Blood Kidney Injury Molecule-1 Is a Biomarker of Acute and Chronic Kidney Injury and Predicts Progression to ESRD in Type I Diabetes

Currently, no blood biomarker that specifically indicates injury to the proximal tubule of the kidney has been identified. Kidney injury molecule-1 (KIM-1) is highly upregulated in proximal tubular cells following kidney injury. The ectodomain of KIM-1 is shed into the lumen, and serves as a urinary biomarker of kidney injury. We report that shed KIM-1 also serves as a blood biomarker of kidney injury. Sensitive assays to measure plasma and serum KIM-1 in mice, rats, and humans were developed and validated in the current study. Plasma KIM-1 levels increased with increasing periods of ischemia (10, 20, or 30 minutes) in mice, as early as 3 hours after reperfusion; after unilateral ureteral obstruction (day 7) in mice; and after gentamicin treatment (50 or 200 mg/kg for 10 days) in rats. In humans, plasma KIM-1 levels were higher in patients with AKI than in healthy controls or post-cardiac surgery patients without AKI (area under the curve, 0.96). In patients undergoing cardiopulmonary bypass, plasma KIM-1 levels increased within 2 days after surgery only in patients who developed AKI (P<0.01). Blood KIM-1 levels were also elevated in patients with CKD of varous etiologies. In a cohort of patients with type 1 diabetes and proteinuria, serum KIM-1 level at baseline strongly predicted rate of eGFR loss and risk of ESRD during 5–15 years of follow-up, after adjustment for baseline urinary albumin-to-creatinine ratio, eGFR, and Hb1Ac. These results identify KIM-1 as a blood biomarker that specifically reflects acute and chronic kidney injury.     

Urinary calprotectin,NGAL, and KIM-1 in the differentiation of primarily inflammatory vs. non-inflammatory stable chronic kidney diseases

Introduction It has been demonstrated that urinary neutrophil gelatinase-associated lipocalin (NGAL) and calprotectin are helpful biomarkers in the differentiation of intrinsic and prerenal acute kidney injury.Objective The present cross-sectional study investigates, whether urinary biomarkers are able to differentiate primarily inflammatory from non-inflammatory entities in chronic kidney disease (CKD).Methods Urinary calprotectin, NGAL, and kidney injury molecule-1 (KIM-1) concentrations were assessed in a study population of 143 patients with stable CKD and 29 healthy controls. Stable renal function was defined as an eGFR fluctuation ≤5 ml/min/1.73 m² in the past 12 months. Pyuria, metastatic carcinoma, and renal transplantation were regarded as exclusion criteria. Diabetic nephropathy, hypertensive nephropathy, and polycystic kidney disease were categorized as ‘primarily non-inflammatory renal diseases’ (NIRD), whereas glomerulonephritis and vasculitis were regarded as ‘primarily inflammatory renal diseases’ (IRD).Results Urinary calprotectin and NGAL concentrations significantly differed between CKD and healthy controls (p < 0.05 each), whereas KIM-1 concentrations did not (p = 0.84). The three biomarkers did neither show significant differences in-between the individual entities, nor the two categories of IRD vs. NIRD (calprotectin 155.7 vs. 96.99 ng/ml; NGAL 14 896 vs. 11 977 pg/ml; KIM-1 1388 vs. 1009 pg/ml; p > 0.05 each). Albumin exceeds the diagnostic power of the investigated biomarkers by far.Conclusions The urinary biomarkers calprotectin, NGAL, and KIM-1 have no diagnostic value in the differentiation of primarily inflammatory vs. non-inflammatory etiologies of CKD.     

Effect of Pentoxifylline on Renal Function and Urinary Albumin Excretion in Patients with Diabetic Kidney Disease: The PREDIAN Trial

Diabetic kidney disease (DKD) is the leading cause of ESRD. We conducted an open-label, prospective, randomized trial to determine whether pentoxifylline (PTF), which reduces albuminuria, in addition to renin-angiotensin system (RAS) blockade, can slow progression of renal disease in patients with type 2 diabetes and stages 3–4 CKD. Participants were assigned to receive PTF (1200 mg/d) (n=82) or to a control group (n=87) for 2 years. All patients received similar doses of RAS inhibitors. At study end, eGFR had decreased by a mean±SEM of 2.1±0.4 ml/min per 1.73 m² in the PTF group compared with 6.5±0.4 ml/min per 1.73 m² in the control group, with a between-group difference of 4.3 ml/min per 1.73 m² (95% confidence interval [95% CI], 3.1 to 5.5 ml/min per 1.73 m²; P<0.001) in favor of PTF. The proportion of patients with a rate of eGFR decline greater than the median rate of decline (0.16 ml/min per 1.73 m² per month) was lower in the PTF group than in the control group (33.3% versus 68.2%; P<0.001). Percentage change in urinary albumin excretion was 5.7% (95% CI, −0.3% to 11.1%) in the control group and −14.9% (95% CI, −20.4% to −9.4%) in the PTF group (P=0.001). Urine TNF-α decreased from a median 16 ng/g (interquartile range, 11–20.1 ng/g) to 14.3 ng/g (interquartile range, 9.2–18.4 ng/g) in the PTF group (P<0.01), with no changes in the control group. In this population, addition of PTF to RAS inhibitors resulted in a smaller decrease in eGFR and a greater reduction of residual albuminuria.     

Delayed Skeletal Muscle Mitochondrial ADP Recovery in Youth With Type 1 Diabetes Relates to Muscle Insulin Resistance

Insulin resistance (IR) increases cardiovascular morbidity and is associated with mitochondrial dysfunction. IR is now recognized to be present in type 1 diabetes; however, its relationship with mitochondrial function is unknown. We determined the relationship between IR and muscle mitochondrial function in type 1 diabetes using the hyperinsulinemic-euglycemic clamp and ³¹P-MRS before, during, and after near-maximal isometric calf exercise. Volunteers included 21 nonobese adolescents with type 1 diabetes and 17 nondiabetic control subjects with similar age, sex, BMI, Tanner stage, and activity levels. We found that youths with type 1 diabetes were more insulin resistant (median glucose infusion rate 10.1 vs. 18.9 mg/kglean/min; P < 0.0001) and had a longer time constant of the curve of ADP conversion to ATP (23.4 ± 5.3 vs. 18.8 ± 3.9 s, P < 0.001) and a lower rate of oxidative phosphorylation (median 0.09 vs. 0.21 mmol/L/s, P < 0.001). The ADP time constant (β = −0.36, P = 0.026) and oxidative phosphorylation (β = 0.02, P < 0.038) were related to IR but not HbA_1c. Normal-weight youths with type 1 diabetes demonstrated slowed postexercise ATP resynthesis and were more insulin resistant than control subjects. The correlation between skeletal muscle mitochondrial dysfunction in type 1 diabetes and IR suggests a relationship between mitochondrial dysfunction and IR in type 1 diabetes.     

Effects of High-Dose Creatine Supplementation on Kidney and Liver Responses In Sedentary and Exercised Rats

This study evaluated the effects of high-dose of short-term creatine supplementation (5g.kg^-1.day^-1 to 1 week) and long-term creatine supplementation (1g.kg^-1. day^-1 to 4-8 weeks) on kidney and liver structure and function of sedentary and exercised Wistar rats (Exercise sessions consisted of swimming at 80% of maximal work load supported during 5 days per week with daily sessions of 60 minutes throughout the duration of the supplementation). Seventy- two animals (245 ± 5g) were divided into four groups (n = 18): control diet Sedentary (SED), Creatine diet Sedentary (CRE), control diet Exercised (EXE), and Creatine diet Exercised (EXECRE). Histological and blood biochemical studies were performed after one, four, and eight weeks of creatine supplementation and exercise (n = 6). No differences were found when comparing SED, EXE and EXECRE groups for kidney and liver structure and function at one, four and eight weeks. However, the CRE group showed higher levels of creatinine (1.1 ± 0.2 vs. 0.4 ± 0.1 mg.dl^-1; p < 0.05), and urea (37 ± 3 vs. 19 ± 1 mg.dl^-1; p < 0.05) when compared with all others groups at four and eight weeks. At eight weeks, the CRE group presented increased levels of ALT (41 ± 7 vs. 23 ± 7 U.L^-1; p < 0.05), AST (89 ± 6 vs. 62 ± 5 U.L^-1; p < 0.05), GGT (8.0 ± 0.9 vs. 3.9 ± 1.0 U.L^-1; p < 0.05), and AP (125 ± 10 vs. 69 ± 9 U.L^-1; p < 0.05) also when compared with all others groups. Moreover, the CRE group demonstrated some structural alterations indicating renal and hepatic damage at four and eight weeks, respectively. These results suggest that long-term creatine supplementation (up to 4-8 weeks) may adversely affect kidney and liver structure and function of sedentary but not of exercised rats.

Key points

• Creatine supplementation is an established ergogenic aid in sports and is now claimed to have therapeutical applications in a variety of diseases.
• Although acknowledged, this nutritional supplement is rarely monitored precisely about their possible side effects.
• Previous studies indicated that short-term creatine supplementation associate with the physical exercise may be safe, but the effect of long-term creatine supplementation is still unknown.
• There is a need for further research to elucidate the controversial points refers to renal and hepatic function after creatine supplementation.
• The results of the current study indicate that supraphysiological long-term creatine supplementation (up to 4-8 weeks) may adversely affect kidney and liver structure and function of sedentary but not of exercised rats.

Key words: Creatine monohydrate, hepatic metabolism, kidney metabolism, swimming training, sports supplements, toxicology.     

Influence of the Burn Wound on Local and Systemic Responses to Injury

Total resting leg blood flow, measured by venous occlusion plethysmography; leg oxygen consumption; substrate turnover; and leg surface temperature were determined in 21 nonseptic burn patients and four normals. The patients studied during the second to third week postinjury sustained total body surface injuries averaging 45% (range 12-86%) and leg injuries of 35% total leg surface (0-82.5%). To integrate the peripheral metabolic and circulatory events with the systemic responses to injury, total body oxygen consumption, cardiac output, rectal and mean skin temperatures were also measured. Leg blood flow and leg surface temperature generally increased with total burn size but did not correlate with cardiac output, total body oxygen consumption, or body temperature. However, leg blood flow was closely related to the extent of the leg burn (r² = 0.73). To evaluate the metabolic determinants of the wound blood flow, patients were matched for burn size (40.5% total body surface in one group vs. 42%), resulting in similar systemic responses to injury (cardiac index 7.8 ± 0.7 L/min m² vs. 7.5 ± 0.8, VO₂ 204 ± 12 ml/min m² vs. 241 ± 22, rectal temperature 38.5 ± 0.3° vs. 38.3 ± 0.3°, NS). One group (n = 7) had extensive leg burns (58% of the leg surface), the other (n = 9) minimal leg injuries (9.5%). Leg oxygen consumption was similar in the two groups (0.24 ± 0.01 ml/100 ml leg min vs. 0.19 ± 0.04, NS), although leg blood flow was markedly increased in the injured extremities (8.0 ± 0.5 ml/100 ml leg min vs. 4.2 ± 0.4, p < 0.001). Glucose uptake and lactate production were enhanced in the burned extremities (glucose 0.34 ± 0.08 mg/100 ml leg mmn vs. 0.04 ± 0.03, p < 0.01, lactate 0.30 ± 0.08 mg/100 ml leg min vs. 0.06 ± 0.06, p < 0.05) and related in a general manner with size of the leg burn. Increased peripheral blood flow following injury is directed to the wound and unrelated to aerobic metabolic demands of the extremity. The selectively perfused wound consumes glucose and produces lactate. The increased systemic cardiovascular and metabolic responses to thermal injury are essential for the enhanced circulatory and anaerobic demands of the healing wound.     

Underestimation of Bone Loss of the Spine With Posterior-Anterior Dual-Energy X-Ray Absorptiometry in Patients With Spinal Cord Injury

Background:

Bone mineral density (BMD) of the lumbar spine (L-spine) has been reported to be normal by routine posterior-anterior (PA) bone density imaging in patients with chronic spinal cord injury (SCI).

Objective:

To determine BMD of the L-spine by PA and lateral (LAT) dual-energy radiographic absorptiometry (DXA) in patients with chronic SCI.

Design:

Prospective study.

Setting:

Veterans Affairs Medical Center and a private rehabilitation facility.

Methods:

Measurements of the PA and LAT L-spine and hip were performed in 15 patients with SCI: 9 with tetraplegia and 6 with paraplegia. The DXA (GE Lunar Advance DXA) images were obtained using standard software. Results are reported as mean ± SD.

Results:

The mean age was 35 ± 15 years (range  =  20–62 years), and the duration of injury was 57 ± 74 months (range  =  3–240 months). T- and Z-scores were lower for the LAT L-spine than those for PA L-spine (T-scores L2: −0.7 ± 1.2 vs 0.0 ± 1.4, P < 0.01; L3: −0.9 ± 1.6 vs 0.3 ± 1.3, P < 0.002; L2-L3: −0.8 ± 1.3 vs 0.2 ± 1.3, P < 0.001; Z-scores L2: −0.3 ± 1.1 vs 0.2 ± 1.2, P < 0.05; L3: −0.6 ± 1.3 vs 0.5 ± 1.3, P < 0.01; L2-L3: −0.4 ± 1.1 vs 0.4 ± 1.2, P < 0.005). The T- and Z-scores for the total hip (−1.1 ± 1.0 and −1.0 ± 1.0, respectively) and L2-L3 LAT L-spine demonstrated remarkable similarity, whereas the L2-L3 PA L-spine scores were not reduced. Bone mineral density of the LAT L-spine, but not the PA L-spine, was significantly reduced with increasing duration of injury.

Conclusions:

Individuals with SCI may have bone loss of the L-spine that is evident on LAT DXA that may be misdiagnosed by PA DXA, underestimating the potential risk of fracture.     

Sphingomyelinase-Like Phosphodiesterase 3b Expression Levels Determine Podocyte Injury Phenotypes in Glomerular Disease

Diabetic kidney disease (DKD) is the most common cause of ESRD in the United States. Podocyte injury is an important feature of DKD that is likely to be caused by circulating factors other than glucose. Soluble urokinase plasminogen activator receptor (suPAR) is a circulating factor found to be elevated in the serum of patients with FSGS and causes podocyte αVβ₃ integrin-dependent migration in vitro. Furthermore, αVβ₃ integrin activation occurs in association with decreased podocyte-specific expression of acid sphingomyelinase-like phosphodiesterase 3b (SMPDL3b) in kidney biopsy specimens from patients with FSGS. However, whether suPAR-dependent αVβ₃ integrin activation occurs in diseases other than FSGS and whether there is a direct link between circulating suPAR levels and SMPDL3b expression in podocytes remain to be established. Our data indicate that serum suPAR levels are also elevated in patients with DKD. However, unlike in FSGS, SMPDL3b expression was increased in glomeruli from patients with DKD and DKD sera-treated human podocytes, where it prevented αVβ₃ integrin activation by its interaction with suPAR and led to increased RhoA activity, rendering podocytes more susceptible to apoptosis. In vivo, inhibition of acid sphingomyelinase reduced proteinuria in experimental DKD but not FSGS, indicating that SMPDL3b expression levels determined the podocyte injury phenotype. These observations suggest that SMPDL3b may be an important modulator of podocyte function by shifting suPAR-mediated podocyte injury from a migratory phenotype to an apoptotic phenotype and that it represents a novel therapeutic glomerular disease target.     

Four-and-a-Half LIM Domains Protein 2 Is a Coactivator of Wnt Signaling in Diabetic Kidney Disease

Diabetic kidney disease (DKD) is a microvascular complication that leads to kidney dysfunction and ESRD, but the underlying mechanisms remain unclear. Podocyte Wnt-pathway activation has been demonstrated to be a trigger mechanism for various proteinuric diseases. Notably, four-and-a-half LIM domains protein 2 (FHL2) is highly expressed in urogenital systems and has been implicated in Wnt/β-catenin signaling. Here, we used in vitro podocyte culture experiments and a streptozotocin-induced DKD model in FHL2 gene-knockout mice to determine the possible role of FHL2 in DKD and to clarify its association with the Wnt pathway. In human and mouse kidney tissues, FHL2 protein was abundantly expressed in podocytes but not in renal tubular cells. Treatment with high glucose or diabetes-related cytokines, including angiotensin II and TGF-β1, activated FHL2 protein and Wnt/β-catenin signaling in cultured podocytes. This activation also upregulated FHL2 expression and promoted FHL2 translocation from cytosol to nucleus. Genetic deletion of the FHL2 gene mitigated the podocyte dedifferentiation caused by activated Wnt/β-catenin signaling under Wnt-On, but not under Wnt-Off, conditions. Diabetic FHL2^+/+ mice developed markedly increased albuminuria and thickening of the glomerular basement membrane compared with nondiabetic FHL2^+/+ mice. However, FHL2 knockout significantly attenuated these DKD-induced changes. Furthermore, kidney samples from patients with diabetes had a higher degree of FHL2 podocyte nuclear translocation, which was positively associated with albuminuria and progressive renal function deterioration. Therefore, we conclude that FHL2 has both structural and functional protein-protein interactions with β-catenin in the podocyte nucleus and that FHL2 protein inhibition can mitigate Wnt/β-catenin–induced podocytopathy.     

Adaptation of Insulin Clearance to Metabolic Demand Is a Key Determinant of Glucose Tolerance

With the development of insulin resistance (IR), there is a compensatory increase in the plasma insulin response to offset the defect in insulin action to maintain normal glucose tolerance. The insulin response is the result of two factors: insulin secretion and metabolic clearance rate of insulin (MCR_I). Subjects (104 with normal glucose tolerance [NGT], 57 with impaired glucose tolerance [IGT], and 207 with type 2 diabetes mellitus [T2DM]), divided in nonobese and obese groups, received a euglycemic insulin-clamp (40 mU/m² ⋅ min) and an oral glucose tolerance test (OGTT) (75 g) on separate days. MCR_I was calculated during the insulin-clamp performed with [3-³H]glucose and the OGTT and related to IR: peripheral (glucose uptake during the insulin clamp), hepatic (basal endogenous glucose production × fasting plasma insulin [FPI]), and adipocyte (fasting free fatty acid × FPI). MCR_I during the insulin clamp was reduced in obese versus nonobese NGT (0.60 ± 0.03 vs. 0.73 ± 0.02 L/min ⋅ m², P < 0.001), in nonobese IGT (0.62 ± 0.02, P < 0.004), and in nonobese T2DM (0.68 ± 0.02, P < 0.03). The MCR_I during the insulin clamp was strongly and inversely correlated with IR (r = −0.52, P < 0.0001). During the OGTT, the MCR_I was suppressed within 15–30 min in NGT and IGT subjects and remained suppressed. In contrast, suppression was minimal in T2DM. In conclusion, the development of IR in obese subjects is associated with a decline in MCR_I that represents a compensatory response to maintain normal glucose tolerance but is impaired in individuals with T2DM.     

Reduced Production of Creatinine Limits Its Use as Marker of Kidney Injury in Sepsis

Although diagnosis and staging of acute kidney injury uses serum creatinine, acute changes in creatinine lag behind both renal injury and recovery. The risk for mortality increases when acute kidney injury accompanies sepsis; therefore, we sought to explore the limitations of serum creatinine in this setting. In mice, induction of sepsis by cecal ligation and puncture in bilaterally nephrectomized mice increased markers of nonrenal organ injury and serum TNF-α. Serum creatinine, however, was significantly lower in septic animals than in animals subjected to bilateral nephrectomy and sham cecal ligation and puncture. Under these conditions treatment with chloroquine decreased nonrenal organ injury markers but paradoxically increased serum creatinine. Sepsis dramatically decreased production of creatinine in nephrectomized mice, without changes in body weight, hematocrit, or extracellular fluid volume. In conclusion, sepsis reduces production of creatinine, which blunts the increase in serum creatinine after sepsis, potentially limiting the early detection of acute kidney injury. This may partially explain why small absolute increases in serum creatinine levels are associated with poor clinical outcomes. These data support the need for new biomarkers that provide better measures of renal injury, especially in patients with sepsis.Serum creatinine is used clinically to detect and evaluate acute kidney injury (AKI) and chronic kidney disease (CKD),^1,2 although the limitations of serum creatinine for the early detection and accurate estimation of renal injury are widely known. In AKI, serum creatinine does not accurately reflect the GFR because the patient is not in steady state.³ Furthermore, serum creatinine is also influenced by tubular creatinine secretion and by nonrenal factors such as muscle mass, liver function, and nonrenal (gastrointestinal) elimination.Sepsis remains a serious problem in critically ill patients, and mortality from sepsis is increased dramatically when complicated by AKI; therefore, early detection and accurate evaluation of AKI is important in patients with sepsis. We modified the cecal ligation and puncture (CLP) mouse sepsis model to make it more clinically relevant, and our model developed sepsis-induced AKI.^4–6 We found that serum creatinine increased to a lesser extent in CLP (approximately 0.5 mg/dl) than ischemia/reperfusion or cisplatin administration (>1 mg/dl) within 24 h of injury. To explore nonrenal factors that might account for this disparity, we used bilateral nephrectomy (BNx), a technique used recently by others to study the contribution of the kidney to cytokine metabolism and acute lung injury.⁷First, we evaluated sepsis in bilaterally nephrectomized mice. We induced sepsis by CLP surgery with 8-mm cecal ligation, which causes modest sublethal sepsis in normal outbred CD-1 mice.⁸ All animals survived until they were killed at 18 h after surgery. CLP surgery in non-nephrectomized mice caused a numerically small, but not significant increase of serum creatinine (sham_BNx+CLP group). As expected, we found large increases of serum creatinine in the BNx+sham_CLP group; however, induction of sepsis at the time of BNx significantly decreased serum creatinine (BNx+CLP group) compared with nonseptic BNx alone (BNx+sham_CLP group; Figure 1A), raising doubt about whether serum creatinine accurately reflects impaired kidney function during sepsis. In contrast, nonrenal organ injury markers (aspartate aminotransferase [AST], alanine aminotransferase [ALT], and lactate dehydrogenase [LDH]) and serum TNF-α were higher in the BNx+CLP group than in the BNx+sham_CLP group, confirming the presence of severe sepsis (Figure 1, B through E).Open in a separate windowFigure 1.BNx and subsequent sepsis induced by CLP. (A through E) Serum creatinine (A), AST (B), ALT (C), LDH (D), and serum TNF-α (E) were measured at 18 h after surgery (n = 5 to 6 per group). Data are means ± SEM. *P < 0.05 versus Sham BNx/Sham CLP; #P < 0.05 versus BNx.We next examined whether treatment of sepsis would affect this observation by using chloroquine, which decreases mortality and improves organ function in a model of CLP-induced sepsis.⁹ Chloroquine treatment of septic bilaterally nephrectomized mice (BNx+CLP+CQ) decreased nonrenal organ injury markers (AST, ALT, and LDH) and TNF-α compared with vehicle-treated mice (BNx+CLP); however, serum creatinine was paradoxically higher in treated animals (Figure 2). As a control, chloroquine did not cause any changes in the BNx+sham_CLP group. We reported recently that chloroquine reduced mortality and sepsis-induced AKI, including renal pathologic damage.⁹ At first glance, these results seem contradictory: Serum creatinine is reduced by chloroquine when kidneys are intact yet increased by chloroquine in the absence of any kidneys. In the septic state, chloroquine alters nonrenal metabolism of creatinine, which is confirmed by completely removing urinary creatinine excretion by BNx; therefore, in both untreated and treated septic mice, serum creatinine changes in the opposite direction as markers of injury from other organs, challenging the underlying assumptions behind the use of serum creatinine as a biomarker for AKI.Open in a separate windowFigure 2.Effects of chloroquine treatment on BNx+CLP. (A through E) Serum creatinine (A), AST (B), ALT (C), LDH (D), and serum TNF-α (E) were measured at 18 h after surgery (n = 8 to 9 per each BNx group; n = 5 per sham group). Sham is Sham BNx/Sham CLP. Data are mean ± SEM. #P < 0.05 versus BNx+CLP.To determine whether changes in fluid compartments could account for these observations, we measured body weight changes and hematocrit, as well as volume of distribution (Vd) for FITC-inulin and creatinine. There was no significant difference in body weight change or hematocrit (body weight change: BNx+sham_CLP 2.3 ± 1.0% [n = 5], BNx+CLP 1.7 ± 0.6% [n = 5]; hematocrit: BNx+sham_CLP 36.0 ± 0.9% [n = 6], BNx+CLP 34.6 ± 1.3% [n = 6]). Creatinine space, a marker of total body water, was not different between the BNx+CLP and the BNx+sham_CLP groups (Figure 3A). FITC-inulin space, a marker of extracellular fluid volume, was only modestly decreased in the BNx+CLP group (Figure 3A). We then estimated creatinine production from the increase in serum creatinine between time 0 and 14 h and the creatinine Vd. We found that sepsis significantly decreased estimated creatinine production by 29.7 ± 4.3% (Figure 3B). Direct measurement of creatinine production¹⁰ would be necessary to confirm our result.Open in a separate windowFigure 3.Volume of distribution (Vd) and estimated creatinine production. (A) Vd of creatinine and FITC-inulin were measured from 14 to 18 h after surgery. (B) Estimated creatinine production was measured at 0 to 14 h after surgery. Data are means ± SEM (n = 5 to 6 per group). #P < 0.05 versus BNx.Creatinine is the end product of creatine metabolism. Alteration in creatinine production can alter serum creatinine levels. For example, N-acetylcysteine, which has been shown to protect from radiocontrast medium–induced nephropathy,¹¹ decreases serum creatinine levels of healthy humans without changing serum cystatin C levels.¹² This suggests that N-acetylcysteine directly reduces creatinine production. We suspected that creatinine production was reduced, on the basis of the small increase in serum creatinine after CLP sepsis versus renal ischemia-reperfusion. In this study, we found that sepsis reduced creatinine production, which would largely account for the slow rise in serum creatinine. Serum creatinine could fall as a result of reduced production or renal or extrarenal clearance (creatinine degradation or gastrointestinal excretion).¹⁰ Creatine is synthesized from guanidinoacetic acid primarily in the liver,¹³ enters skeletal muscle via a membrane transporter, and accumulates there because it is phosphorylated by creatine kinase. Creatine is converted into creatinine by a nonenzymatic cyclization throughout the body but especially in skeletal muscle as a result of the high abundance of creatine. Creatinine production can fall because of reductions in lean body mass, dietary intake of creatine, or liver disease.¹⁴ Indeed, the BNx+CLP group showed significantly higher liver enzymes and pathologic changes such as extensive loss of hepatocyte glycogen stores and bland cytoplasm (data not shown).⁴ Intensive care unit patients have a progressive decline in creatinine production as a result of a loss of muscle mass worsened by subclinical hepatic injury.¹⁵Sepsis reduces energy production and metabolic rate because of hormonal and inflammatory mediators,¹⁶ which could reduce muscle production of creatinine. As we previously reported, CLP induced severe septic shock with hypodynamic circulation failure and reduced microvascular capillary perfusion,^6,8,17 which could reduce muscle creatinine release, liver creatine-to-creatinine conversion, and/or release into the circulation. In this study, the numerical decrease in creatinine space in the BNx+CLP group is consistent with reduced cellular perfusion in sepsis. Because our CLP model has a prolonged period of hypodynamic shock,⁸ our results may not translate to patients with hyperdynamic shock. Examination of creatinine production in hyperdynamic animal sepsis models^18–20 would be needed to determine the extent that microvascular perfusion may contribute to the decrease in creatinine production during hyperdynamic sepsis.Sepsis-induced hypothermia may also decrease nonenzymatic conversion of creatine to creatinine. CLP sepsis causes profound hypothermia, which is a marker of reduced metabolism.²¹ In this study, we found that the severe hypothermia in the BNx+CLP animals was attenuated by chloroquine treatment (data not shown). Creatinine is converted from creatine and creatine phosphate nonenzymatically, and the rate of nonenzymatic conversion of creatine to creatinine depends on the pH and temperature; a 3°C decrease would reduce this conversion by 15 to 20%.²² In addition, decreasing metabolism by systemic injection of drugs such as 5′-adenosine monophosphate (AMP)²³ or 2-deoxy-d-glucose (2-DG)²⁴ instead of sepsis to bilaterally nephrectomized mice also caused hypothermia and decreases of serum creatinine 18 h after surgery (Supplemental Figure 1).Our findings may have direct clinical significance. As a result of decreased creatinine production, serum creatinine is an even poorer indicator of renal damage in sepsis because its reduced production further magnifies the kinetic disparity between apparent and actual renal function; serum creatinine underestimates renal damage to a greater extent in sepsis than in other forms of AKI. The reduced creatinine production may also explain why such small increases in serum creatinine are associated with dramatic increases in morbidity and mortality of human patients.^25,26 Higher serum creatinine was paradoxically associated with better survival in AKI in several clinical studies.^27–29 Underestimation of renal function changes by “inappropriately low” serum creatinine will delay the early diagnosis of AKI, impede recognition of an additional organ injury for prognostic purposes, and suppress entry into clinical trials. Although the timing of renal replacement therapy initiation (including prophylactic dialysis)³⁰ is controversial, renal replacement therapy may need to be started at relatively lower serum creatinine than other types of AKI.The paradoxic effect of chloroquine on creatinine in bilaterally nephrectomized animals also has implications for clinical trial design. Chloroquine improved sepsis and sepsis-induced AKI by survival analysis and pathologic examination.⁹ We hypothesize that chloroquine restores creatinine metabolism indirectly by reducing the severity of sepsis, perhaps by improvement in muscle metabolism and/or liver function. Although serum creatinine is widely used as an end point for AKI clinical studies, the effects of treatment of sepsis and sepsis-induced AKI might be incorrectly ascertained if kidney damage in sepsis is evaluated only by serum creatinine. Confirmation with other end points such as injury biomarkers and mortality and morbidity rate should be required, especially for clinical studies on sepsis.In conclusion, we demonstrated that sepsis reduced creatinine production, thereby blunting the expected increases of serum creatinine in bilaterally nephrectomized mice. Moreover, treatment with chloroquine reduced improved sepsis but paradoxically increased serum creatinine in bilaterally nephrectomized mice, suggesting a normalization of creatinine production. Our data indicate that evaluation of kidney injury by serum creatinine alone would cause a severe underestimation of renal injury, serious failure of early diagnosis of sepsis-induced AKI, and incorrect ascertainment of drug effects. Newer biomarkers that more accurately measure renal injury are needed, especially in patients with sepsis.     

Clinical Predictors and Long-term Impact of Acute Kidney Injury on Progression of Diabetic Kidney Disease in Chinese Patients With Type 2 Diabetes

We aim to assess the long-term impact of acute kidney injury (AKI) on progression of diabetic kidney disease (DKD) and all-cause mortality and investigate determinants of AKI in Chinese patients with type 2 diabetes (T2D). A consecutive cohort of 9,096 Chinese patients with T2D from the Hong Kong Diabetes Register was followed for 12 years (mean ± SD age 57 ± 13.2 years; 46.9% men; median duration of diabetes 5 years). AKI was defined based on the Kidney Disease: Improving Global Outcomes (KDIGO) criteria using serum creatinine. Estimated glomerular filtration rate measurements were used to identify the first episode with chronic kidney disease (CKD) and end-stage renal disease (ESRD). Polygenic risk score (PRS) composed of 27 single nucleotide polymorphisms (SNPs) known to be associated with serum uric acid (SUA) in European populations was used to examine the role of SUA in pathogenesis of AKI, CKD, and ESRD. Validation was sought in an independent cohort including 6,007 patients (age 61.2 ± 10.9 years; 59.5% men; median duration of diabetes 10 years). Patients with AKI had a higher risk for developing incident CKD (hazard ratio 14.3 [95% CI 12.69–16.11]), for developing ESRD (12.1 [10.74–13.62]), and for all-cause death (7.99 [7.31–8.74]) compared with those without AKI. Incidence rate for ESRD among patients with no episodes of AKI and one, two, and three or more episodes of AKI was 7.1, 24.4, 32.4, and 37.3 per 1,000 person-years, respectively. Baseline SUA was a strong independent predictor for AKI. A PRS composed of 27 SUA-related SNPs was associated with AKI and CKD in both discovery and replication cohorts but not ESRD. Elevated SUA may increase the risk of DKD through increasing AKI. The identification of SUA as a modifiable risk factor and PRS as a nonmodifiable risk factor may facilitate the identification of individuals at high risk to prevent AKI and its long-term impact in T2D.     

Prognosis and risk factors of chronic kidney disease progression in patients with diabetic kidney disease and non-diabetic kidney disease: a prospective cohort CKD-ROUTE study

Diabetic kidney disease (DKD) is emerging rapidly as the leading cause of chronic kidney disease (CKD) worldwide. In this 3-year prospective, multicenter cohort study, a total of 1138 pre-dialysis CKD patients were recruited. Patients were categorized into two groups according to the etiologies of DKD and non-diabetic kidney disease (NDKD). Propensity score matching was performed to adjust for confounding factors, resulting in 197 patients being assigned to DKD and NDKD groups, respectively. The primary endpoints were 50% estimated glomerular filtration rate (eGFR) decline and initiation of kidney replacement therapy (KRT). The secondary endpoints were all-cause death and the development of cardiovascular disease (CVD) events. We found that DKD patients have a higher risk to develop 50% eGFR decline endpoint (HR:2.30, 95%CI [1.48–3.58], p < 0.001) and KRT endpoint (HR:1.64, 95%CI [1.13–2.37], p < 0.05) than NDKD patients. The 3-year cumulative incidence of 50% eGFR decline and KRT endpoint was significantly higher in DKD patients (26.90% vs.13.71% and 35.03% vs. 22.34%, respectively). The Cox regression analyses showed that the increased systolic blood pressure (SBP), DKD, decreased serum albumin (Alb), and higher CKD stages were risk factors for the 50% eGFR decline endpoint; the increased SBP, DKD, decreased serum Alb, serum creatinine (Scr), higher CKD stages, presence of proteinuria and CVD were risk factors for KRT endpoint; the increased age, decreased hemoglobin (Hb), decreased serum Alb were risk factors for all-cause death endpoint; the increased age, decreased serum Alb were risk factors for CVD events endpoint. Appropriate preventive or therapeutic interventions should be taken to control these predictive factors to delay the development of CKD complications, thereby improving the prognosis and reducing the disease burden of the high-risk populations.     

The Association of Predonation Hypertension with Glomerular Function and Number in Older Living Kidney Donors

The effect of preexisting hypertension on living donor nephron number has not been established. In this study, we determined the association between preexisting donor hypertension and glomerular number and volume and assessed the effect of predonation hypertension on postdonation BP, adaptive hyperfiltration, and compensatory glomerular hypertrophy. We enrolled 51 living donors to undergo physiologic, morphometric, and radiologic evaluations before and after kidney donation. To estimate the number of functioning glomeruli (N_FG), we divided the whole-kidney ultrafiltration coefficient (K_f) by the single-nephron ultrafiltration coefficient (SNK_f). Ten donors were hypertensive before donation. We found that, in donors ages >50 years old, preexisting hypertension was associated with a reduction in N_FG. In a comparison of 10 age- and sex-matched hypertensive and normotensive donors, we observed more marked glomerulopenia in hypertensive donors (N_FG per kidney, 359,499±128,929 versus 558,239±205,152; P=0.02). Glomerulopenia was associated with a nonsignificant reduction in GFR in the hypertensive group (89±12 versus 95±16 ml/min per 1.73 m²). We observed no difference in the corresponding magnitude of postdonation BP, hyperfiltration capacity, or compensatory renocortical hypertrophy between hypertensive and normotensive donors. Nevertheless, we propose that the greater magnitude of glomerulopenia in living kidney donors with preexisting hypertension justifies the need for long-term follow-up studies.     

Reduction in Bone Volume Resection with a Newer Posterior Stabilized Total Knee Arthroplasty Design

Background

Posterior stabilized total knee arthroplasty requires an intercondylar notch to accommodate the cam housing that articulates with the tibial post to create femoral rollback required for deep flexion. The volume of bone resected for the intercondylar notch varies with implant design, and newer designs may accommodate high flexion with less bone resection.

Questions/Purposes

This study aims to analyze the bone volume and density resected from the intercondylar notch for three posterior stabilized implants from a single company: a Posterior Stabilized (PS) system, a Hi-Flex system (HF), and a rounded new box-reamer (RB) system and to further assess whether the newer RB with a cylindrical cutting tool would preserve more native bone.

Materials and Methods

Using a computer model, the PS, HF, and RB femoral components were digitally implanted into CT scans of 19 cadaver femurs. Nine cadavers were fit with a size 4 implant, six with size 3, and four with a size 2. The volume of intercondylar bone resected digitally for femoral preparation was measured. Bone density was measured by CT scans in Hounsfield units (HU). A paired t test was used to compare the mean volume of bone resected for each implant.

Results

For the size 4 femurs, the newer RB design removed 8% less intercondylar bone than the PS design (7,832 ± 501 vs. 8,547 ± 377 mm³, p < 0.001) and 28% less bone than the HF design (7,832 ± 501 vs. 10,897 ± 444 mm³, p < 0.001). The average HU for size 4 femurs for RB design was 427 ± 72 (PS = 399 ± 69, p < 0.001; HF = 379 ± 66, p < 0.001). For the size 3 femurs, the RB design removed 12% less intercondylar bone than the PS (6,664 ± 786 vs. 7,516 ± 648 mm³, p < 0.001) and 27% less bone than the HF (6,664 ± 786 vs. 9,078 ± 713 mm³, p < 0.001). HU for size 3 femurs for the RB design was 452 ± 70 (PS = 422 ± 53, p < 0.1; HF = 410 ± 59, p < 0.01). For the size 2 femurs, the RB design removed 5% less intercondylar bone than the PS (5,730 ± 552 vs. 6,009 ± 472 mm³, p < 0.01) and 22% less bone than the HF (5,730 ± 552 vs. 7,380 ± 532 mm³, p < 0.001). HU for size 2 femurs for the RB design was 430 ± 48 (PS = 408 ± 55, p < 0.01; HF = 385 ± 56, p < 0.01).

Conclusions

The newer RB design removes less bone from the intercondylar notch than the classic PS and HF designs in all sizes tested. The bone-conserving cuts incorporated into this newer implant design appear to preserve native bone without compromising design objectives.

Electronic supplementary material

The online version of this article (doi:10.1007/s11420-013-9340-1) contains supplementary material, which is available to authorized users.     

Cycling Efficiency in Trained Male and Female Competitive Cyclists

The aim of this study was to examine differences in cycling efficiency between competitive male and female cyclists. Thirteen trained male (mean ± SD: 34 ± 8 yr, 74.1 ± 6.0 kg, Maximum Aerobic Power (MAP) 414 ± 40 W, VO₂max 61.3 ± 5.4 ml·kg^-1·min^-1) and 13 trained female (34 ± 9 yr, 60.1 ± 5.2 kg, MAP 293 ± 22 W, VO₂max 48.9 ± 6.1 ml·kg^-1·min^-1) competitive cyclists completed a cycling test to ascertain their gross efficiency (GE). Leg and lean leg volume of all cyclists was also measured. Calculated GE was significantly higher in female cyclists at 150W (22.5 ± 2.1 vs 19.9 ± 1. 8%; p < 0.01) and 180W (22.3 ± 1.8 vs 20.4 ± 1.5%; p = 0.01). Cadence was not significantly different between the groups (88 ± 6 vs 91 ± 5 rev·min^-1). Lean leg volume was significantly lower for female cyclists (4.04 ± 0.5 vs 5.51 ± 0.8 dm³; p < 0.01) and was inversely related to GE in both groups at 150 and 180W (r = -0.59 and -0.58; p < 0.05). Lean leg volume was shown to account for the differences in GE between the males and females. During an “unloaded ”pedalling condition, male cyclists had a significantly higher O₂ cost than female cyclists (1.0 ± 0.1 vs 0.7 ± 0.1 L·min^-1; p < 0.01), indicative of a greater non-propulsive cost of cycling. These results suggest that differences in efficiency between trained male and female cyclists can be partly accounted for by sex-specific variation in lean leg volume.

Key points

• Differences in GE exist between male and female cyclists.
• Males have a higher oxygen cost of “unloaded ”cycling, as predicted by the intercept of the O2 cost-power output relationship
• This suggests that in addition to work rate, leg volume/mass may be an important determinant of observed differences in oxygen cost and therefore GE, between male and female competitive cyclists.

Key words: Gross efficiency, endurance performance, sex-related differences, power output, leg volume     

Advanced glycation end-product expression is upregulated in the gastrointestinal tract of type 2 diabetic rats

AIM:To investigate changes in advanced glycation end products(AGEs) and their receptor(RAGE) expression in the gastrointestinal(GI) tract in type 2 diabetic rats.METHODS:Eight inherited type 2 diabetic rats GotoKakizak(GK) and ten age-matched normal rats were used in the study.From 18 wk of age,the body weight and blood glucose were measured every week and 2 wk respectively.When the rats reached 32 wk,twocentimeter segments of esophagus,duodenum,jejunum,ileum,and colon were excised and the wet weight was measured.The segments were fixed in 10% formalin,embedded in paraffin and five micron sections were cut.The layer thickness was measured in Hematoxylin and Eosin-stained slides.AGE [N epsilon-(carboxymethyl) lysine and N epsilon-(carboxyethyl)lysine] and RAGE were detected by immunohistochemistry staining and image analysis was done using Sigmascan Pro 4.0 image analysis software.RESULTS:The blood glucose concentration(mmol/L) at 18 wk age was highest in the GK group(8.88 ± 1.87 vs 6.90 ± 0.43,P 0.001),a difference that continued to exist until the end of the experiment.The wet weight per unit length(mg/cm) increased in esophagus,jejunum and colon from the normal to the GK group(60.64 ± 9.96 vs 68.56 ± 11.69,P 0.05 for esophagus; 87.01 ± 9.35 vs 105.29 ± 15.45,P 0.01 for jejunum; 91.37 ± 7.25 vs 97.28 ± 10.90,P 0.05 for colon).Histologically,the layer thickness of the GItract was higher for esophagus,jejunum and colon in the GK group [full thickness(μm):575.37 ± 69.22 vs 753.20 ± 150.41,P 0.01 for esophagus; 813.51 ± 44.44 vs 884.81 ± 45.31,P 0.05 for jejunum; 467.12 ± 65.92 vs 572.26 ± 93.60,P 0.05 for colon].In esophagus,the AGE and RAGE mainly distributed in striated muscle cells and squamous epithelial cells.The AGE distribution was much stronger in the GK group compared to the normal group both in the striated muscle layer and mucosa layer(immuno-positive area/ total measuring area %:4.52 ± 0.89 vs 10.96 ± 1.34,P 0.01 for muscle; 8.90 ± 2.62 vs 22.45 ± 1.26,P 0.01 for mucosa).No visible difference was found for RAGE distribution between the two groups.In the intestine AGE and RAGE distributed in epithelial cells of villi and crypt.RAGE was also found in neurons in the myenteric and submucosal plexus.The intensity of AGE staining in mucosa of all segments and RAGE staining in neurons in all segments were strongest in the diabetes group.Significant difference for AGE was found in the epithelial cells of villi and crypt in duodenum(immunopositive area/total measuring area %:13.37 ± 3.51 vs 37.48 ± 8.43,P 0.05 for villi; 0.38 ± 0.12 vs 1.87 ± 0.53,P 0.05 for crypt) and for RAGE in neurons of all segments(e.g.,for jejunum:no staining neurons% 0 vs 0,mild 36.0 ± 5.2 vs 28.7 ± 3.5,moderate 53.2 ± 4.8 vs 55.8 ± 5.4,strong 10.7 ± 1.1 vs 15.4 ± 2.0,P 0.05).In the colon,RAGE was primarily found in neurons in the myenteric and submucosal plexus.It was stronger in the diabetes group than in the normal group(no staining neurons% 6.2 ± 0.2 vs 0.3 ± 0.04,mild 14.9 ± 2.1 vs 17.6 ± 1.5,moderate 53.1 ± 4.6 vs 44.7 ± 4.4,strong 25.6 ± 18 vs 43.6 ± 4.0,P 0.05).In the rectum,RAGE was primarily found in the mucosa epithelial cells.CONCLUSION:The AGE and RAGE expression was upregulated in the GI tract of GK diabetic rats and may contribute to GI dysfunction in type 2 diabetic patients.     

Increased Skeletal Muscle Capillarization Independently Enhances Insulin Sensitivity in Older Adults After Exercise Training and Detraining

Intramuscular signaling and glucose transport mechanisms contribute to improvements in insulin sensitivity after aerobic exercise training. This study tested the hypothesis that increases in skeletal muscle capillary density (CD) also contribute to exercise-induced improvements in whole-body insulin sensitivity (insulin-stimulated glucose uptake per unit plasma insulin [M/I]) independent of other mechanisms. The study design included a 6-month aerobic exercise training period followed by a 2-week detraining period to eliminate short-term effects of exercise on intramuscular signaling and glucose transport. Before and after exercise training and detraining, 12 previously sedentary older (65 ± 3 years) men and women underwent research tests, including hyperinsulinemic-euglycemic clamps and vastus lateralis biopsies. Exercise training increased Vo_2max (2.2 ± 0.2 vs. 2.5 ± 0.2 L/min), CD (313 ± 13 vs. 349 ± 18 capillaries/mm²), and M/I (0.041 ± 0.005 vs. 0.051 ± 0.007 μmol/kg fat-free mass/min) (P < 0.05 for all). Exercise training also increased the insulin activation of glycogen synthase by 60%, GLUT4 expression by 16%, and 5′ AMPK-α1 expression by 21%, but these reverted to baseline levels after detraining. Conversely, CD and M/I remained 15% and 18% higher after detraining, respectively (P < 0.05), and the changes in M/I (detraining minus baseline) correlated directly with changes in CD in regression analysis (partial r = 0.70; P = 0.02). These results suggest that an increase in CD is one mechanism contributing to sustained improvements in glucose metabolism after aerobic exercise training.     

Competitive Interaction Between Fibroblast Growth Factor 23 And Asymmetric Dimethylarginine in Patients With CKD

Both fibroblast growth factor 23 (FGF-23) and asymmetric dimethylarginine (ADMA) are associated with progression of CKD. We tested the hypothesis that ADMA and FGF23 are interactive factors for CKD progression in a cohort of 758 patients with CKD in Southern Europe (mean eGFR±SD, 36±13 ml/min per 1.73 m²) and in a central European cohort of 173 patients with CKD (MMKD study, mean eGFR, 64±39 ml/min per 1.73 m²). In the first cohort, 214 patients had renal events (decrease in eGFR of >30%, dialysis, or kidney transplantation) during a 3-year follow-up. Both intact FGF-23 and ADMA predicted the incidence rate of renal events in unadjusted and adjusted analyses (P<0.001). There was a strong competitive interaction between FGF-23 and ADMA in the risk of renal events (P<0.01 in adjusted analyses); the risk associated with raised ADMA levels was highest in patients with low FGF-23 levels. These results were confirmed in the MMKD cohort, in which FGF-23 level was again an effect modifier of the relationship between plasma ADMA level and renal events (doubling of baseline serum creatinine, dialysis, or kidney transplantation) in the adjusted analyses (P<0.01). Furthermore, in the MMKD cohort there was a parallel, independent competitive interaction between symmetric dimethylarginine level and c-terminal FGF-23 level for the risk for renal events (P=0.001). These findings indicate that the association of ADMA level with the risk of CKD progression is modified by FGF-23 level and provide further evidence that dysregulation of the nitric oxide system is involved in CKD progression.