Pericyte TIMP3 and ADAMTS1 modulate vascular stability after kidney injury

Abstract

Kidney pericytes are progenitors of scar-forming interstitial myofibroblasts that appear after injury. The function of kidney pericytes as microvascular cells and how these cells detach from peritubular capillaries and migrate to the interstitial space, however, are poorly understood. Here, we used an unbiased approach to identify genes in kidney pericytes relevant to detachment and differentiation in response to injury in vivo, with a particular focus on genes regulating proteolytic activity and angiogenesis. Kidney pericytes rapidly activated expression of a disintegrin and metalloprotease with thrombospondin motifs-1 (ADAMTS1) and downregulated its inhibitor, tissue inhibitor of metalloproteinase 3 (TIMP3) in response to injury. Similarly to brain pericytes, kidney pericytes bound to and stabilized capillary tube networks in three-dimensional gels and inhibited metalloproteolytic activity and angiogenic signaling in endothelial cells. In contrast, myofibroblasts did not have these vascular stabilizing functions despite their derivation from kidney pericytes. Pericyte-derived TIMP3 stabilized and ADAMTS1 destabilized the capillary tubular networks. Furthermore, mice deficient in Timp3 had a spontaneous microvascular phenotype in the kidney resulting from overactivated pericytes and were more susceptible to injury-stimulated microvascular rarefaction with an exuberant fibrotic response. Taken together, these data support functions for kidney pericytes in microvascular stability, highlight central roles for regulators of extracellular proteolytic activity in capillary homoeostasis, and identify ADAMTS1 as a marker of activation of kidney pericytes.

Figure 1.

Global analysis of kidney pericyte transcriptome in response to UUO injury. Temporal changes in gene expression map to two distinct clusters characterized by progressively increasing or decreasing patterns. In each graph, the dark gray line corresponds to the average change in gene expression in response to injury relative to baseline expression (day 0) and the light gray lines identify the 90th and 10th percentiles of each group. The temporal expression patterns of two genes, Adamts1 and Timp3, are depicted with red lines. Gene members of each cluster underwent functional analysis based on Gene Ontology database annotations. Enriched functional categories among upregulated and downregulated genes are shown using color-coded bars corresponding to enrichment P values with the most highly significant shown in red and the least significant shown in dark blue.

Figure 2.

Characterization of primary kidney pericyte cultures. (A) Fluorescent confocal images of kidney pericyte cultures for typical pericyte markers and markers of endothelium or podocytes (bar=25 µm). (B) Histogram plots and dot plot from FACS analysis of primary kidney pericyte cultures for typical pericyte markers and markers of potential contaminating cells (gray areas are the isotype controls and white areas are the specific antibody). (C) Quantitative PCR for typical pericyte markers and myofibroblast markers in primary kidney pericyte compared with myofibroblast cultures (n=3–6/group; ***P<0.001).

Figure 3.

Kidney pericytes stabilize capillary tubes in a 3D gel assay. (A) Schema showing the addition of ECs (red) to gel in wells that spontaneously form capillary networks. Addition of pericytes to this assay permits migration and binding of pericytes to capillary tubes. (B) Toluidine blue–stained gel showing capillary tube network (ECs only) within the gel (bar=100 μm). (C) Kidney pericyte (GFP+) in culture (bar=25 μm). Note cell processes extending the length of several cell bodies. (D) Low-power light images of gels containing capillary tubes (ECs only). Under the influence of the coagulation cascade serine protease, KLK, endothelial tubes are destabilized in the gel and vessels become disorganized leading to progressive collapse of the gel (bar=100 μm). (E) Confocal image of 3D gel with YZ and XZ stacks showing capillary tube (red, CD34) and kidney pericyte (green, Coll-GFP). Note the attachment of kidney pericytes to the capillary tube and numerous processes attached to the capillary tube. Z stacks (arrowheads) show yellow color at points of direct interaction of pericyte processes with capillary tubes, suggestive of peg and socket junctions (bar=25 μm). (F) Dose-response curves measuring collapse of collagen gel (ECs only) induced by KLK. (G) Gel collapse curves induced by KLK (625 ng/ml) in the presence of increasing numbers of kidney pericytes. Note 30% kidney pericytes completely prevent gel collapse (n=12–16/timepoint). (H) Gel collapse curves in response to KLK (625 ng/ml) in the presence of 30% human vascular brain pericytes (HVBPs) or 30% mouse kidney myofibroblasts (kMF). (I) Gelatin zymography of supernatants from collagen gels in the presence of KLK showing multiple bands of gelatinase activity. Note that an approximately 72-kD band of activity, representing MMP9, is completely lost by the addition of 20% or 30% pericytes and the major approximately 60-kD band of MMP2 is attenuated by the presence of 30% pericytes. (J) Phosphoblot for proteins from the collagen gels of KLK-activated gels detecting phospho-VEGFR2. Note that kidney pericytes complete suppress signaling at VEGFR2.

Figure 4.

Characterization of TIMP3 and ADAMTS1 expression in pericytes, in vivo and in vitro. (A) In situ hybridization of kidney sections from normal kidney (day 0) and on day 2 and day 7 after injury onset (disease) for Timp3, Adamts1, and control epithelial marker Ngfa. Positive-stained pericytes and interstitial cells are shown (arrows) and positive-stained epithelial cells are shown (arrowheads) (bar=50 µm). (B) Split panels showing immunostaining for Adamts1 in normal kidneys (day 0) and on day 2 and day 7 after injury kidney sections from Coll-GFP mice. Coll-GFP+ pericytes do not express Adamts1 (arrows) but early after injury onset (day 2) pericytes and cellular processes (arrowheads) now express Adamts1. Kidney tubule expression is also increased. (C and D) Quantitative PCR for Timp3 comparing (C) Coll-GFP+ purified pericytes (normal kidney) with Coll-GFP+ purified myofibroblasts (disease kidney day 2) or (D) primary pericyte cultures with primary myofibroblast cultures. (E and F) Quantitative PCR for Adamts1 comparing (E) Coll-GFP+ purified pericytes (normal kidney) with Coll-GFP+ purified myofibroblasts (disease kidney day 2) or (F) primary pericyte cultures with primary myofibroblast cultures. (G) Western blot showing expression of TIMP3 (upper blot) in normal kidney compared with diseased kidney (day 7) or (lower blot) pericyte cultures compared with myofibroblasts. Note that the 24-kD Timp3 band is expressed at higher levels in pericytes (n=3–5/group; *P< 0.05, ***P<0.001).

Figure 5.

Characterization of TIMP3 and ADAMTS1 functions on capillary tube stability in a 3D tube assay. (A and B) Graph of KLK-induced capillary tube regression (ECs only) showing (A) retardation of collapse in the presence of low concentrations of recombinant TIMP3 and (B) complete prevention of collapse (ECs only) in the presence of high concentrations of recombinant TIMP3 (doses stated in ng/ml). (C) Graph of KLK-induced capillary tube regression (ECs only) showing modest acceleration by recombinant ADAMTS1 (dose stated in ng/ml). (D) Graph of KLK-induced capillary tube regression in the presence of kidney pericytes and recombinant ADAMTS1. Thirty percent pericyte addition is no longer able to stabilize capillaries when ADAMTS1 is present (doses stated in ng/ml). (E) Western blots showing Timp3 levels in pericytes at time points after introduction of siRNA by transfection. (F) Graph of KLK-induced capillary tube regression in the presence of 30% kidney pericytes after Timp3 silencing or mock silencing (n=12–16/timepoint; *P<0.05, **P<0.01, ***P<0.001).

Figure 6.

TIMP3 deficiency in vivo predisposes to microvascular instability of kidney peritubular capillaries due to overactivated pericytes. (A) Confocal images showing microvascular density and pericyte coverage of normal and day 5 post-IRI kidneys. (B) Confocal images showing areas of expanded pericytes (Pdfgfrß+) in normal kidneys of Timp3^−/− mice, some of which express αSMA (arrows) and some of which do not (arrowheads). Five days after IRI, the pericyte/myofibroblasts (Pdgfrβ+) show greater expansion and higher levels of αSMA, particularly in Timp3^−/− kidneys. (C) Low-power images of Sirius red–stained kidneys. (D) Fluorescence images of Adamts1. Note that normal Timp3^−/− kidneys have pericytes that express Adamts1 (arrowheads). (E through K) Graphs showing quantification of (E) vascular density, (F) endothelial cell proliferation, (G) pericytes, (H) myofibroblasts, and (I) fibrosis in interstitial (J) Adamts1+ cells. (K) Western blot showing αSMA and Pdgfrβ expression in kidneys from Timp3^−/− and Timp3^+/+ mice. (L) Western blot quantifying Adamts1 in kidney lysates after IRI. (M) Graphs showing primary pericyte cultures undergoing migration in a scratch closure assay, proliferation, and quantitative PCR expression of Acta2. (N and O) Images of (N) Evans blue–labeled mouse skin and sectioned kidneys and (O) Evans blue quantification indicating a microvascular leak 5 days after IRI. (P) pVEGFR2 expression in normal plus day 5 post-IRI kidneys (bar=50 µm; n=6–8/group; *P<0.05, **P<0.01, ***P<0.001). g, glomerulus; a, arteriole.