Polymerization-Incompetent Uromodulin in the Pregnant Stroke-Prone Spontaneously Hypertensive Rat

Abstract

The kidney is centrally involved in blood pressure regulation and undergoes extensive changes during pregnancy. Hypertension during pregnancy may result in an altered urinary peptidome that could be used to indicate new targets of therapeutic or diagnostic interest. The stroke-prone spontaneously hypertensive rat (SHRSP) is a model of maternal chronic hypertension. Capillary electrophoresis-mass spectrometry was conducted to interrogate the urinary peptidome in SHRSP and the control Wistar-Kyoto strain at three time points: prepregnancy and gestational days 12 and 18. The comparison within and between the Wistar-Kyoto and SHRSP peptidome at all time points detected 123 differentially expressed peptides (fold change >1.5; P<0.05). Sequencing of these peptides identified fragments of collagen α-chains, albumin, prothrombin, actin, serpin A3K, proepidermal growth factor, and uromodulin. Uromodulin peptides showed a pregnancy-specific alteration in SHRSP with a 7.8-fold (P<0.01) and 8.8-fold (P<0.05) increase at gestational days 12 and 18, respectively, relative to the Wistar-Kyoto. Further investigation revealed that these peptides belonged to the polymerization-inhibitory region of uromodulin. Two forms of uromodulin (polymerization competent and polymerization incompetent) were found in urine from both Wistar-Kyoto and SHRSP, where the polymerization-incompetent form was increased in a pregnancy-specific manner in SHRSP. Nifedipine-treated pregnant SHRSP showed only polymerization-competent uromodulin, indicating that calcium may be mechanistically involved in uromodulin polymerization. This study highlights, for the first time, a potential role of uromodulin and its polymerization in hypertensive pregnancy.

Keywords: Nifedipine; hypertension; kidney; pregnancy; uromodulin.

Figure 1.

Schematic flowchart representing peptidomics data analysis and identification of peptide markers. The counterplot at the top represents the peptide mass fingerprint pattern of Wistar–Kyoto (WKY; n=7) and stroke-prone spontaneously hypertensive rat (SHRSP); n=7) at prepregnancy (nonpregnant [NP]) and gestational days (GDs) 12 and 18 observed in capillary electrophoresis–mass spectrometry. On the plot, X-axis represents the CE-migration time, Y-axis mass:charge ratio, and Z-axis the peptide signal intensity. Various comparisons were made between strains and GDs. The numbers above the arrow represent the number of peptide identified during the comparison with P<0.05. The strain- and pregnancy-specific peptide markers were not used to for analysis. The disease-specific peptide markers were analyzed for pattern of significance, that is, at P<0.05 whether the peptide was significant at a given GD. Star marked pattern of significance represents peptides that were significant at all GD or GDs 12–18 and were further used for analysis.

Figure 2.

Urinary Umod peptides are increased in the stroke-prone spontaneously hypertensive rat (SHRSP) relative to Wistar–Kyoto (WKY) in a pregnancy-dependent manner. Seven peptides detected in the urinary peptidome were derived from Umod protein (A). Of these peptides, they were all increased in a pregnancy-specific manner in SHRSP (n=7) relative to WKY (n=7) at gestational days (GDs) 12, GD18, or GD12 and GD18. Taking into account the sum of all of the 7 peptides (B) showed that Umod peptides were increased in a pregnancy-specific manner at GD12 and GD18 in SHRSP relative to WKY (*P<0.05, **P<0.01 vs WKY analyzed by Wilcoxon rank test).

Figure 3.

Increase in Umod in stroke-prone spontaneously hypertensive rat (SHRSP) validated in urine and kidney tissue. A, Purified Umod from the urine of Wistar–Kyoto (WKY; n=7) and SHRSP (n=7) was run on 10% NuPAGE gel and blotted onto a single polyvinylidene fluoride membrane. Umod showed an increase in SHRSP in pregnancy-dependent manner at gestational days (GDs) 12 and 18. B, Gene expression of Umod was measured in kidney tissue from nonpregnant and pregnant (GD18) WKY and SHRSP (n=5). Umod expression was increased in kidney tissue from SHRSP at both NP (P=n.s.) and GD18 time points (P<0.001). C, Umod protein was measured from kidney tissue extract of pregnant (GD18) SHRSP (n=4) and WKY (n=4). Pregnant SHRSP showed increased Umod expression in kidney tissue (**P<0.01, ***P<0.001 vs WKY analyzed by 1-way ANOVA, 2-way ANOVA, and t test).

Figure 4.

A, Schematic representation of rat Umod structure containing an epidermal growth factor–like domain (orange box I, II, and III), the Zona Pellucida (ZP) domain, internal and external hydrophobic patches (IHP and EHP, respectively), hepsin cleavage site and glycosylphosphatidylinositol (GPI) anchoring site. The zoomed-in sequence represents the C-terminal region identified in mass spectrometry rat data (pink) and previous human studies on preeclampsia (blue). B, Deglycosylation of Umod identified 2 bands in untreated WKY (pool of n=7) and stroke-prone spontaneously hypertensive rat (SHRSP; pool of n=7) at all gestational day (GD), and only single band in nifedipine-treated SHRSP (pool of 3) at GD12 and GD18. C, In the polymerization assay, the pellet fraction (P) represents the polymerization-competent and supernatant (S) the polymerization-incompetent Umod. Polymerization assay showed polymerization-incompetent Umod in the supernatant (S) of untreated WKY (pool of n=7) and SHRSP (pool of n=7) at all GDs, whereas no Umod bands were observed in nifedipine-treated SHRSP (pool of 3). Polymerization-competent Umod in the pellet (P) was observed in untreated WKY and SHRSP, as well as in nifedipine-treated SHRSP. D, Proteasix software predicted few serine proteases and metalloproteases that might cleave the C-terminal of Umod, which resulted in the peptides observed in urine. CTSG indicates cathepsin G; ELANE, neutrophil elastase; GZMA, granzyme A; MEP, Meprin A subunit α; MMP, matrix metalloprotease; NP, nonpregnant; and PLG, plasminogen.