenal.00030.2016. Epub 2016 Jun 22.

Maintenance of vascular integrity by pericytes is essential for normal kidney function

Dario R Lemos  1 , Graham Marsh  1 , Angela Huang  1 , Gabriela Campanholle  2 , Takahide Aburatani  2 , Lan Dang  1 , Ivan Gomez  1 , Ken Fisher  3 , Giovanni Ligresti  1 , Janos Peti-Peterdi  4 , Jeremy S Duffield  5   2
Affiliations

Maintenance of vascular integrity by pericytes is essential for normal kidney function

Dario R Lemos et al. Am J Physiol Renal Physiol. .

Abstract

Pericytes are tissue-resident mesenchymal progenitor cells anatomically associated with the vasculature that have been shown to participate in tissue regeneration. Here, we tested the hypothesis that kidney pericytes, derived from FoxD1+ mesodermal progenitors during embryogenesis, are necessary for postnatal kidney homeostasis. Diphtheria toxin delivery to FoxD1Cre::RsDTR transgenic mice resulted in selective ablation of >90% of kidney pericytes but not other cell lineages. Abrupt increases in plasma creatinine, blood urea nitrogen, and albuminuria within 96 h indicated acute kidney injury in pericyte-ablated mice. Loss of pericytes led to a rapid accumulation of neutral lipid vacuoles, swollen mitochondria, and apoptosis in tubular epithelial cells. Pericyte ablation led to endothelial cell swelling, reduced expression of vascular homeostasis markers, and peritubular capillary loss. Despite the observed injury, no signs of the acute inflammatory response were observed. Pathway enrichment analysis of genes expressed in kidney pericytes in vivo identified basement membrane proteins, angiogenic factors, and factors regulating vascular tone as major regulators of vascular function. Using novel microphysiological devices, we recapitulated human kidney peritubular capillaries coated with pericytes and showed that pericytes regulate permeability, basement membrane deposition, and microvascular tone. These findings suggest that through the active support of the microvasculature, pericytes are essential to adult kidney homeostasis.

Keywords: acute kidney injury; endothelium; microfluidics; pericyte.

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Figures

Fig. 1.
Fig. 1.
Characterization of in vivo ablation of kidney pericytes. A: schema showing simplified gene loci maps for the two alleles that result in FoxD1::iDTR mice expressing the diphtheria toxin (DT) receptor (DTR) in cells of the FoxD1 lineage as well as mice expressing tdTomato in cells of the FoxD1 lineage. B: split panel images showing the expression of tdTomato in adult kidney pericytes derived from FoxD1+ embryonic progenitors identified by coexpression of PDGF receptor (PDGFR)β. DAPI, 4′,6-diamidino-2-phenylindole. Bar = 25 μm. C: detection (left) and quantification (right) of kidney pericytes in FoxD1::iDTR mice that received a single intraperitoneal injection of either vehicle (Veh) or DT. D1–D3, days 1–3 posttreatment; WT, wild type. Values are means ± SD. *P < 0.05 by Student's t-test. Bar = 50 μm. D: determination of tubular injury by periodic acid-Schiff (PAS) staining of kidney sections from vehicle- and DT-recipient FoxD1::iDTR mice (top). Lipid vacuoles are indicated by arrows. The accumulation of proteinaceous fluid in proximal tubules is indicated by an arrowhead. Lipid accumulation was confirmed by oil red O staining (bottom). Values in bar graphs are means ± SD. *P < 0.05 by Student's t-test. Bar = 50 μm. E: TUNEL immunofluorescence showing increased cell death in the kidneys of FoxD1::iDTR mice after DT treatment compared with vehicle. Values are means ± SD; n = 6–7 mice/group. *P < 0.05 by Student's t-test. Bar = 50 μm.
Fig. 2.
Fig. 2.
Effects of DT administration on the glomerulus. A: detection of mesangial cells using anti-desmin antibody in kidney sections of WT and FOXD1-iDTR mice 2 days after DT administration. Bar = 25 μm. B: detection of vascular smooth muscle (arrows) using anti-smooth muscle actin (SMA) antibody. G, glomerulus. Bar in top image = 50 μm; bar in bottom image = 25 μm. C: images of PAS-stained kidney sections showing glomeruli. Bar = 25 μm. D: quantification of glomerular area and volume. E: detection of podocytes using anti-podoplanin antibody. Bar = 25 μm. F: quantification of podoplanin cells by morphometry. G: detection of podocytes using anti-Wilms' tumor 1 (WT1) antibody. Bar = 25 μm. H: quantification of WT1+ cells. Values are means ± SD; n = 3–5 mice/group.
Fig. 3.
Fig. 3.
Functional consequences of pericyte ablation. A–C: values corresponding to plasma creatinine (A), blood urea nitrogen (BUN; B), and the urinary albumin-to-creatinine ratio (C). Values are means ± SD. *P < 0.05 by Student's t-test. D: quantification of daily body weight changes in WT and FoxD1::iDTR mice that received daily intraperitoneal injections of either vehicle or DT. E: survival curves for WT and FoxD1::iDTR mice treated with DT. n = 6–8 mice/group.
Fig. 4.
Fig. 4.
Effect of pericyte ablation on inflammation. Macrophages (arrowheads) in the kidneys of FoxD1::iDTR mice treated with DT were detected by F4/80 immunofluorescence (A) and quantified (B). Values are means ± SD. Bar = 50 μm. C: quantitative PCR analysis of IL-6 and TNF-α expression in whole kidneys of WT and FoxD1::iDTR mice treated with DT. Values are means ± SD; n = 6–8 mice/group.
Fig. 5.
Fig. 5.
Role of pericytes in regulating capillary homeostasis. A, left: representative CD31 immunofluorescence images from FoxD1::iDTR mice at day 2 of either vehicle or DT treatment. Arrows indicate areas of capillary loss. Bar = 50 μm. Right, quantification of capillary density. B: representative CD31 and Ki67 immunofluorescence images (bar = 25 μm) and quantification of endothelial cell proliferation. Arrows indicate proliferating endothelial cells. C: quantitative PCR quantification of Krüppel-like factor (KLF)-2 and VCAM mRNA. Values are means ± SD. *P < 0.05 by Student's t-test. D: electron microscopy images of either vehicle- or DT-treated FoxD1::iDTR mouse kidneys. Pp, pericyte; L, lumen; E, endothelial cell; RBC, red blood cell; m, mitochondria. Bar = 2 μm. E: analysis of pericyte-specific translational profiles from healthy Col1a1::GFP-L10a mouse kidneys by translated ribosomal affinity purification (TRAP). Col, collagen; GFP, green fluorescent protein. Pathway enrichment analysis was performed using pathway analysis software.
Fig. 6.
Fig. 6.
Evaluation of human kidney pericyte function in microphysiological devices. A: schema showing the microfluidic device used. The flow was automated, constant, and unidirectional through a 150-μm lumen. B: representative immunofluorescence images showing capillaries formed by purified human kidney microvascular endothelial (HKMECs) labeled with vascular-endothelial cadherin (red) with (+) or without (−) pericytes (green). Bar = 25 μm. C: electron microscopy scanning of microfabricated human capillaries showing basement membrane matrix deposition on the abluminal face (left; bar = 1 μm) and tight junctions (right; bar = 500 nm). D: immunofluorescence images of fabricated kidney capillaries immunostained with antibodies against laminin and heparan sulfate in the absence or presence of pericytes (PDGFRβ). Top, longitudinal scanning; bottom, cross-sectional scanning. Bar = 50 μm. E, top: fluorescence imaging of dextran diffusion. Bottom, bright-field images of microfluidic channels coated with HKMECs in the absence or presence of pericytes. Bar = 50 μm. F: quantification of vessel permeability (P). Values in the bar graph are means ± SD. *P < 0.05 by Student's t-test. G: quantification of microfluidic vessel contraction. Values in the bar graph are means ± SD. *P < 0.05 by Student's t-test.
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