Urinary Plasminogen Activator Inhibitor-1: A Biomarker of Acute Tubular Injury.

INTRODUCTION: Acute kidney injury (AKI) is a threatening, multiaetiological syndrome encompassing a variety of forms and damage patterns. AKI lacks sufficiently specific diagnostic tools to evaluate the distinct combination of pathophysiological events underlying each case, which limits personalized and optimized handling. Therefore, a pathophysiological diagnosis based on new urinary biomarkers is sought for practical (readiness and noninvasiveness) and conceptual reasons, as the urine is a direct product of the kidneys. However, biomarkers found in the urine may also have extrarenal origin, thus conveying pathophysiological information from other organs or tissues. Urinary plasminogen activator inhibitor-1 (PAI-1) has been associated to AKI, although its origin and traffic to the urine are not known. METHODS: Herein, we studied the blood or renal origin of urinary PAI-1 (uPAI-1) in experimental AKI in Wistar rats, by means of the in situ renal perfusion method. For this purpose, urine was collected while the kidneys of rats with AKI showing increased uPAI-1 excretion, and controls, were in situ perfused with a saline solution. RESULTS: Our results show that during perfusion, PAI-1 remained in the urine of AKI rats, suggesting that renal cells shed this protein directly to the urine. PAI-1 is also significantly increased in the urine of AKI patients. Its low correlation with other urinary markers such as NGAL or NAG suggests that PAI-1 provides complementary and distinct phenotypical information. CONCLUSION: In conclusion, uPAI-1 is a biomarker produced by damaged kidneys following AKI, whose precise pathophysiological meaning in AKI needs to be further investigated.

Combined use of GM2AP and TCP1-eta urinary levels predicts recovery from intrinsic acute kidney injury.

Deficient recovery from acute kidney injury (AKI) has immediate and long-term health, clinical and economic consequences. Pre-emptive recovery estimation may improve nephrology referral, optimize decision making, enrollment in trials, and provide key information for subsequent clinical handling and follow-up. For this purpose, new biomarkers are needed that predict outcome during the AKI episode. We hypothesized that damage pattern-specific biomarkers are expected to more closely associate to outcome within distinct subpopulations (i.e. those affected by specific pathological processes determining a specific outcome), as biomarker pleiotropy (i.e. associated to phenomena unrelated to AKI) introduced by unselected, heterogeneous populations may blur statistics. A panel of urinary biomarkers was measured in patients with AKI and their capacity to associate to normal or abnormal recovery was studied in the whole cohort or after sub-classification by AKI etiology, namely pre-renal and intrinsic AKI. A combination of urinary GM2AP and TCP1-eta best associates with recovery from AKI, specifically within the sub-population of renal AKI patients. This two-step strategy generates a multidimensional space in which patients with specific characteristics (i.e. renal AKI patients with good or bad prognosis) can be identified based on a collection of biomarkers working serially, applying pathophysiology-driven criteria to estimate AKI recovery, to facilitate pre-emptive and personalized handling.

Pathophysiological mechanisms underlying a rat model of triple whammy acute kidney injury.

Simultaneous administration of certain antihypertensive (renin-angiotensin system inhibitors and diuretics) and nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a renal toxicity syndrome known as "triple whammy" acute kidney injury (TW-AKI), yet poorly characterized at the pathophysiological level, as no specific experimental model exists on which to conduct preclinical research. Herein, we generated and characterized a rat model of TW-AKI (0.7 mg/kg/day trandolapril +400 mg/kg/day ibuprofen +20 mg/kg/day furosemide). Double treatments involving the NSAID caused a subclinical acute kidney injury, as they reduced glomerular filtration rate to a significant but not sufficient extent to increase Crpl concentration. Only the triple treatment generated an overt AKI with increased Crpl provided that animals were under partial water ingestion restriction. Histological examination revealed no evidence of tissue renal injury, and no proteinuria or makers of renal damage were detected in the urine. These findings, along with a normal fractional excretion of sodium and glucose, indicated that these drug combinations produce a prerenal type of AKI. In fact, blood pressure and renal blood flow were also reduced (most markedly following the triple combination), although renal dysfunction was more pronounced than expected for the corresponding pressure drop, supporting a key pathological role of the interference with renal autoregulation mechanisms. In summary, prerenal TW-AKI only occurs when volemia is challenged (i.e., by furosemide in partially water-deprived animals) under the effects of renin-angiotensin system inhibitors and NSAIDs. This model will facilitate further pathophysiological knowledge for a better diagnosis and clinical handling of this syndrome.

Impaired Tubular Reabsorption Is the Main Mechanism Explaining Increases in Urinary NGAL Excretion Following Acute Kidney Injury in Rats.

Neutrophil gelatinase-associated lipocalin (NGAL) is a secreted low-molecular weight iron-siderophore-binding protein. NGAL overexpression in injured tubular epithelia partly explains its utility as a sensitive and early urinary biomarker of acute kidney injury (AKI). Herein, we extend mechanistic insights into the source and kinetics of urinary NGAL excretion in experimental AKI. Three models of experimental AKI were undertaken in adult male Wistar rats; renal ischemia-reperfusion injury (IRI) and gentamicin (G) and cisplatin (Cisp) nephrotoxicity. Alongside standard histological and biochemical assessment of AKI, urinary NGAL excretion rate, plasma NGAL concentration, and renal NGAL mRNA/protein expression were assessed. In situ renal perfusion studies were undertaken to discriminate direct shedding of NGAL to the urine from addition of NGAL to the urine secondary to alterations in the tubular handling of glomerular filtrate-derived protein. Renal NGAL expression and urinary excretion increased in experimental AKI. In acute studies in both the IRI and G models, direct renal perfusion with Kreb's buffer eliminated urinary NGAL excretion. Addition of exogenous NGAL to the Kreb's buffer circuit, reestablishment of perfusion with systemic blood or reperfusion with renal vein effluent restored high levels of urinary NGAL excretion. Urinary NGAL excretion in AKI arises in large proportion from reduced reabsorption from the glomerular filtrate. Hence, subclinical cellular dysfunction could increase urinary NGAL, particularly in concert with elevations in circulating prerenal NGAL and/or pharmacological inhibition of tubular reabsorption. More granular interpretation of urinary NGAL measurements could optimize the scope of its clinical utility as a biomarker of AKI.

Urinary TCP1-eta: A Cortical Damage Marker for the Pathophysiological Diagnosis and Prognosis of Acute Kidney Injury.

Acute kidney injury (AKI) is a serious syndrome with increasing incidence and health consequences, and high mortality rate among critically ill patients. Acute kidney injury lacks a unified definition, has ambiguous semantic boundaries, and relies on defective diagnosis. This, in part, is due to the absence of biomarkers substratifying AKI patients into pathophysiological categories based on which prognosis can be assigned and clinical treatment differentiated. For instance, AKI involving acute tubular necrosis (ATN) is expected to have a worse prognosis than prerenal, purely hemodynamic AKI. However, no biomarker has been unambiguously associated with tubular cell death or is able to provide etiological distinction. We used a cell-based system to identify TCP1-eta in the culture medium as a noninvasive marker of damaged renal tubular cells. In rat models of AKI, TCP1-eta was increased in the urine co-relating with renal cortical tubule damage. When kidneys from ATN rats were perfused in situ with Krebs-dextran solution, a portion of the urinary TCP1-eta protein content excreted into urine disappeared, and another portion remained within the urine. These results indicated that TCP1-eta was secreted by tubule cells and was not fully reabsorbed by the damaged tubules, both effects contributing to the increased urinary excretion. Urinary TCP1-eta is found in many etiologically heterogeneous AKI patients, and is statistically higher in patients partially recovered from severe AKI. In conclusion, urinary TCP1-eta poses a potential, substratifying biomarker of renal cortical damage associated with bad prognosis.

Preventive Effect of Cardiotrophin-1 Administration before DSS-Induced Ulcerative Colitis in Mice.

Ulcerative colitis is a relatively frequent, chronic disease that impacts significantly the patient's quality of life. Although many therapeutic options are available, additional approaches are needed because many patients either do not respond to current therapies or show significant side effects. Cardiotrophin-1 (CT-1) is a cytokine with potent cytoprotective, anti-inflammatory, and antiapoptotic properties. The purpose of this study was to assess if the administration of CT-1 could reduce colon damage in mice with experimental colitis was induced with 5% dextran sulfate sodium (DSS) in the drinking water. Half of the mice received an i.v. dose of CT-1 (200 µg/kg) 2 h before and 2 and 4 days after DSS administration. Animals were followed during 7 days after DSS administration. The severity of colitis was measured by standard scores. Colon damage was assessed by histology and immunohistochemistry. Inflammatory mediators were measured by Western blot and PCR. CT-1 administration to DSS-treated mice ameliorated both the clinical course (disease activity index), histological damage, inflammation (colon expression of TNF-α, IL-17, IL-10, INF IFN-γ, and iNOS), and apoptosis. Our results suggest that CT-1 administration before induction of colitis improves the clinical course, tissue damage, and inflammation in DSS-induced colitis in mice.

Cardiotrophin-1 Improves Kidney Preservation, Graft Function, and Survival in Transplanted Rats.

BACKGROUND: Cold ischemia-reperfusion injury is unavoidable during organ transplantation, and prolonged preservation is associated with poorer function recovery. Cardiotrophin-1 (CT-1) is an IL-6 family cytokine with cytoprotective properties. This preclinical study in rats tested whether CT-1 mitigates cold renal ischemia-reperfusion injury in the context of the transplantation of long-time preserved kidneys. METHODS: Kidneys were flushed with cold (4°C) University of Wisconsin solution containing 0.2 µg/mL CT-1 and stored for several periods of time at 4°C in the same solution. In a second approach, kidneys were first cold-preserved for 6 hours and then were perfused with University of Wisconsin solution containing CT-1 (0, 16, 32, or 64 µg/mL) and further cold-preserved. Organ damage markers were measured in the kidneys at the end of the storage period. For renal transplantation, recipient consanguineous Fischer rats underwent bilateral nephrectomy and received a previously cold-preserved (24 hours) kidney as described above. Survival and creatinine clearance were monitored over 30 days. RESULTS: Cardiotrophin-1 in perfusion and preservation fluids reduced oxidative stress markers (superoxide anion and inducible nitric oxide synthase), inflammation markers (NF-κB and tumor necrosis factor-α), and vascular damage (vascular cell adhesion molecule-1) and activated leukemia inhibitory factor receptor and STAT-3 survival signaling. Transplantation of kidneys cold-preserved with CT-1 increased rat survival and renal function (ie, lower plasma creatinine and higher creatinine clearance) and improved kidney damage markers after transplantation (ie, lower superoxide anion, tumor necrosis factor-α, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 and higher NF-κB). CONCLUSIONS: Cardiotrophin-1 represents a novel therapeutic strategy to reduce ischemia-reperfusion and cold preservation injury to rescue suboptimal kidneys and, consequently, to improve the clinical outcomes of renal transplantation.

Cardiotrophin-1 attenuates experimental colitis in mice.

Cardiotrophin-1 (CT-1) holds potent anti-inflammatory, cytoprotective, and anti-apoptotic effects in the liver, kidneys, and heart. In the present study, the role of endogenous CT-1 and the effect of exogenous CT-1 were evaluated in experimental ulcerative colitis. Colitis was induced in CT-1 knockout and wild-type (WT) mice by administration of dextran sulphate sodium (DSS) in the drinking water during 7 days. CT-1 knockout mice showed higher colon damage and disease severity than WT mice. In addition, CT-1 (200 µg/kg/day, iv) or vehicle (as control) was administered during 3 days to WT, colitic mice, starting on day 4 after initiation of DSS. Disease activity index (DAI), inflammatory markers (tumor necrosis factor α (TNF-α), INFγ, IL-17, IL-10, inducible nitric oxide synthase (iNOS)), colon damage, apoptosis (cleaved caspase 3), nuclear factor κB (NFκB) and STAT-3 activation, and bacterial translocation were measured. Compared with mice treated with DSS, mice also treated with exogenous CT-1 showed lower colon damage, DAI, plasma levels of TNFα, colon expression of TNF-α, INFγ, IL-17, iNOS and cleaved caspase 3, higher NFκB and signal transducer and activator of transcription 3 (STAT3) pathways activation, and absence of bacterial translocation. We conclude that endogenous CT-1 plays a role in the defense and repair response of the colon against ulcerative lesions through an anti-inflammatory and anti-apoptotic effect. Supplementation with exogenous CT-1 ameliorates disease symptoms, which opens a potentially new therapeutic strategy for ulcerative colitis.

Cardiotrophin-1 therapy prevents gentamicin-induced nephrotoxicity in rats.

Aminoglycosides are very effective antibiotics for the treatment of severe infections, but they rank among the most frequent causes of drug-induced nephrotoxicity. Thus, prevention of aminoglycoside nephrotoxicity is an unmet therapeutic objective. Cardiotrophin-1 (CT-1), a member of the IL-6 family of cytokines, has been reported to protect the kidney against toxic and ischemic acute kidney injury (AKI). We have assessed the effect of rat CT-1 in the severity of gentamicin (G)-induced AKI. Groups of male Wistar rats received the following for 6 consecutive days: i) isotonic saline solution (group CONT), ii) G, 150mg/kg/day, i.p. (group G), iii) CT-1, 100µg/kg/day i.v. (group CT-1), or iv) G and CT-1 at the doses described above. The G group showed a manifest AKI characterized by low creatinine clearance, high plasma creatinine and urea levels, increased urinary excretion of proteins, glucose and AKI markers such as N-acetyl-glucosaminidase, neutrophil gelatinase-associated lipocalin, kidney-injury molecule-1 and T-gelsolin, increased kidney levels of CD-68, iNOS, IL-1ß and TNF-α, and markedly higher histological renal damage and leukocyte infiltration than the CONT and CT-1 groups. Administration of CT-1 together with G reduced almost all of the above-described manifestations of G-induced AKI. The results of this study have potential clinical application, as CT-1 is near to being used as a drug for organ protection.

Increased Klk9 Urinary Excretion Is Associated to Hypertension-Induced Cardiovascular Damage and Renal Alterations.

Early detection of hypertensive end-organ damage and secondary diseases are key determinants of cardiovascular prognosis in patients suffering from arterial hypertension. Presently, there are no biomarkers for the detection of hypertensive target organ damage, most outstandingly including blood vessels, the heart, and the kidneys.We aimed to validate the usefulness of the urinary excretion of the serine protease kallikrein-related peptidase 9 (KLK9) as a biomarker of hypertension-induced target organ damage.Urinary, plasma, and renal tissue levels of KLK9 were measured by the Western blot in different rat models of hypertension, including angiotensin-II infusion, DOCA-salt, L-NAME administration, and spontaneous hypertension. Urinary levels were associated to cardiovascular and renal injury, assessed by histopathology. The origin of urinary KLK9 was investigated through in situ renal perfusion experiments.The urinary excretion of KLK9 is increased in different experimental models of hypertension in rats. The ACE inhibitor trandolapril significantly reduced arterial pressure and the urinary level of KLK9. Hypertension did not increase kidney, heart, liver, lung, or plasma KLK9 levels. Hypertension-induced increased urinary excretion of KLK9 results from specific alterations in its tubular reabsorption, even in the absence of overt nephropathy. KLK9 urinary excretion strongly correlates with cardiac hypertrophy and aortic wall thickening.KLK9 appears in the urine in the presence of hypertension as a result of subtle renal handling alterations. Urinary KLK9 might be potentially used as an indicator of hypertensive cardiac and vascular damage.

Urinary proteomics in renal pathophysiology: Impact of proteinuria.

Urinary differential proteomics is used to study renal pathophysiological mechanisms, find novel markers of biological processes and renal diseases, and stratify patients according to proteomic profiles. The proteomic procedure determines the pathophysiological meaning and clinical relevance of results. Urine samples for differential proteomic studies are usually normalized by protein content, regardless of its pathophysiological characteristics. In the field of nephrology, this approach translates into the comparison of a different fraction of the total daily urine output between proteinuric and nonproteinuric samples. Accordingly, alterations in the level of specific proteins found by this method reflect the relative presence of individual proteins in the urine; but they do not necessarily show alterations in their daily excretion, which is a key parameter for the understanding of the pathophysiological meaning of urinary components. For renal pathophysiology studies and clinical biomarker identification or determination, an alternative proteomic concept providing complementary information is based on sample normalization by daily urine output, which directly informs on changes in the daily excretion of individual proteins. This is clinically important because daily excretion (rather than absolute or relative concentration) is the only self-normalized way to evaluate the real meaning of urinary parameters, which is also independent of urine concentration.

Sub-nephrotoxic cisplatin sensitizes rats to acute renal failure and increases urinary excretion of fumarylacetoacetase.

Nephrotoxicity limits the therapeutic efficacy of the antineoplastic drug cisplatin. Due to dosage adjustment and appropriate monitoring, most therapeutic courses with cisplatin produce no or minimal kidney damage. However, we studied whether even sub-nephrotoxic dosage of cisplatin poses a potential risk for the kidneys by predisposing to acute kidney injury (AKI), specifically by lowering the toxicity threshold for a second nephrotoxin. With this purpose rats were treated with a single sub-nephrotoxic dosage of cisplatin (3mg/kg, i.p.) and after two days, with a sub-nephrotoxic regime of gentamicin (50mg/kg/day, during 6 days, i.p.). Control groups received only one of the drugs or the vehicle. Renal function and renal histology were monitored throughout the experiment. Cisplatin treatment did not cause any relevant functional or histological alterations in the kidneys. Rats treated with cisplatin and gentamicin, but not those under single treatments, developed an overt renal failure characterized by both renal dysfunction and massive tubular necrosis. In addition, the urinary excretion of fumarylacetoacetase was increased in cisplatin-treated animals at subtoxic doses, which might be exploited as a cisplatin-induced predisposition marker. In fact, the urinary level of fumarylacetoacetase prior to the second nephrotoxin correlated with the level of AKI triggered by gentamicin in predisposed animals.

Hypertension and hyperglycemia synergize to cause incipient renal tubular alterations resulting in increased NGAL urinary excretion in rats.

BACKGROUND: Hypertension and diabetes are the two leading causes of chronic kidney disease (CKD) eventually leading to end stage renal disease (ESRD) and the need of renal replacement therapy. Mortality among CKD and ESRD patients is high, mostly due to cardiovascular events. New early markers of risk are necessary to better anticipate the course of the disease, to detect the renal affection of additive risk factors, and to appropriately handle patients in a pre-emptive and personalized manner. METHODS: Renal function and NGAL urinary excretion was monitored in rats with spontaneous (SHR) or L-NAME induced hypertension rendered hyperglycemic (or not as controls). RESULTS: Combination of hypertension and hyperglycemia (but not each of these factors independently) causes an increased urinary excretion of neutrophil gelatinase-associated lipocalin (NGAL) in the rat, in the absence of signs of renal damage. Increased NGAL excretion is observed in diabetic animals with two independent models of hypertension. Elevated urinary NGAL results from a specific alteration in its tubular handling, rather than from an increase in its renal expression. In fact, when kidneys of hyperglycaemic-hypertensive rats are perfused in situ with Krebs-dextran solution containing exogenous NGAL, they excrete more NGAL in the urine than hypertensive rats. We also show that albuminuria is not capable of detecting the additive effect posed by the coexistence of these two risk factors. CONCLUSIONS: Our results suggest that accumulation of hypertension and hyperglycemia induces an incipient and quite specific alteration in the tubular handling of NGAL resulting in its increased urinary excretion.