SOD1 is a novel prognostic biomarker of acute kidney injury following cardiothoracic surgery.

BACKGROUND: Acute kidney injury (AKI) is a major burden among hospitalized and critical care patients. Among hospitalized patients that progress to severe AKI there is increased risk for morbidity, mortality, and the need for renal replacement therapy (RRT). As there are no specific treatments for AKI, the discovery of novel biomarkers that predict the progression of AKI may aid in timely implementation of supportive care to improve outcomes. METHODS: We collected urine from 204 patients that developed Stage 1 AKI by AKIN criteria within 72 h following cardiothoracic surgery. Urine samples were collected at the time of the initial diagnosis of AKI and stored at -80° C. Among the 204 patients, 25 progressed to a composite primary outcome of Stage 3 AKI, requirement of RRT, or 30-day mortality. The remaining 179 patients did not progress beyond Stage 2 AKI and were considered controls. Urinary concentrations of SOD1 and SOD1 activity were measured following collection of all samples. Samples were thawed and urinary superoxide dismutase 1 (SOD1) concentrations were measured by sandwich ELISA and urinary SOD1 activity was measured through a commercially available colorimetric assay. RESULTS: Urinary concentrations of SOD1 were significantly elevated (67.0 ± 10.1 VS 880.3 ± 228.8 ng/ml, p < 0.0001) in patients that progressed to severe AKI and were able to predict the progression to severe AKI (AUC - 0.85, p < 0.0001). Furthermore, total SOD activity also increased in the urine of patients that required RRT (77.6% VS 49.81% median inhibition, p < 0.01) and was able to predict the need for RRT (AUC: 0.83, p < 0.01). CONCLUSION: These findings show that urinary SOD1 concentrations and SOD activity are novel prognostic biomarkers for severe AKI following cardiothoracic surgery.

Discovery of seven novel putative antigens in membranous nephropathy and membranous lupus nephritis identified by mass spectrometry.

Multiple autoantigens have been identified in membranous nephropathy (MN) by tissue-based proteomics. However, antigenic targets of disease are unknown for over 10% of patients with MN and over half of those with membranous lupus nephritis (MLN). Here, we identified multiple new targets in PLA2R-/THSD7A-/EXT-/NELL1-quadruple negative MN biopsies through mass spectrometry of immune complexes recovered from biopsy tissue of patients with MN. Patients with MN negative for these four antigens were identified from Arkana Laboratories case archives. Protein G immunoprecipitation recovered immune complexes from frozen biopsy tissue from 142 quadruple-negative cases and 278 cases of known antigen type, followed by interrogation by mass spectrometry. Potential putative antigens were confirmed through paraffin immunofluorescence and co-localization with IgG within immune deposits. Consecutive series of 165 cases of PLA2R-negative MN and 142 MLN biopsies were screened to determine the frequency for each potential antigen. Seven putative antigens were discovered within immune complexes from biopsies of patients with MN including FCN3, CD206, EEA1, SEZ6L2, NPR3, MST1, and VASN. Peptides from these proteins were not enriched in the 278 cases of known antigen type. Between three to 30 unique peptides were detected for each new target. Frequencies of each biomarker, determined by staining consecutive case series, ranged from under 1 to 4.9%. NPR3 and CD206 were only positive in index cases. All cases showed co-localization of IgG within the immune deposits. Thus, seven putative antigens were newly identified in MN and MLN. Due to the number of antigens identified, it is becoming impractical to type PLA2R-negative MN or MLN cases through immunostaining alone. A multiplex approach is needed for subtyping of these diseases.

Ticagrelor inhibits platelet aggregation and reduces inflammatory burden more than clopidogrel in patients with stages 4 or 5 chronic kidney disease.

BACKGROUND: No study has compared pharmacologic properties of ticagrelor and clopidogrel in non-dialysis patients with stage 4-5 chronic kidney disease (CKD). METHODS: We conducted a double-blind RCT to compare effects of ticagrelor and clopidogrel in 48 CKD, with the primary outcome of ADP-induced platelet aggregation (WBPA) after 2 weeks of DAPT. In a parallel arm, we compared effects of 2 weeks of ticagrelor plus aspirin on mean changes in WBPA and markers of thromboinflammation among non-CKD controls (n = 26) with that of CKD in the ticagrelor-arm. RESULTS: Average age of CKD was 53.7 years, with 62% women, 54% African American, and 42% with stage 5 CKD. Ticagrelor generated statistically lower WBPA values post treatment [median 0 Ω (IQR 0, 2)] vs. clopidogrel [median 0 Ω (IQR 0, 5)] (P = 0.002); percent inhibition of WBPA was greater (87 ± 22% vs. 63 ± 50%; P = 0.04; and plasma IL-6 levels were much lower (8.42 ± 1.73 pg/ml vs. 18.48 ± 26.56 pg/ml; P = 0.04). No differences in mean changes in WBPA between CKD-ticagrelor and control groups were observed. Ticagrelor- DAPT reduced levels of IL-1α and IL-1ß in CKD-ticagrelor and control groups, attenuated lowering of TNFα and TRAIL levels in CKD-ticagrelor (vs controls), and had global changes in correlation between various cytokines in a subgroup of CKD-ticagrelor subjects not on statins (n = 10). Peak/trough levels of ticagrelor/metabolite were not different between CKD-ticagrelor and control groups. CONCLUSIONS: We report significant differences in platelet aggregation and anti-inflammatory properties between ticagrelor- and clopidogrel-based DAPT in non-dialysis people with stage 4-5 CKD. These notable inflammatory responses suggest ticagrelor-based DAPT might lower inflammatory burden of asymptomatic patients with stage 4 or 5 CKD. (clinicaltrials.gov # NCT03649711).

Are Undergraduates Familiar with Nephrology as a Medical Specialty? A Single Site Survey of Undergraduate Students.

Background: Over the past decade, nephrology has experienced a 43% decline in the number of fellowship applicants. Previous studies examining why residents choose a fellowship program cite lack of exposure as a main factor having an effect against a career in nephrology; however, no studies have surveyed the undergraduate population to inquire whether they recognize nephrology as a medical specialty compared with other medical specialties. We conducted a survey at a primarily undergraduate institution in the Southeast United States to test whether undergraduate students identified the word "nephrology." Methods: A total of 274 undergraduates responded to a survey that requested them to select every medical specialty that they recognized by name (15 real specialties and one fictitious specialty). Demographics regarding sex, race, collegiate level, high school location, premedical track, and household income were collected. Correlations between survey findings and rates of application and average salary per specialty were assessed. Results: Out of 15 medical specialties, nephrology (29%) and pulmonology (40%) were the least recognized. Pediatrics (97%) and surgery (97%) ranked highest. Sex, race, collegiate level, and household income were not different between those students who recognized "nephrology" and those who did not. Premedical students were about twice as likely to have recognized nephrology versus nonpremedical students (49% versus 22%, respectively; P<0.001). STEM majors were about twice as likely to identify nephrology versus non-STEM majors (40% versus 20%, respectively; P<0.001). The proportion of undergraduate students who recognized a specific medical specialty significantly correlated only with the number of US applicants per fellowship position across different medical specialties in 2020 (P<0.05). Conclusions: On the basis of word association alone, nephrology is the one of the least recognized specialties by undergraduates. The discrepancy between nephrology and other specialties highlights a gap in name recognition at an early career stage, even among premedical students.

Platelet-Dependent Inflammatory Dysregulation in Patients with Stages 4 or 5 Chronic Kidney Disease: A Mechanistic Clinical Study.

Background: Chronic kidney disease (CKD) is characterized by dysregulated inflammation that worsens with CKD severity. The role of platelets in modulating inflammation in stage 4 or 5 CKD remains unexplored. We investigated whether there are changes in platelet-derived thromboinflammatory markers in CKD with dual antiplatelet therapy (DAPT; aspirin 81 mg/d plus P2Y12 inhibitor). Methods: In a mechanistic clinical trial, we compared platelet activation markers (aggregation and surface receptor expression), circulating platelet-leukocyte aggregates, leukocyte composition (monocyte subtypes and CD11b surface expression), and plasma cytokine profile (45 analytes) of non-CKD controls (n=26) and CKD outpatients (n=48) with a glomerular filtration rate (GFR) <30 ml/min per 1.73 m2 on 2 weeks of DAPT. Results: Patients with CKD demonstrated a reduced mean platelet count, elevated mean platelet volume, reduced platelet-leukocyte aggregates, reduced platelet-bound monocytes, higher total non-classic monocytes in the circulation, and higher levels of IL-1RA, VEGF, and fractalkine (all P<0.05). There were no differences in platelet activation markers between CKD and controls. Although DAPT reduced platelet aggregation in both groups, it had multifaceted effects on thromboinflammatory markers in CKD, including a reduction in PDGF levels in all CKD individuals, reductions in IL-1ß and TNF-α levels in select CKD individuals, and no change in a number of other cytokines. Significant positive correlations existed for baseline IL-1ß, PDGF, and TNF-α levels with older age, and for baseline TNF-α levels with presence of diabetes mellitus and worse albuminuria. Mean change in IL-1ß and PDGF levels on DAPT positively correlated with younger age, mean change in TNF-α levels with higher GFR, and mean changes in PDGF, and TRAIL levels correlated with worse albuminuria. Minimum spanning trees plot of cytokines showed platelet-derived CD40L had a large reduction in weight factor after DAPT in CKD. Additionally, platelet-derived IL-1ß and PDGF were tightly correlated with other cytokines, with IL-1ß as the hub cytokine. Conclusions: Attenuated interactions between platelets and leukocytes in the CKD state coincided with no change in platelet activation status, an altered differentiation state of monocytes, and heightened inflammatory markers. Platelet-derived cytokines were one of the central cytokines in patients with CKD that were tightly correlated with others. DAPT had multifaceted effects on thromboinflammation, suggesting that there is platelet-dependent and -independent inflammation in stage 4 or 5 CKD.

Development of ACE2 autoantibodies after SARS-CoV-2 infection.

BACKGROUND: Activation of the immune system is implicated in the Post-Acute Sequelae after SARS-CoV-2 infection (PASC) but the mechanisms remain unknown. Angiotensin-converting enzyme 2 (ACE2) cleaves angiotensin II (Ang II) resulting in decreased activation of the AT1 receptor and decreased immune system activation. We hypothesized that autoantibodies against ACE2 may develop after SARS-CoV-2 infection, as anti-idiotypic antibodies to anti-spike protein antibodies. METHODS AND FINDINGS: We tested plasma or serum for ACE2 antibodies in 67 patients with known SARS-CoV-2 infection and 13 with no history of infection. None of the 13 patients without history of SARS-CoV-2 infection and 1 of the 20 outpatients that had a positive PCR test for SARS-CoV-2 had levels of ACE2 antibodies above the cutoff threshold. In contrast, 26/32 (81%) in the convalescent group and 14/15 (93%) of patients acutely hospitalized had detectable ACE2 antibodies. Plasma from patients with antibodies against ACE2 had less soluble ACE2 activity in plasma but similar amounts of ACE2 protein compared to patients without ACE2 antibodies. We measured the capacity of the samples to inhibit ACE2 enzyme activity. Addition of plasma from patients with ACE2 antibodies led to decreased activity of an exogenous preparation of ACE2 compared to patients that did not have antibodies. CONCLUSIONS: Many patients with a history of SARS-CoV-2 infection have antibodies specific for ACE2. Patients with ACE2 antibodies have lower activity of soluble ACE2 in plasma. Plasma from these patients also inhibits exogenous ACE2 activity. These findings are consistent with the hypothesis that ACE2 antibodies develop after SARS-CoV-2 infection and decrease ACE2 activity. This could lead to an increase in the abundance of Ang II, which causes a proinflammatory state that triggers symptoms of PASC.

Role of Platelets in Chronic Kidney Disease.

Platelet-dependent mechanisms for excessive clotting and bleeding in CKD remain undefined. Moreover, platelets' contribution to inflammation, and specifically to CKD, are equally elusive. To date, descriptions of changes in the functional properties of circulating platelets during CKD have provided confusing interpretations. Experimental approaches that can advance our understanding of platelet dysfunction in CKD are needed, and studies that provide mechanistic insights into the dynamic relationships between thrombosis, bleeding, and inflammation associated with CKD will be essential to improve clinical management and outcomes for this vulnerable population. This article summarizes existing literature characterizing platelets in CKD and identifies areas that need further investigation.

APOL1 Risk Variants and Acute Kidney Injury in Black Americans with COVID-19.

BACKGROUND AND OBJECTIVES: Black Americans have a higher incidence of kidney disease compared with populations that do not have recent African ancestry. Two risk variants in the APOL1 are responsible for a portion of this higher risk. We sought to assess the odds of AKI conferred by APOL1 risk alleles in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Black Americans who tested positive for coronavirus disease 2019 (COVID-19) were genotyped to determine APOL1 risk allele status. We assessed the incidence of AKI, persistent AKI, and AKI requiring KRT within 21 days of the PCR-based diagnosis. Outcomes were adjusted for age, sex, body mass index, hypertension, eGFR, and use of angiotensin-converting enzyme inhibitor/angiotensin receptor blocker. RESULTS: In total, 126 cases of SARS-CoV-2 infection were included within a 5-month period, with 16 (13%) and 110 (87%) cases with two and zero/one APOL1 high-risk alleles, respectively. AKI occurred in 11 (69%) patients with two APOL1 high-risk alleles and 39 (35%) patients with zero/one high-risk alleles (adjusted odds ratio, 4.41; 95% confidence interval, 1.11 to 17.52; P=0.04). Persistent AKI occurred in eight (50%) patients with two APOL1 high-risk alleles and 21 (19%) of those with zero/one high-risk alleles (adjusted odds ratio, 3.53; 95% confidence interval, 1.8 to 11.57; P=0.04). AKI KRT occurred in four (25%) of those with two APOL1 high-risk alleles and eight (7%) of those with zero/one high-risk alleles (adjusted odds ratio, 4.99; 95% confidence interval, 1.02 to 24.4, P=0.05). CONCLUSIONS: APOL1 high-risk alleles are associated with greater odds of AKI in Black American patients with COVID-19.

Transforming Growth Factor Beta Receptor 3 (TGFBR3)-Associated Membranous Nephropathy.

Background: Membranous lupus nephritis (MLN) comprises 10%-15% of lupus nephritis and increases morbidity and mortality of patients with SLE through complications of nephrotic syndrome and chronic kidney failure. Identification of the target antigens in MLN may enable noninvasive monitoring of disease activity, inform treatment decisions, and aid in prognostication, as is now possible for idiopathic MN caused by antibodies against the phospholipase A2 receptor. Here, we show evidence for type III TGF-ß receptor (TGFBR3) as a novel biomarker expressed in a subset of patients with MLN. Methods: Mass spectrometry was used for protein discovery through enrichment of glomerular proteins by laser capture microdissection and through elution of immune complexes within MLN biopsy specimens. Colocalization with IgG within glomerular immune deposits from patients and disease controls was evaluated by confocal microscopy. Immunostaining of consecutive case series was used to determine the overall frequency in MN and MLN. Results: TGFBR3 was found to be enriched in glomeruli and coimmunoprecipitated with IgG within a subset of MLN biopsy specimens by mass spectrometry. Staining of consecutive MN cases without clinical evidence of SLE did not show TGFBR3 expression (zero of 104), but showed a 6% prevalence in MLN (11 of 199 cases). TGFBR3 colocalized with IgG along the glomerular basement membranes in TGFBR3-associated MN, but not in controls. Conclusions: Positive staining for TGFBR3 within glomerular immune deposits represents a distinct form of MN, substantially enriched in MLN. A diagnosis of TGFBR3-associated MN can alert the clinician to search for an underlying autoimmune disease.

Home run-results of a chronic kidney disease Telemedicine Patient Education Study.

BACKGROUND: Chronic kidney disease (CKD) incidence is increasing and associated mortality and morbidity are high. Educating patients is effective in delaying progression and establishing optimal renal replacement therapy (RRT). Tele-education/telemedicine (TM) can be an effective tool to provide such education, but there are no available data quantifying its effectiveness. We attempted to establish such evidence correlating the effect of education in patient choices and with the start of actual RRT. We present results from a 3-year pilot study evaluating the effectiveness of comprehensive predialysis education (CPE) through TM for CKD patients compared with a standard care group [face to face (FTF)]. The patient's ability to choose RRT was the primary endpoint. METHODS: This was a randomized controlled study providing CPE over three classes at nine sites (one FTF and eight TM). Three assessment tools were utilized to compare groups: CKD knowledge, literacy and quality of life. RESULTS: A total of 47.1% of FTF and 52.2% of TM patients reported not having enough information to choose a modality. This decreased by the third visit (FTF 7.4%, TM 13.2%). Home modality choices more than doubled in both groups (FTF 25.8-67.7%, TM 22.2-50.1%). In patients that completed one visit and needed to start RRT, 47% started on a home modality or received a pre-emptive transplant (home hemodialysis 6%, peritoneal dialysis 38%, transplant 3%). CONCLUSIONS: Results show almost 90% (TM 87%, FTF 95%) of the attendees could choose a modality after education. Home modality choices doubled. Patients were able to make an informed choice regardless of the modality of education.

Serum amyloid P deposition is a sensitive and specific feature of membranous-like glomerulopathy with masked IgG kappa deposits.

Membranous-like glomerulopathy with masked IgG kappa deposits (MGMID) is a recently described pattern of glomerulonephritis with a unique histopathology. The pattern is characterized by subepithelial and/or mesangial immune deposits that are "masked", to immunoglobulin staining by routine immunofluorescence but strongly stain for IgG and kappa light chain after protease digestion. Patients with this pattern of glomerulonephritis are most commonly young females presenting with proteinuria and a vague history of autoimmune disease such as low titer antinuclear antibodies. Here we compared the mass spectrometry profile of laser capture microdissected glomeruli from nine MGMID renal biopsies with eight biopsies showing other patterns of membranous glomerulopathy. The protein most significantly increased in MGMID was serum amyloid P. Immunostaining showed serum amyloid P colocalized with IgG in the glomeruli of MGMID but not with PLA2R-associated membranous glomerulopathy. Serum amyloid P was positive in the glomeruli of all 32 MGMID biopsies but negative in biopsies of other types of membranous glomerulopathies such as those associated with PLA2R and THSD7A. There were four biopsies with glomerular serum amyloid P staining among the 173 biopsies that did not fulfill criteria for MGMID or amyloidosis. All four of these biopsies with positive serum amyloid P staining had a membranous pattern of glomerulopathy with IgG kappa deposits that only differed from MGMID by the lack of "masking". Thus, positive staining within glomerular deposits for serum amyloid P identifies a unique form of glomerulonephritis likely sharing a common pathophysiologic mechanism of disease.

Chronic kidney disease and the gut microbiome.

The gut microbiome is composed of a diverse population of bacteria that have beneficial and adverse effects on human health. The microbiome has recently gained attention and is increasingly noted to play a significant role in health and a number of disease states. Increasing urea concentration during chronic kidney disease (CKD) leads to alterations in the intestinal flora that can increase production of gut-derived toxins and alter the intestinal epithelial barrier. These changes can lead to an acceleration of the process of kidney injury. A number of strategies have been proposed to interrupt this pathway of injury in CKD. The purpose of this review is to summarize the role of the gut microbiome in CKD, tools used to study this microbial population, and attempts to alter its composition for therapeutic purposes.

Metaproteomics reveals potential mechanisms by which dietary resistant starch supplementation attenuates chronic kidney disease progression in rats.

BACKGROUND: Resistant starch is a prebiotic metabolized by the gut bacteria. It has been shown to attenuate chronic kidney disease (CKD) progression in rats. Previous studies employed taxonomic analysis using 16S rRNA sequencing and untargeted metabolomics profiling. Here we expand these studies by metaproteomics, gaining new insight into the host-microbiome interaction. METHODS: Differences between cecum contents in CKD rats fed a diet containing resistant starch with those fed a diet containing digestible starch were examined by comparative metaproteomics analysis. Taxonomic information was obtained using unique protein sequences. Our methodology results in quantitative data covering both host and bacterial proteins. RESULTS: 5,834 proteins were quantified, with 947 proteins originating from the host organism. Taxonomic information derived from metaproteomics data surpassed previous 16S RNA analysis, and reached species resolutions for moderately abundant taxonomic groups. In particular, the Ruminococcaceae family becomes well resolved-with butyrate producers and amylolytic species such as R. bromii clearly visible and significantly higher while fibrolytic species such as R. flavefaciens are significantly lower with resistant starch feeding. The observed changes in protein patterns are consistent with fiber-associated improvement in CKD phenotype. Several known host CKD-associated proteins and biomarkers of impaired kidney function were significantly reduced with resistant starch supplementation. Data are available via ProteomeXchange with identifier PXD008845. CONCLUSIONS: Metaproteomics analysis of cecum contents of CKD rats with and without resistant starch supplementation reveals changes within gut microbiota at unprecedented resolution, providing both functional and taxonomic information. Proteins and organisms differentially abundant with RS supplementation point toward a shift from mucin degraders to butyrate producers.

Perfluoroalkyl substances and kidney function in chronic kidney disease, anemia, and diabetes.

BACKGROUND: Anemia often complicates chronic kidney disease (CKD), leading to insufficient tissue oxygenation and hypoxic injury, the factor thought to underlie progression from CKD to renal failure. Perfluorocarbons are potent oxygen transporters used in organ preservation and synthetic blood development. Data are scarce on their relationship with kidney function, especially in diabetes where anemia and hypoxia are more prevalent. We investigated the relationship of perfluoroalkyl acids (PFAS) with kidney function and variation by diabetes and anemia status. METHODS: Data on 53,650 adults (5,210 with diabetes) were obtained from the C8 Health Project. CKD was defined as an estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m2. Four PFAS were investigated: perfluorohexane sulfonate (PFHxS), perfluorooctanoic acid, perfluorooctane sulfonate, and perfluorononanoic acid. FINDINGS: Each PFAS was positively associated with eGFR among those with CKD or anemia; this was the strongest among those with both CKD and anemia, followed by those with CKD uncomplicated by anemia. These relationships were more pronounced among those with diabetes (all P<0.01). In the absence of both CKD and anemia, PFAS was inversely associated with eGFR. Among persons with both anemia and diabetes, when further stratified by CKD stage, compared to an eGFR <30, ORs (95% CI) for being in the eGFR ≥ 90, 60-89, 45-59, and 30-45 range, respectively, were 3.20 (2.00-5.13), 2.64 (1.83-3.80), 3.18 (2.17-4.67), and 1.99 (1.38-2.86) for each ng/dL increase in PFHxS. Results were similar for each PFAS. INTERPRETATION: PFAS are inversely associated with kidney function in CKD and diabetes, with a stronger relation observed when anemia is present.

A Novel CLCN5 Mutation Associated With Focal Segmental Glomerulosclerosis and Podocyte Injury.

INTRODUCTION: Tubular dysfunction is characteristic of Dent's disease; however, focal segmental glomerulosclerosis (FSGS) can also be present. Glomerulosclerosis could be secondary to tubular injury, but it remains uncertain whether the CLCN5 gene, which encodes an endosomal chloride and/or hydrogen exchanger, plays a role in podocyte biology. Here, we implicate a role for CLCN5 in podocyte function and pathophysiology. METHODS: Whole exome capture and sequencing of the proband and 5 maternally-related family members was conducted to identify X-linked mutations associated with biopsy-proven FSGS. Human podocyte cultures were used to characterize the mutant phenotype on podocyte function. RESULTS: We identified a novel mutation (L521F) in CLCN5 in 2 members of a Hispanic family who presented with a histologic diagnosis of FSGS and low-molecular-weight proteinuria without hypercalciuria. Presence of CLCN5 was confirmed in cultured human podocytes. Podocytes transfected with the wild-type or the mutant (L521F) CLCN5 constructs showed differential localization. CLCN5 knockdown in podocytes resulted in defective transferrin endocytosis and was associated with decreased cell proliferation and increased cell migration, which are hallmarks of podocyte injury. CONCLUSIONS: The CLCN5 mutation, which causes Dent's disease, may be associated with FSGS without hyercalcuria and nepthrolithiasis. The present findings supported the hypothesis that CLCN5 participates in protein trafficking in podocytes and plays a critical role in organizing the components of the podocyte slit diaphragm to help maintain normal cell physiology and a functional filtration barrier. In addition to tubular dysfunction, mutations in CLCN5 may also lead to podocyte dysfunction, which results in a histologic picture of FSGS that may be a primary event and not a consequence of tubular damage.

Proteomic Analysis for Identification of Biomarkers that Predict Severe Acute Kidney Injury.

The search for acute kidney injury (AKI) biomarkers has identified a number of urine proteins that can be used to predict the presence of AKI but has struggled to identify proteins that are prognostic for severe AKI. In this review, we discuss 2 currently available biomarkers and the designs of the studies in which they were identified and relate this to the AKI characteristics they predict clinically. We discuss recent advances in mass spectrometry and sample preparation, which have improved the ability to identify low abundance proteins as well as the ability to characterize more of the protein by mass spectrometry. We show how these changes can lead to a deeper and more thorough analysis of the urine proteome. Finally, we highlight 2 important issues that can help in the identification of these biomarkers, appropriate study design and adequate technical characteristics in the analysis.