Urinary Glycosaminoglycans Predict Outcomes in Septic Shock and Acute Respiratory Distress Syndrome

Abstract

Rationale: Degradation of the endothelial glycocalyx, a glycosaminoglycan (GAG)-rich layer lining the vascular lumen, is associated with the onset of kidney injury in animal models of critical illness. It is unclear if similar pathogenic degradation occurs in critically ill patients.

Objectives: To determine if urinary indices of GAG fragmentation are associated with outcomes in patients with critical illnesses such as septic shock or acute respiratory distress syndrome (ARDS).

Methods: We prospectively collected urine from 30 patients within 24 hours of admission to the Denver Health Medical Intensive Care Unit (ICU) for septic shock. As a nonseptic ICU control, we collected urine from 25 surgical ICU patients admitted for trauma. As a medical ICU validation cohort, we obtained serially collected urine samples from 70 patients with ARDS. We performed mass spectrometry on urine samples to determine GAG (heparan sulfate, chondroitin sulfate, and hyaluronic acid) concentrations as well as patterns of heparan sulfate/chondroitin sulfate disaccharide sulfation. We compared these indices to measurements obtained using dimethylmethylene blue, an inexpensive, colorimetric urinary assay of sulfated GAGs.

Measurements and main results: In septic shock, indices of GAG fragmentation correlated with both the development of renal dysfunction over the 72 hours after urine collection and with hospital mortality. This association remained after controlling for severity of illness and was similarly observed using the inexpensive dimethylmethylene blue assay. These predictive findings were corroborated using urine samples previously collected at three consecutive time points from patients with ARDS.

Conclusions: Early indices of urinary GAG fragmentation predict acute kidney injury and in-hospital mortality in patients with septic shock or ARDS. Clinical trial registered with www.clinicaltrials.gov (NCT01900275).

Keywords: chondroitin sulfate; glycocalyx; glycomics; heparan sulfate; hyaluronic acid.

Figure 1.

Endothelial glycocalyx glycosaminoglycans. (A) The endothelial glycocalyx (and its in vivo equivalent, the endothelial surface layer) is composed of glycosaminoglycans including proteoglycan-bound heparan sulfate (HS) and chondroitin sulfate (CS) as well as proteoglycan-independent hyaluronic acid (HA). (B) Glycosaminoglycans are composed of linear polymers of disaccharide repeats. CS and HS may be additionally modified by sulfation (X = SO− or H; Y = SO− or COCH3). n = number of times this disaccharide sequence repeats.

Figure 2.

Urinary glycosaminoglycans (GAGs) in septic shock and trauma. (A) Urine GAGs (including heparan sulfate [HS], chondroitin sulfate [CS], and hyaluronic acid [HA]) were significantly elevated in patients with septic shock (collected within 24 h of shock diagnosis) in comparison to surgical intensive care unit–admitted trauma patients. (B) Urine HS was not associated with severity of illness (Acute Physiology and Chronic Health Evaluation [APACHE] II) in the combined surgical/medical population. In contrast, urine HA (C) and CS (D) were associated with severity of illness. Mass spectrometry measurements revealed that patients with septic shock had distinct patterns of urine HS (E) and CS (F) disaccharide sulfation (inset). *P < 0.05. NS = N-sulfated.

Figure 3.

Urine glycosaminoglycans (GAGs) predict the development/progression of renal dysfunction in septic shock. (A) In urine collected within 24 hours of the diagnosis of septic shock, GAG concentrations (heparan sulfate [HS], chondroitin sulfate [CS], and hyaluronic acid [HA]) were significantly elevated in patients who developed new Acute Kidney Injury Network 2 criteria (>twofold increase in serum creatinine or <0.5 ml/kg/h urine output) between 24 and 72 hours after urine collection. (B–E) Urine HS (B), CS (C), HA (D), and total GAGs (E) have significant predictive value for the development of renal dysfunction, as demonstrated by receiver operating characteristic curves. Mass spectrometry measurements revealed that patients with septic shock who developed new/progressive renal dysfunction had distinct patterns of urine HS (F) and CS (G) disaccharide sulfation. *P < 0.05. AUC = area under the receiver operating characteristic curve; NS = N-sulfated.

Figure 4.

Urine glycosaminoglycans (GAGs) predict hospital mortality in septic shock. (A) In urine collected within 24 hours of the diagnosis of septic shock, urine heparan sulfate (HS) and hyaluronic acid (HA) were significantly elevated in patients who died during their hospitalization. Urine HS (B) and HA (D) have significant predictive value for mortality, as demonstrated by receiver operating characteristic curves. Conversely, urine chondroitin sulfate (CS) (C) and total GAGs (E) did not predict mortality. Mass spectrometry measurements revealed that patients with septic shock who died had distinct patterns of urine HS (F) and CS (G) disaccharide sulfation. *P < 0.05. AUC = area under the receiver operating characteristic curve; NS = N-sulfated.

Figure 5.

Dimethylmethylene blue (DMMB) colorimetric assay of urinary glycosaminoglycans (GAGs). (A) DMMB, a colorimetric assay that identifies sulfated glycosaminoglycans (GAGs), correlates with urine GAGs as measured by mass spectrometry. Accordingly, DMMB predicted the onset/progression of renal dysfunction (B) and in-hospital mortality (C) in patients with septic shock. AUC = area under the receiver operating characteristic curve.

Figure 6.

Urinary indices of glycosaminoglycan (GAG) degradation and acute kidney injury (AKI) in acute respiratory distress syndrome. Urine was collected from patients with acute respiratory distress syndrome 0, 1, and 3 days after study enrollment and analyzed by mass spectrometry for (A) heparan sulfate (HS), (B) chondroitin sulfate (CS), (C) hyaluronic acid (HA), or (D) total GAGs (reflecting the sum of HS, CS, and HA). In addition, sulfated glycosaminoglycans were measured using the colorimetric dimethylmethylene blue (DMMB) assay (E). At baseline, all patients had normal renal function. A subset of patients later developed AKI; the remainder of patients remained with normal renal function. *P < 0.05 between AKI and no AKI groups.

Figure 7.

Urinary indices of glycosaminoglycan (GAG) degradation and hospital mortality in acute respiratory distress syndrome. Urine was collected from patients with acute respiratory distress syndrome 0, 1, and 3 days after study enrollment and analyzed by mass spectrometry for (A) heparan sulfate (HS), (B) chondroitin sulfate (CS), (C) hyaluronic acid (HA), or (D) total GAGs (reflecting the sum of HS, CS, and HA). In addition, sulfated glycosaminoglycans were measured using the colorimetric dimethylmethylene blue (DMMB) assay (E). *P < 0.05 between nonsurvivors and survivors.