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AP214, an analogue of alpha-melanocyte-stimulating hormone, ameliorates sepsis-induced acute kidney injury and mortality.

K Doi — 2008-08-12T09:36:33-00:00

Kidney international, Vol. 73, No. 11. (June 2008), pp. 1266-1274.

Sepsis remains a serious problem in critically ill patients with the mortality increasing to over half when there is attendant acute kidney injury. alpha-Melanocyte-stimulating hormone is a potent anti-inflammatory cytokine that inhibits many forms of inflammation including that with acute kidney injury. We tested whether a new alpha-melanocyte-stimulating hormone analogue (AP214), which has increased binding affinity to melanocortin receptors, improves sepsis-induced kidney injury and mortality using a cecal ligation and puncture mouse model. In the lethal cecal ligation-puncture model of sepsis, severe hypotension and bradycardia resulted and AP214 attenuated acute kidney injury of the lethal model with a bell-shaped dose-response curve. An optimum AP214 dose reduced acute kidney injury even when it was administered 6 h after surgery and it significantly improved blood pressure and heart rate. AP214 reduced serum TNF-alpha and IL-10 levels with a bell-shaped dose-response curve. Additionally; NF-kappaB activation in the kidney and spleen, and splenocyte apoptosis were decreased by the treatment. AP214 significantly improved survival in both lethal and sublethal models. We have shown that AP214 improves hemodynamic failure, acute kidney injury, mortality and splenocyte apoptosis attenuating pro- and anti-inflammatory actions due to sepsis.

Association of achieved dialysis dose with mortality in the hemodialysis study: an example of "dose-targeting bias".

T Greene — 2008-07-08T06:33:25-00:00

Journal of the American Society of Nephrology : JASN, Vol. 16, No. 11. (November 2005), pp. 3371-3380.

In the intention-to-treat analysis of the Hemodialysis Study, all-cause mortality did not differ significantly between the high versus standard hemodialysis dose groups. The association of mortality with delivered dose within each of the two randomized treatment groups was examined, and implications for observational studies were considered. Time-dependent Cox regression was used to relate the relative risk (RR) for mortality to the running mean of the achieved equilibrated Kt/V (eKt/V) over the preceding 4 mo. eKt/V was categorized by quintiles within each dose group. Analyses were controlled for case-mix factors and baseline anthropometric volume. Within each randomized dose group, mortality was elevated markedly when achieved eKt/V was in the lowest quintile (RR, 1.93; 95% confidence interval [CI], 1.40 to 2.66; P < 0.0001 in the standard-dose group; RR, 2.04; 95% CI, 1.50 to 2.76; P < 0.0001 in the high-dose group; RR relative to the middle quintiles). The mortality rate in the lowest eKt/V quintile of the high-dose group was higher than in the full standard-dose group (RR, 1.59; 95% CI, 1.29 to 1.96; P < 0.0001). Each 0.1 eKt/V unit below the group median was associated with a 58% higher mortality in the standard-dose group (P < 0.001) and a 37% higher mortality in the high-dose group (P < 0.001). The magnitude of these dose-mortality effects was seven- to 12-fold higher than the upper limit of the 95% CI from the intention-to-treat analysis. The effects were attenuated in lagged analyses but did not disappear. When dialysis dose is targeted closely, as under the controlled conditions of the Hemodialysis Study, patients with the lowest achieved dose relative to their target dose experience markedly increased mortality, to a degree that is not compatible with a biologic effect of dose. The possibility of similar (albeit smaller) biases should be considered when analyzing observational data sets relating mortality to achieved dose of dialysis.

Dialysis dose and the effect of gender and body size on outcome in the HEMO Study.

T Depner — 2008-07-01T02:59:28-00:00

Kidney international, Vol. 65, No. 4. (April 2004), pp. 1386-1394.

BACKGROUND: Gender and body size have been associated with survival in hemodialysis populations. In recent observational studies, overall mortality was similar in men and women and higher in small patients. The effect of dialysis dose in each of these subgroups has not been tested in a clinical trial. METHODS: The HEMO Study was a controlled trial of dialysis dose and membrane flux in 1846 hemodialysis patients followed up for 6.6 years in 15 centers throughout the United States. We examined the effect of dialysis dose on mortality and on selected secondary outcomes in subgroups of patients. RESULTS: Adjusting for age only, overall mortality was lower in patients with higher body weight (P < 0.001), higher body mass index (P < 0.001), and higher body water content determined by the Watson formula (Vw) (P < 0.001), but was not associated with gender (P= 0.27). The RR of mortality comparing the high dose with the standard dose group was related to gender (P= 0.014). Women randomized to the high dose had a lower mortality rate than women randomized to the standard dose (RR = 0.81, P= 0.02), while men randomized to the high dose had a nonsignificant trend for a higher mortality rate than men randomized to the standard dose (RR = 1.16, P= 0.16). Analysis of both genders combined showed no overall dose effect (R = 0.96, P= 0.52), as reported previously. Vw was greater than 35 L in 84% of men compared with 17% of women. However, the RR of mortality for the high versus standard dose remained lower in women than in men after adjustment for the interaction of dose with Vw or with other size parameters, including weight and body mass index. Conversely, the dose effect was not significantly related to size parameters after controlling for the relationship of the dose comparison with gender. CONCLUSION: The data suggest that mortality and morbidity might be reduced by increasing the dialysis dose above the current standard in women but not in men. This effect was not explained by differences between men and women in age, race, or in several indices of body size. Because multiple comparisons were considered in this analysis, the role of gender on the effect of dialysis dose is suggestive and invites further study.

Urinary exosomal transcription factors, a new class of biomarkers for renal disease.

Hua Zhou — 2008-06-26T02:53:59-00:00

Kidney international (28 May 2008)

Urinary exosomes are excreted from all nephron segments and constitute a rich source of intracellular kidney injury biomarkers. To study whether they contain transcription factors, we collected urine from two acute kidney injury models (cisplatin or ischemia-reperfusion), two podocyte injury models (puromycin-treated rats or podocin-Vpr transgenic mice) and from patients with focal segmental glomerulosclerosis, acute kidney injury and matched controls. Exosomes were isolated by differential centrifugation and found to contain activating transcription factor 3 (ATF3) and Wilms Tumor 1 (WT-1) proteins detected by Western blot. These factors were found in the concentrated exosomal fraction, but not in whole urine. ATF3 was continuously present in urine exosomes of the rat models following acute injury at times earlier than the increase in serum creatinine. ATF3 was found in exosomes isolated from patients with acute kidney injury but not from patients with chronic kidney disease or controls. Urinary WT-1 was present in animal models before significant glomerular sclerosis and in 9/10 patients with focal segmental glomerulosclerosis but not in 8 controls. Our findings suggest that transcription factor ATF3 may provide a novel renal tubular cell biomarker for acute kidney injury while WT-1 may detect early podocyte injury. Measurement of urinary exosomal transcription factors may offer insight into cellular regulatory pathways.Kidney International advance online publication, 28 May 2008; doi:10.1038/ki.2008.206.

Liver proteomics for therapeutic drug discovery: inhibition of the cyclophilin receptor CD147 attenuates sepsis-induced acute renal failure.

JW Dear — 2008-02-19T04:14:31-00:00

Crit Care Med, Vol. 35, No. 10. (October 2007), pp. 2319-2328.

OBJECTIVE: Sepsis-induced multi-organ failure continues to have a high mortality. The liver is an organ central to the disease pathogenesis. The objective of this study was to identify the liver proteins that change in abundance with sepsis and subsequently identify new drug targets. DESIGN: Proteomic discovery study and drug target validation. For the proteomics study, three biological replicate mice were used per group. SETTING: Research institute laboratory. SUBJECTS: Three-month-old C57BL/6 mice. INTERVENTIONS: We used a mouse model of sepsis based on cecal ligation and puncture, but with fluid and antibiotic resuscitation. Liver proteins that changed in abundance were identified by difference in gel electrophoresis. We compared liver proteins from 6-hr post-cecal ligation and puncture to sham-operated mice ("early proteins") and 24-hr post-cecal ligation and puncture with 6-hr post-cecal ligation and puncture ("late proteins"). Proteins that changed in abundance were identified by tandem mass spectrometry. We then inhibited the receptor for one protein and determined the effect on sepsis-induced organ dysfunction. RESULTS: The liver proteins that changed in abundance after sepsis had a range of functions such as acute phase response, coagulation, endoplasmic reticulum stress, oxidative stress, apoptosis, mitochondrial electron transfer proteins, and nitric oxide metabolism. We found that cyclophilin increased in abundance after cecal ligation and puncture. When the receptor for this protein, CD147, was inhibited, sepsis-induced renal dysfunction was reduced. There was also a significant reduction in serum cytokine production when CD147 was inhibited. CONCLUSION: By applying proteomics to a clinically relevant mouse model of sepsis, we identified a number of novel proteins that changed in abundance. The inhibition of the receptor for one of these proteins, cyclophilin, attenuated sepsis-induced acute renal failure. The application of proteomics to sepsis research can facilitate the discovery of new therapeutic targets.

Discovery of protein biomarkers for renal diseases.

SM Hewitt — 2006-02-27T21:21:38-00:00

J Am Soc Nephrol, Vol. 15, No. 7. (July 2004), pp. 1677-1689.

Animal models and human studies have been useful in dissecting the molecular mechanisms of renal disease and finding new disease targets; however, translation of these findings to new clinical therapeutics remains challenging. Difficulties with detecting early disease, measuring drug effectiveness, and the daunting cost of clinical trials hampers the development of new therapeutics for renal diseases. Many existing laboratory tests were discovered because of inspired recognition that a particular protein might prove useful in clinical practice. New unbiased genomic and proteomic techniques identify many constituents present in biologic samples and thus may greatly accelerate biomarker research. This review focuses on the steps needed to develop new biomarkers that are useful in laboratory and clinical investigations, with particular focus on new proteomic screening technologies. New biomarkers will speed the laboratory and clinical development of new treatments for renal diseases through mechanistic insights, diagnoses that are more refined, early detection, and enhanced proof of concept testing.

Simvastatin improves sepsis-induced mortality and acute kidney injury via renal vascular effects.

H Yasuda — 2007-12-14T15:25:36-00:00

Kidney Int, Vol. 69, No. 9. (May 2006), pp. 1535-1542.

Acute kidney injury (AKI) occurs in about half of patients in septic shock and the mortality of AKI with sepsis is extremely high. An effective therapeutic intervention is urgently required. Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors that also have pleiotropic actions. They have been reported to increase the survival of septic or infectious patients. But the effect of simvastatin, a widely used statin, on sepsis-induced AKI is unknown. The effects of simvastatin and tumor necrosis factor (TNF)-alpha neutralizing antibody were studied in a clinically relevant model of sepsis-induced AKI using cecal ligation and puncture (CLP) in elderly mice. Simvastatin significantly improved CLP-induced mortality and AKI. Simvastatin attenuated CLP-induced tubular damage and reversed CLP-induced reduction of intrarenal microvascular perfusion and renal tubular hypoxia at 24 h. Simvastatin also restored towards normal CLP-induced renal vascular protein leak and serum TNF-alpha. Neither delayed simvastatin therapy nor TNF-alpha neutralizing antibody improved CLP-induced AKI. Simvastatin improved sepsis-induced AKI by direct effects on the renal vasculature, reversal of tubular hypoxia, and had a systemic anti-inflammatory effect.

Imprecision of the hemodialysis dose when measured directly from urea removal. Hemodialysis Study Group.

TA Depner — 2007-10-20T06:26:11-00:00

Kidney Int, Vol. 55, No. 2. (February 1999), pp. 635-647.

Bootstrap BACKGROUND: The postdialysis blood urea nitrogen (BUN; Ct) is a pivotal parameter for assessing hemodialysis adequacy by conventional blood-side methods, but Ct is relatively unstable because of hemodialysis-induced disequilibrium. The uncertainty associated with this method is potentially reduced or eliminated by measuring urea removed on the dialysate side, a more direct approach that can determine adequacy from the fraction of urea removed and by substituting an estimate of the equilibrated postdialysis BUN (Ceq) for Ct. For a patient with a known urea volume (V), Ceq, the equilibrated Kt/V (eKt/V), and the solute removal index (SRI) can be calculated from the predialysis BUN (C0), total urea nitrogen removed (A), and V from simple mass balance calculations (dialysate/volume method). However, a theoretical error analysis showed that relatively small errors in A, C0, or V are magnified when SRI or eKt/V is calculated using this method, especially at higher eKt/V values (for example, if eKt/V = 1.4 per dialysis, a 7% dialysate collection error causes a 20% error in eKt/V). METHODS: During three to four baseline dialyses in each of 39 patients enrolled in the pilot phase of the HEMO Study, "A" was measured using an instrument that sampled dialysate frequently (Biostat), and V was calculated from A, C0, and Ceq (median CV for V = 5.6%). The mean V was then applied to the dialysate/volume method to estimate eKt/V and SRI during two to five subsequent dialyses per patient (comparison dialyses). The accuracy and precision of these estimates were assessed by comparing them with eKt/V and SRI derived from a direct measurement of Ceq drawn 30 minutes after dialysis (reference method), from mathematical curve-fitting of sequential dialysate urea concentrations (dialysate curve-fit method), and from another blood-side method that estimates eKt/V from single pool Kt/V and the fractional rate of solute removal (rate method): eKt/V = spKt/V - 0.6.K/V + 0.03. RESULTS: During 128 comparison dialyses, median absolute errors for calculated eKt/V compared with the reference method were 0.169, 0.061, and 0.071 for the dialysate/volume method, the rate method, and the dialysate curve-fitting method, respectively. The corresponding correlation coefficients were 0.47, 0.88, and 0.81. For SRI, median absolute errors were 0.044, 0.018, and 0.027, and the correlation coefficients were 0.54, 0.85, and 0.74 for the three methods. CONCLUSIONS: The precision of eKt/V and SRI measurements was significantly lower for the dialysate/volume method compared with the blood-side methods. Inclusion of the dialysate curve analysis provided by the Biostat restored precision to the dialysate method to a level comparable to that of the blood-side methods. New techniques employing dialysate urea analysis should include a concentration profile to avoid these inherent methodological errors and assure the accuracy of eKt/V and SRI.

Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery.

H Zhou — 2006-03-01T14:57:01-00:00

Kidney Int (22 February 2006)

Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80 degrees C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20 degrees C caused a major loss in exosomes compared to fresh urine. In contrast, recovery after freezing at -80 degrees C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80 degrees C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80 degrees C with extensive vortexing after thawing maximizes the recovery of urinary exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts.Kidney International advance online publication, 22 February 2006; doi:10.1038/sj.ki.5000273.

Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator.

A Cheruvanky — 2007-06-27T02:41:15-00:00

Am J Physiol Renal Physiol, Vol. 292, No. 5. (May 2007)

Urinary exosomes are excreted from all nephron segments and may serve as biomarkers for classifying renal diseases. Isolation of urinary exosomes by the established ultracentrifugation method has some limitations for use in a clinical laboratory. We sought a rapid and simple way to obtain urinary exosomes. We used a commercially available nanomembrane concentrator to enrich exosomes from urine by centrifugation at 3,000 g for 10-30 min. Urinary exosomal markers tumor susceptibility gene 101, aquaporin-2, neuron-specific enolase, annexin V, angiotensin-converting enzyme, and podocalyxin (PODXL) were recovered from the nanomembrane concentrator and detected by Western blotting, and typical features of urinary vesicles were found by electron microscopy. Exosomal markers were detected in as little as 0.5 ml of urine. By the nanomembrane method, exosomal proteins could be recovered from urine samples frozen at -80 degrees C or refrigerated overnight at 4 degrees C then stored at -80 degrees C. By enriching exosomes we could detect PODXL, a podocyte marker, which decreased by 71% in five male patients with focal segmental glomerulosclerosis and abundant proteinuria. We conclude that 1) use of a nanomembrane concentrator simplifies and accelerates the enrichment of urinary exosomes; and 2) the nanomembrane concentrator can concentrate exosomal proteins from clinical urine samples. This enhanced method may accelerate the translation of urinary exosomal biomarkers from bench to bedside for the diagnosis, classification, and prognostication of renal diseases.

Exosomal Fetuin-A identified by proteomics: a novel urinary biomarker for detecting acute kidney injury.

H Zhou — 2007-06-27T02:33:16-00:00

Kidney Int, Vol. 70, No. 10. (November 2006), pp. 1847-1857.

Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We aimed to discover biomarkers in urinary exosomes to detect acute kidney injury (AKI), which has a high mortality and morbidity. Animals were injected with cisplatin. Urinary exosomes were isolated by differential centrifugation. Protein changes were evaluated by two-dimensional difference in gel electrophoresis and changed proteins were identified by mass spectrometry. The identified candidate biomarkers were validated by Western blotting in individual urine samples from rats subjected to cisplatin injection; bilateral ischemia and reperfusion (I/R); volume depletion; and intensive care unit (ICU) patients with and without AKI. We identified 18 proteins that were increased and nine proteins that were decreased 8 h after cisplatin injection. Most of the candidates could not be validated by Western blotting. However, exosomal Fetuin-A increased 52.5-fold at day 2 (1 day before serum creatinine increase and tubule damage) and remained elevated 51.5-fold at day 5 (peak renal injury) after cisplatin injection. By immunoelectron microscopy and elution studies, Fetuin-A was located inside urinary exosomes. Urinary Fetuin-A was increased 31.6-fold in the early phase (2-8 h) of I/R, but not in prerenal azotemia. Urinary exosomal Fetuin-A also increased in three ICU patients with AKI compared to the patients without AKI. We conclude that (1) proteomic analysis of urinary exosomes can provide biomarker candidates for the diagnosis of AKI and (2) urinary Fetuin-A might be a predictive biomarker of structural renal injury.

Prospects for urinary proteomics: Exosomes as a source of urinary biomarkers (Review Article)

Ewou Hoorn — 2005-06-09T10:22:18-00:00

Nephrology, Vol. 10, No. 3. (June 2005), pp. 283-290.