Urinary Extracellular Vesicles of Podocyte Origin and Renal Injury in Preeclampsia

Abstract

Renal histologic expression of the podocyte-specific protein, nephrin, but not podocin, is reduced in preeclamptic compared with normotensive pregnancies. We hypothesized that renal expression of podocyte-specific proteins would be reflected in urinary extracellular vesicles (EVs) of podocyte origin and accompanied by increased urinary soluble nephrin levels (nephrinuria) in preeclampsia. We further postulated that podocyte injury and attendant formation of EVs are related mechanistically to cellfree fetal hemoglobin (HbF) in maternal plasma. Our study population included preeclamptic (n=49) and normotensive (n=42) pregnant women recruited at delivery. Plasma measurements included HbF concentrations and concentrations of the endogenous chelators haptoglobin, hemopexin, and α_1- microglobulin. We assessed concentrations of urinary EVs containing immunologically detectable podocyte-specific proteins by digital flow cytometry and measured nephrinuria by ELISA. The mechanistic role of HbF in podocyte injury was studied in pregnant rabbits. Compared with urine from women with normotensive pregnancies, urine from women with preeclamptic pregnancies contained a high ratio of podocin-positive to nephrin-positive urinary EVs (podocin⁺ EVs-to-nephrin⁺ EVs ratio) and increased nephrinuria, both of which correlated with proteinuria. Plasma levels of hemopexin, which were decreased in women with preeclampsia, negatively correlated with proteinuria, urinary podocin⁺ EVs-to-nephrin⁺ EVs ratio, and nephrinuria. Administration of HbF to pregnant rabbits increased the number of urinary EVs of podocyte origin. These findings provide evidence that urinary EVs are reflective of preeclampsia-related altered podocyte protein expression. Furthermore, renal injury in preeclampsia associated with an elevated urinary podocin⁺ EVs-to-nephrin⁺ EVs ratio and may be mediated by prolonged exposure to cellfree HbF.

Keywords: extracellular vesicles; podocyte; preeclampsia.

Graphical abstract

Figure 1.

Centrifugation of urine samples from women with preeclampsia results in decrease in the number of extracellular vesicles. (A) Podocin-positive EVs, expressed as median (25th–75th percentile), in women with normotensive pregnancies: unspun urine, 348.5 (187–2473) versus spun urine, 401.5 (85–919); in women with preeclamptic pregnancies: unspun urine, 3150 (2800–3400) versus spun urine, 274 (256–539). (B) Nephrin-positive EVs, expressed as median (25th–75th percentile), in women with normotensive pregnancies: unspun urine, 185 (61–538) versus spun urine, 114.5 (32–281); in women with preeclamptic pregnancies: unspun urine, 997 (525–1209) versus spun urine, 111 (77–129). PE, preeclampsia.

Figure 2.

Freeze/thaw cycles of urine samples from women with either normotensive or preeclamptic pregnancies do not affect the number of extracellular vesicles. (A) Podocin-positive EVs, expressed as median (25th–75th percentile), in freshly collected unspun urine and after freezing at −80°C and thawing on two occasions, 1 and 2 weeks after collection. Normotensive pregnancies: 348.5 (187–2473), 357 (189–2390), and 355 (194–2462), respectively; preeclamptic pregnancies: 3150 (2800–400), 3178 (3040–3178), and 3393 (3332–3393), respectively. (B) Nephrin-positive EVs, expressed as median (25th–75th percentile), in freshly collected unspun urine and after freezing at −80°C and thawing on two occasions, 1 and 2 weeks after collection. Normotensive pregnancies: 185 (61–538), 181 (63–539), and 171 (66–477), respectively; preeclamptic pregnancies: 997 (525–1209), 1002 (577–1379), and 1022 (622–1423), respectively. PE, preeclampsia.

Figure 3.

Administration of fetal hemoglobin to pregnant rabbits results in increase in urinary extracellular vesicles. (A) Annexin-V–positive and (B) podocin-positive EVs, expressed as median (25th–75th percentile), were significantly increased in HbF compared with control animals: 39,094 (21,719–43,537) versus 8808 (5760–10,525); P=0.04, and 1850 (1241–3569) versus 543 (230–719); P=0.04, respectively.

Figure 4.

Electron microscopy analysis of renal sections from HbF-treated rabbits showing endotheliosis. HbF-treated rabbit (A) demonstrated endotheliosis (arrow), which was absent in the control (B).

Figure 5.

Renal injury in preeclampsia is associated with the presence of urinary extracellular vesicles of podocyte origin and is related to fetal hemoglobin.

Figure 6.

Digital flow cytometry analysis of urinary EVs indicating an elevated podocin-positive to nephrin-postive EVs ratio in preeclampsia. Normotensive pregnant woman on the left, preeclamptic pregnant woman on the right, with a calculated podocin-positive to nephrin-positive EVs ratio of 1.56 and 17.57, respectively.