Loss of Renal Peritubular Capillaries in Hypertensive Patients Is Detectable by Urinary Endothelial Microparticle Levels

Abstract

Hypertension, an important cause of chronic kidney disease, is characterized by peritubular capillary (PTC) loss. Circulating levels of endothelial microparticles (EMPs) reflect systemic endothelial injury. We hypothesized that systemic and urinary PTC-EMPs levels would reflect renal microvascular injury in hypertensive patients. We prospectively measured by flow cytometry renal vein, inferior vena cava, and urinary levels of EMPs in essential (n=14) and renovascular (RVH; n=24) hypertensive patients and compared them with peripheral blood and urinary levels in healthy volunteers (n=14). PTC-EMPs were identified as urinary exosomes positive for the PTC marker plasmalemmal-vesicle-associated protein. In 7 RVH patients, PTC and fibrosis were also quantified in renal biopsy, and in 18 RVH patients, PTC-EMPs were measured again 3 months after continued medical therapy with or without stenting (n=9 each). Renal vein and systemic PTC-EMPs levels were not different among the groups, whereas their urinary levels were elevated in both RVH and essential hypertension versus healthy volunteers (56.8%±12.7% and 62.8%±10.7% versus 34.0%±17.8%; both P≤0.001). Urinary PTC-EMPs levels correlated directly with blood pressure and inversely with estimated glomerular filtration rate. Furthermore, in RVH, urinary PTC-EMPs levels correlated directly with stenotic kidney hypoxia, histological PTC count, and fibrosis and inversely with cortical perfusion. Three months after treatment, the change in urinary PTC-EMPs levels correlated inversely with a change in renal function ( r=-0.582; P=0.011). Therefore, urinary PTC-EMPs levels are increased in hypertensive patients and may reflect renal microcirculation injury, whereas systemic PTC-EMPs levels are unchanged. Urinary PTC-EMPs may be useful as novel biomarkers of intrarenal capillary loss.

Keywords: exosomes; glomerular filtration rate; hypertension; microvascular rarefaction; urine.

Figure 1

(A) Computed tomography (CT) angiogram with a renal artery stenosis in left kidney. 3D-CT angiography shows left renal artery stenosis in the proximal portion. The left kidney has smaller volume than that of right kidney. (B) Representative T2* images and R₂* parametric maps for subjects with essential hypertension (EH) (a) or renovascular hypertension (RVH) (b) obtained using the same color scale for R₂*. Red areas represent hypoxic regions. Fractional hypoxia >30/s in RVH was greater than in EH (29.8% vs 12.1%).

Figure 2

Levels of exosomes in urine of hypertensive patients. (A) There were no differences among the groups in percent of urinary PL-VAP⁺/CD31⁺, PL-VAP⁺/CD144⁺, and PL-VAP⁺/CD31⁺/CD144⁺ exosomes. (B) PTC-EMPs were identified using flow cytometry as PL-VAP⁺/CD31⁻/CD144⁻ as shown in representative fluorescent images. Scale bar =20 µm. (C) Renal vein and systemic levels of PL-VAP⁺/CD31⁻/CD144⁻ EMPs were not different among the groups, whereas their urinary levels were elevated in both EH and RVH compared to HVs (p<0.001 and p=0.001, respectively). EMPs, endothelial microparticles; HVs, healthy volunteers; EH, essential hypertension; RVH; renovascular hypertension ^*p <0.05 versus HV

Figure 3

Correlation of PTC-EMPs with clinical parameters in hypertensive patients (n=38). Urinary PTC-EMPs levels correlated directly with MAP and inversely with eGFR. In patients with RVH (n=24), PTC-EMPs showed inverse correlation with stenotic RBF and cortical perfusion, and direct correlation with cortex R₂* and fractional hypoxia. PTC-EMPs, peritubular capillary-derived endothelial microparticles; eGFR, estimated glomerular filtration rate; RVH, renovascular hypertension; RBF, renal blood flow

Figure 4

Correlation of PTC-EMPs with renal histology. (A) Representative H&E and trichrome staining in stenotic kidney biopsies. The PTC number and the degree of interstitial fibrosis were higher and lower, respectively, in patients with low compared to patients with high urinary PTC-EMPs percent. (B) Urinary PTC-EMPs levels were inversely correlated with the number of PTC and directly with interstitial fibrosis in this subset of seven RVH patients. PTC-EMPs, peritubular capillary-derived endothelial microparticles; RVH, renovascular hypertension

Figure 5

Change in the levels of PTC-EMPs 3 months after medical therapy (n=9) or stenting (n=9). Changes in individual PTC-EMPs from baseline to 3 months follow-up were not statistically significant in either stented (A) or medically treated (B) patients with RVH. (C) Change of urinary PTC-EMPs inversely correlated with change of eGFR. PTC-EMPs, peritubular capillary-derived endothelial microparticles; eGFR, estimated glomerular filtration rate; RVH, renovascular hypertension