Gene Expression Analysis by Reverse Transcription Quantitative Polymerase Chain Reaction
RNA was isolated from 20 × 5 µm kidney cryosections using the RNeasy Mini Plus Kit (Qiagen, Leusden, The Netherlands) according to the manufacturer’s instructions. RNA integrity was analyzed, complementary DNA synthesized, and quantitative reverse transcription polymerase chain reaction performed as described (Supplemental Digital Content 1, http://links.lww.com/CCX/A103).
Immunohistochemistry and Morphometric Analysis
Immunohistochemical staining of NGAL, KIM-1, and Neutrophil Elastase on formalin-fixed paraffin-embedded human kidney tissue was performed as described (Supplemental Digital Content 1, http://links.lww.com/CCX/A103). To quantify NGAL and KIM-1 immunostaining, the sections were first scanned using a Nanozoomer HT (Hamamatsu Photonics, Hamamatsu, Japan). Morphometric analysis was performed using the Aperio Imagescope positive pixel analysis v9.1 algorithm (Aperio Technologies, Vista, CA) as described previously (23). Neutrophil infiltration was quantified by counting the number of neutrophils (neutrophil Elastase positive) present in all glomeruli of the kidney sections.
Cryosections (9 µm) were mounted on PolyEthylene Naphthalate–membrane slides (Carl Zeiss B.V., Breda, The Netherlands), fixed, stained with Mayer hematoxylin, washed with diethylpyrocarbonate-treated water, and air-dried. Glomeruli were laser microdissected using the LMD6500 system (Leica Microsystems, Wetzlar, Germany) using LMD6500 software v7.0 (Leica Microsystems). Glomeruli (80–100) with a total area of 2 × 106 µm2 were dissected and collected in a 0.5-mL adhesive cap (Carl Zeiss B.V.) and stored at –80°C until gene expression analysis by reverse transcription quantitative polymerase chain reaction (RT-qPCR).
All statistical analyses were performed using GraphPad Prism Software v8 (San Diego, CA). Data are presented as mean ± sd. Statistical analysis was performed using a two-tailed unpaired Student t test, assuming unequal variances to compare two replicate means. Correlations between selected groups were assessed by Pearson tests. Differences were considered significant when p values were less than 0.05.
Renal NGAL and KIM-1 Levels Are Increased in Critically Ill Patients With Sepsis-AKI
Early detection of AKI and subtyping of AKI patients based on early pathophysiological parameters is critical to enable fast individualized therapeutic options for patients. We found significantly higher renal NGAL and KIM-1 messenger RNA levels in sepsis-AKI biopsies when compared with control subjects (Fig. 1A). However, NGAL and KIM-1 messenger RNA levels did not correlate with each other (Fig. 1B).
We proceeded by investigating the localization of NGAL and KIM-1 staining in sepsis-AKI and control biopsies. NGAL staining was present in the distal tubules, collecting ducts, adventitia of the renal arterioles, and, surprisingly, in the glomerular tuft of renal biopsies from Sepsis-AKI patients. In control biopsies, NGAL was absent in the glomeruli and present in the distal tubules and collecting ducts, but to a lesser extent than in Sepsis-AKI biopsies (Fig. 1C; and Supplemental Fig. 1, Supplemental Digital Content 1, http://links.lww.com/CCX/A103). NGAL was virtually absent in proximal tubules in Sepsis-AKI and control biopsies (Fig. 1C; and Supplemental Fig. 1, Supplemental Digital Content 1, http://links.lww.com/CCX/A103). KIM-1 staining was primarily localized at the brush border of the proximal tubular epithelium (Fig. 1C; and Supplemental Fig, 1, Supplemental Digital Content 1, http://links.lww.com/CCX/A103). Morphometric analysis of the kidney biopsy sections found a clear increase in renal NGAL and KIM-1 protein levels in sepsis-AKI biopsies when compared with control (Fig. 1D). Thus, both increased NGAL and KIM-1 messenger RNA and protein levels were found in sepsis-AKI compared with control biopsies, yet both injury markers were distributed differently within the kidney. No correlation was found between NGAL and KIM-1 messenger RNA or protein levels (Fig. 1, B and E) suggesting a heterogeneous and independent up-regulation of both markers in patients with sepsis.
Renal NGAL and KIM-1 Protein Levels Do Not Correlate With the Severity of Critical Illness or the Extent of Renal Injury
Using the APACHE IV or the SAPS II scoring system, we found that NGAL and KIM-1 levels did not correlate with the severity of critical illness (Fig. 2A and data not shown). Additionally, the NGAL and KIM-1 protein levels were not dependent on the degree of renal injury, as characterized in the sepsis-AKI patients using the RIFLE criteria (Fig. 2B). They were also not related to serum creatinine levels at ICU admission (Fig. 2C) or on the day of expiration (Fig. 2D). The origin and type of infection also had no influence on the extent of renal NGAL or KIM-1 protein levels (Supplemental Fig. 2, Supplemental Digital Content 1, http://links.lww.com/CCX/A103). Taken together, these findings indicate that renal NGAL and KIM-1 expression levels are increased in sepsis-AKI patients but are not associated with the severity of critical illness, AKI severity, or the type of infection.
Glomerular NGAL Protein Levels Identify Glomerular Heterogeneity in Sepsis-AKI Patients
We found NGAL staining in the glomeruli of sepsis-AKI patients (Fig. 3A). This was an unexpected finding, which, to our knowledge, has not been described before. We morphometrically determined the amount of NGAL staining in the glomerular tuft for all glomeruli in all biopsies. The size of the glomerular tufts in the control and sepsis-AKI biopsies did not significantly differ (Fig. 3B). Glomerular NGAL staining intensity was significantly higher in sepsis-AKI patients than in control subjects (Fig. 3C). Minimal glomerular NGAL staining intensity was found in control subjects (Fig. 3D). In contrast, glomerular NGAL staining intensity in kidneys of sepsis-AKI patients was highly variable (Fig. 3D). Some sepsis-AKI biopsies presented similar glomerular NGAL levels as control subjects (n = 9), whereas very high glomerular NGAL levels were found in 11 sepsis-AKI patients. Intermediate levels of glomerular NGAL were found in seven sepsis-AKI patients (Fig. 3D). Glomerular NGAL appeared to be associated with sepsis in particular because staining of renal biopsies from patients with acute and chronic organ rejection, in which inflammation plays an important role, was devoid of NGAL positive glomeruli (data not shown).
To elucidate whether NGAL is trapped in glomeruli, for example, due to impaired filtration, or, alternatively, is produced by endogenous cells, we laser microdissected glomeruli and subjected them to RT-qPCR. NGAL messenger RNA levels were not detectable in glomeruli of control subjects but could be detected in four of the five sepsis-AKI patients (Fig. 3E). Furthermore, high NGAL messenger RNA levels paralleled high glomerular NGAL protein staining (Fig. 3E). Intriguingly, NGAL protein levels did not correlate with the number of neutrophils present in the glomeruli of sepsis-AKI patients (Fig. 3F), which suggests that glomerular NGAL expression is not derived from neutrophils present in the glomeruli. Taken together, our data indicate that NGAL protein is produced in the glomeruli of sepsis-AKI patients to varying degrees within one kidney and is not associated with the number of infiltrating neutrophils.
In critically ill patients, sepsis-AKI is associated with high morbidity and mortality (1). Many patients leaving the ICU are dialysis dependent or at risk of developing or accelerating progression of chronic kidney disease (24). In sepsis, many processes in multiple cell types act in unison to cause kidney injury. Among these processes are acute inflammation resulting from bacteria or cellular products due to end-organ damage, microvascular dysfunction, and ischemic injury due to low blood pressure, blood shunting, and intravascular coagulation (25). Paradoxically, therapies such as antibiotics and high chloride resuscitation fluid might also be nephrotoxic (26). However, not all of these detrimental processes will occur in all sepsis patients and to the same extent. Furthermore, not all of these injurious cellular processes will induce the same renal response because damage can be inflicted at the glomerular, microvascular, and/or tubular level (25). For example, renal tubules are known to respond differently to ischemia than to toxin or LPS exposure, all of which are prevalent in sepsis (15).
We investigated whether NGAL and KIM-1 could differentiate AKI patient phenotypes. NGAL is a N-glycosylated protein with partly elucidated functions (27). It has iron-chelating capacity and might play a role in iron-mediated bacteriostasis (28), and in endogenous metabolic processes mediating cell growth (29), apoptosis (30), metabolism, and rescue from ischemia (31). There is a low-grade continuous NGAL production in the liver (32), yet most organs can produce NGAL in response to toxic, inflammatory, or infectious cellular injury. Serum NGAL is significantly increased in patients with sepsis-AKI when compared with patients with nonseptic AKI (33). We found increased NGAL expression in the kidney from sepsis-AKI patients compared with control subjects. This expression was localized in the distal tubules and collecting ducts, corroborating previous findings (34). Surprisingly, we also found that glomeruli in kidneys from sepsis-AKI patients produced NGAL. However, not all glomeruli expressed NGAL to the same extent, even when they were adjacent. This observation implies that even within a short distance range, glomerular heterogeneity in human kidneys exists. It is well known that adjacent glomeruli vary in structure and possibly also in function. Previous studies have shown that some glomeruli appear normal, whereas others are sclerotic, despite a clinical picture of increased proteinuria and diminished glomerular filtration rate (35). Our data indicate that NGAL staining in the glomerulus was not a result of impaired glomerular filtration but a consequence of cells localized within the glomerulus producing NGAL. Neutrophils are known to produce and secrete NGAL; however, our previous study found only a few neutrophils in the glomerulus (18). Furthermore, we found no correlation between the amount of glomerular NGAL and the number of neutrophils localized in the glomerulus. Hence, glomerulus-specific cells, endothelial cells, podocytes, or mesangial cells may produce NGAL under sepsis conditions. LPS was previously found to induce NGAL expression in podocytes in culture and in glomeruli in vivo (36). Likewise, NGAL expression was previously found in macrophages, smooth muscle cells, and endothelial cells in human carotid atherosclerotic arteries (37). Hence, glomerular endothelial cells and podocytes may be able to produce NGAL under sepsis conditions.
KIM-1 is a transmembrane glycoprotein that is undetectable in normal healthy kidneys. However, the expression of KIM-1 is specifically induced in the kidney after ischemic or toxic injury and can be detected in the plasma and urine, highlighting the specificity of KIM-1 for kidney injury (3839). KIM-1 is thought to be important for the removal of dead cells and the regeneration of tubular epithelial cells after injury (13). We found increased and considerable diverse expression of both KIM-1 messenger RNA and protein in sepsis-AKI patients. KIM-1 was found primarily localized on the apical surface of a limited number of proximal tubular epithelial cells as well as in the cytoplasm in flat and stretched tubular epithelial cells within dilated tubules, corroborating previous findings (40).
Both renal injury markers were increased in the kidney of patients with sepsis, but a correlation between NGAL and KIM-1 messenger RNA or protein expression was absent. The trigger inducing up-regulation of these biomarkers is known to be different (15), and the lack of correlation suggests that in different patients with the same consensus diagnosis, the balance between both response mechanisms is different which may be related to the kinetics of biomarker production. Hence, the pathophysiological mechanisms of kidney injury likely differ between sepsis-AKI patients.
A strength of our study is the use of kidney biopsies from sepsis-AKI patients, which has allowed us to make associations among clinical data, pathology, and molecular changes albeit in a small cohort of patients. However, several limitations need to be considered. We analyzed a subpopulation of sepsis-AKI patients, namely nonsurvivors, which probably represent the most severe critically ill patients. Furthermore, the onset and duration of sepsis varied per patient, as did the length of ICU stay. Hence, the kinetics of renal biomarker production in the kidney is unknown. Ideally, we would have correlated plasma and urine levels of NGAL and KIM-1 with the data presented here because this would have provided an insight into the relationship between the amounts of NGAL and KIM-1 found in the serum and/or urine and kidney pathophysiology. Unfortunately, we currently do not have plasma or urine samples from this sepsis cohort, yet we plan to expand this cohort and will include blood and urine samples from the same patients.
We have shown that there is a difference in the expression of renal NGAL and KIM in patients with the same syndrome “sepsis-AKI.” The fact that the expression differs means that there is no single pathway leading to sepsis-AKI. This underscores the beliefs that there are many/different pathophysiological pathways that can cause sepsis-AKI. Hence, patients with criteria that meet the definitions of both AKI and sepsis can be divided into subtypes based on pathophysiological features.
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Supplemental Digital Content
Copyright © 2019 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine.