Title Renal kallikrein excretion and epigenetics in human acute kidney injury : Expression , mechanisms and consequences Permalink

Background: Renal kallikrein (KLK1) synthesis and urinary excretion are reportedly diminished during AKI (acute kidney injury) in animal models, and provision of kallikrein abrogates renal injury in this setting, but data in human AKI is limited. Therefore we first examined KLK1 renal excretion in human AKI, and then probed potential endocrine and epigenetic mechanisms for its alterations. Methods: KLK1 enzymatic activity excretion was evaluated in urine from patients with established or incipient AKI, versus healthy/non-hospital as well as ICU controls. Endocrine control of KLK1 excretion was then probed by catecholamine and aldosterone measurements in established AKI versus healthy controls. To examine epigenetic control of KLK1 synthesis, we tested blood and urine DNA for changes in promoter CpG methylation of the KLK1 gene, as well as LINE-1 elements, by bisulfite sequencing. Results: Patients with early/incipient AKI displayed a modest reduction of KLK1 excretion, but unexpectedly, established AKI displayed substantially elevated urine KLK1 excretion, ~11-fold higher than healthy controls, and ~3fold greater than ICU controls. We then probed potential mechanisms of the change. Established AKI patients had lower SBP, higher heart rate, and higher epinephrine excretion than healthy controls, though aldosterone excretion was not different. Promoter KLK1 CpG methylation was higher in blood than urine DNA, while KLK1 methylation in blood DNA was significantly higher in established AKI than healthy controls, though KLK1 methylation in urine tended to be higher in AKI, directionally consistent with earlier/incipient but not later/established changes in KLK1 excretion in AKI. On multivariate ANOVA, AKI displayed coordinate changes in KLK1 excretion and promoter methylation, though directionally opposite to expectation. Control (LINE-1 repetitive element) methylation in blood and urine DNA was similar between AKI and controls. Conclusions: Unexpectedly, increased KLK1 excretion in AKI patients was found; this increase is likely to be due in part to increments in adrenergic tone during BP depression. Epigenetic changes at KLK1 may also play a role in early changes of KLK1 expression and thus AKI susceptibility or recovery. * Correspondence: rmehta@ucsd.edu; doconnor@ucsd.edu † Contributed equally Departments of Medicine and Pharmacology, and Institute for Genomic Medicine, University of California at San Diego, CA, USA Division of Nephrology and Hypertension, Department of Medicine, University of California San Diego Medical Center, San Diego, CA, USA Full list of author information is available at the end of the article Kang et al. BMC Nephrology 2011, 12:27 http://www.biomedcentral. com/1471-2369/12/27 © 2011 Kang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The incidence of acute kidney injury (AKI) in hospitalized patients is estimated to be 5-10%, and is much higher in the critically ill [1,2]. Despite the potential for recovery of kidney function, acute kidney injury is associated with substantial morbidity and even mortality. AKI, due to ischemia or nephrotoxic agent exposure, may lead to death or sublethal injury of proximal tubular cells, after which surviving cells may repolarize and/or de-differentiate, proliferate, migrate to denuded areas, re-differentiate, and restore nephron structure (including the tubular epithelium) and function [3,4]. The serine protease kallikrein (KLK1; E.C.-3.4.21.35; OMIM 147910), excreted from kidney into urine, catalyzes the cleavage of low molecular weight kininogen to lysylbradykinin (kallidin), which exhibits both vasodilator and natriuretic pharmacological properties in the kidney; if these properties occur in vivo, the potential of the system for regulating blood pressure is clear [5]. Renal kallikrein levels were markedly reduced in an aminoglycoside-induced AKI animal model [6], and KLK1 gene transfer protected against aminoglycoside-induced nephropathy by diminishing apoptosis and inflammation [7]. In addition, KLK1 infusion during aminoglycoside treatment attenuated drug-induced renal dysfunction, cortical damage, and apoptosis in the rat [8]. Previously, we have reported that urinary KLK1 excretion was diminished in renal allograft recipients with a clinical diagnosis of acute tubular necrosis (ATN) [9]; since urinary KLK1 originates in the kidney, reduced urinary kallikrein levels may reflect impaired renal function. However, this finding has not been pursued in humans. In mammals, cytosine methylation occurs almost exclusively at CpG dinucleotides, which are enriched at CpG islands, often are located at 5’-/promoter regions of functional genes [10]. Cytosine methylation may result in transcriptional repression either by interfering with transcription factor binding or by inducing a repressive chromatin structure [11]. Apoptotic pathways are targets for such “epigenetic” silencing, and several apoptosis-linked genes that are regulated directly or indirectly by methylation have been described [12]. In this study, we first probed whether KLK1 excretion is altered in human AKI, and if so what mechanisms (endocrine or epigenetic) might be driving the change. Since kidney repair after injury may recapitulate normal morphogenesis, we hypothesized that urinary kallikrein levels would be associated with severity of AKI and with epigenetic changes in the renal kallikrein-1 (KLK1) promoter. We considered that changes in kallikrein excretion or the KLK1 promoter might predict renal functional recovery and thus serve as biomarkers of recovery from AKI, thereby facilitating timely diagnosis and treatment. We therefore examined patients with AKI for urinary expression of KLK1 enzymatic activity, as well as genomic DNA from blood and urine for CpG methylation pattern at the KLK1 gene promoter.