The Emerging Role of Biomarkers in Diabetic and Hypertensive Chronic Kidney Disease

Currently used measures to assess kidney function and injury are largely inadequate. Markers such as serum creatinine, formulas to estimate glomerular filtration rate, cystatin C, and proteinuria largely identify an underlying disease process that is well established. Thus, there has been a recent effort to identify new biomarkers that reflect kidney function, early injury, and/or repair that ultimately can relate to progression or regression of damage. Several biomarkers emerged recently that are able to detect kidney damage earlier than is currently possible with traditional biomarkers such as serum creatinine and proteinuria. Identification of urine biomarkers has proven to be beneficial in recent years because of ease of handling, stability, and the ability to standardize the various markers to creatinine or other peptides generally already present in the urine. Recent markers such as neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), and podocin have garnered a lot of attention. The emergence of these and other biomarkers is largely because of the evolution of novel genomic and proteomic applications in investigations of acute kidney injury and chronic kidney disease. In this article, we focus on the applications of these biomarkers in disease.     

Pilot randomized, controlled trial of pramipexole to augment antipsychotic treatment

The preferential dopamine D(3)-agonist pramipexole (4.25±0.38 mg/day) or placebo were added for up to 12 weeks to ongoing antipsychotic treatment for 24 adult patients with DSM-IV schizophrenia or schizoaffective disorder. Pramipexole was generally well-tolerated (82% trial-completion), and yielded greater decreases in PANSS-total scores (drug/placebo=2.1; p=0.04), with similar decreases in PANSS positive and negative scores and 6.7-fold greater reduction of serum prolactin concentrations compared to placebo. There were no differences in ratings of mood, cognition or extrapyramidal symptoms, all of which were low at intake.     

Colony-Stimulating Factor-1 Promotes Kidney Growth and Repair via Alteration of Macrophage Responses

Colony-stimulating factor (CSF)-1 controls the survival, proliferation, and differentiation of macrophages, which are recognized as scavengers and agents of the innate and the acquired immune systems. Because of their plasticity, macrophages are endowed with many other essential roles during development and tissue homeostasis. We present evidence that CSF-1 plays an important trophic role in postnatal organ growth and kidney repair. Notably, the injection of CSF-1 postnatally enhanced kidney weight and volume and was associated with increased numbers of tissue macrophages. Moreover, CSF-1 promotes postnatal renal repair in mice after ischemia-reperfusion injury by recruiting and influencing macrophages toward a reparative state. CSF-1 treatment rapidly accelerated renal repair with tubular epithelial cell replacement, attenuation of interstitial fibrosis, and functional recovery. Analysis of macrophages from CSF-1-treated kidneys showed increased expression of insulin-like growth factor-1 and anti-inflammatory genes that are known CSF-1 targets. Taken together, these data suggest that CSF-1 is important in kidney growth and the promotion of endogenous repair and resolution of inflammatory injury.Macrophages are versatile cells that have been long recognized as immune effectors where their recruitment to sites of injury is a fundamental feature of inflammation. Although their role in host defense has been well documented, macrophages and their precursors are also important during embryogenesis, normal tissue maintenance, and postnatal organ repair.^1,2 Almost all developing organs contain a population of resident monocytes that infiltrate very early during organogenesis and persist throughout adult life.^3–6 In addition to their phagocytic capabilities during tissue remodeling-associated apoptosis,^5,7 fetal macrophages have many trophic effects that promote tissue and organ growth.^6,8,9Colony-stimulating factor (CSF)-1 controls the differentiation, proliferation, and survival of macrophages by binding to a high-affinity cell-surface tyrosine kinase receptor (CSF-1R), encoded by the c-fms proto-oncogene that is expressed on macrophages and their progenitors.⁶ CSF-1 is critical for both adult and embryonic macrophage development. This is manifested by multiple organ growth deficiencies observed in osteopetrotic (Csf1^op/Csf1^op) mice that have a spontaneous mutation in the csf-1 gene. These mice show growth restriction and developmental abnormalities of the bones, brain, and reproductive and endocrine organs,^10–13 a phenotype that can be rescued by injection of exogenous CSF-1 or insertion of a csf-1 transgene.^14–16In adult organs, there is considerable heterogeneity of monocytes and macrophages with distinct subsets defined by phenotype, function, and the differential expression of cell surface markers.^17–19 Subpopulations of macrophages directly contribute to wound healing and tissue repair, supporting the concept that some macrophage phenotypes can promote organ regeneration after a pro-inflammatory state of injury.²⁰ The concept of macrophage polarization states has emerged; the M1 “classically activated” pro-inflammatory cell type apparently opposed by an M2 “alternatively activated” immune regulatory macrophage.¹⁸ In general, these two states are thought to be analogous to the opposing T helper 1 and T helper 2 immune responses, although in both cases this model is probably too simplistic. Functionally, it is more likely that distinct subpopulations of macrophages may exist in the same tissue and play critical roles in both the injury and recovery phases of inflammatory scarring.²⁰Our previous study provided evidence that the addition of CSF-1 to a developing murine kidney promotes a growth and differentiation response that is accompanied by increased numbers of macrophages.³ Furthermore, with the use of expression profiling we demonstrated that fetal kidney, lung, and brain macrophages share a characteristic gene expression profile that includes the production of factors important in the suppression of inflammation and the promotion of proliferation.³ Embryonic macrophages appear to play a positive trophic role that may have parallel reparative functions in many adult tissues undergoing repair and cellular replacement.^1,20 A number of studies have suggested that infiltrating macrophages along with the trophic factors they release participate in tissue repair of the kidney,^20–22 brain,²³ skin,^24,25 lung,²⁶ liver,²⁷ heart,²⁸ gastrointestinal tract,^29,30 and skeletal muscle.^31,32 Indeed, the pleiotrophic roles for CSF-1 in reproduction, development of multiple organ systems, and maternal-fetal interactions during pregnancy by macrophage-mediated processes have also been well defined.^2,33,34To determine the physiological relevance of CSF-1 as a component of the mammalian growth regulatory axis, CSF-1 was administered to neonatal mice. We report that CSF-1 administration to newborn mice increased body weight and kidney weight and volume and was associated with increased numbers of macrophages. Our results also establish that CSF-1 injection into mice after ischemia-reperfusion (IR) injury promoted endogenous repair with characteristic rapid re-epithelialization of the damaged tubular epithelium, leading to functional recovery. Flow cytometric and gene expression analyses were used to delineate the macrophage profile present in the kidneys during the early and resolution phase of IR injury with and without CSF-1 therapy. We thus provide evidence that CSF-1 recruits macrophages to the reparative site and influences their phenotype, partly through an insulin-like growth factor (IGF)-1 signaling response. Therefore, macrophages under the stimulus of CSF-1 in an acute setting of renal disease markedly accelerate renal cell replacement and tissue remodeling while attenuating downstream interstitial extracellular matrix accumulation.     

Microglia/macrophages migrate through retinal epithelium barrier by a transcellular route in diabetic retinopathy: role of PKCζ in the Goto Kakizaki rat model

Diabetic retinopathy is associated with ocular inflammation, leading to retinal barrier breakdown, macular edema, and visual cell loss. We investigated the molecular mechanisms involved in microglia/macrophages trafficking in the retina and the role of protein kinase Cζ (PKCζ) in this process. Goto Kakizaki (GK) rats, a model for spontaneous type 2 diabetes were studied until 12 months of hyperglycemia. Up to 5 months, sparse microglia/macrophages were detected in the subretinal space, together with numerous pores in retinal pigment epithelial (RPE) cells, allowing inflammatory cell traffic between the retina and choroid. Intercellular adhesion molecule-1 (ICAM-1), caveolin-1 (CAV-1), and PKCζ were identified at the pore border. At 12 months of hyperglycemia, the significant reduction of pores density in RPE cell layer was associated with microglia/macrophages accumulation in the subretinal space together with vacuolization of RPE cells and disorganization of photoreceptors outer segments. The intraocular injection of a PKCζ inhibitor at 12 months reduced iNOS expression in microglia/macrophages and inhibited their migration through the retina, preventing their subretinal accumulation. We show here that a physiological transcellular pathway takes place through RPE cells and contributes to microglia/macrophages retinal trafficking. Chronic hyperglycemia causes alteration of this pathway and subsequent subretinal accumulation of activated microglia/macrophages.     

Implementation and workflow for PET monitoring of therapeutic ion irradiation: a comparison of in-beam, in-room, and off-line techniques

An independent assessment of the dose delivery in ion therapy can be performed using positron emission tomography (PET). For that a distribution of positron emitters which appear as the result of interaction between ions of the therapeutic beam and the irradiated tissue is measured during or after the irradiation. Three concepts for PET monitoring implemented in various therapy facilities are considered in this paper. The in-beam PET concept relies on the PET measurement performed simultaneously to the irradiation by means of a PET scanner which is completely integrated into the irradiation site. The in-room PET concept allows measurement immediately after irradiation by a standalone PET scanner which is installed very close to the irradiation site. In the off-line PET scenario the measurement is performed by means of a standalone PET/CT scanner 10-30 min after the irradiation. These three concepts were evaluated according to image quality criteria, integration costs, and their influence onto the workflow of radiotherapy. In-beam PET showed the best performance. However, the integration costs were estimated as very high for this modality. Moreover, the performance of in-beam PET depends heavily on type and duty cycle of the accelerator. The in-room PET is proposed for planned therapy facilities as a good compromise between the quality of measured data and integration efforts. For facilities which are close to the nuclear medicine departments off-line PET can be suggested under several circumstances.     

Genetic Variant in HK1 Is Associated With a Proanemic State and A1C but Not Other Glycemic Control�CRelated Traits

OBJECTIVE

A1C is widely considered the gold standard for monitoring effective blood glucose levels. Recently, a genome-wide association study reported an association between A1C and rs7072268 within HK1 (encoding hexokinase 1), which catalyzes the first step of glycolysis. HK1 deficiency in erythrocytes (red blood cells [RBCs]) causes severe nonspherocytic hemolytic anemia in both humans and mice.

RESEARCH DESIGN AND METHODS

The contribution of rs7072268 to A1C and the RBC-related traits was assessed in 6,953 nondiabetic European participants. We additionally analyzed the association with hematologic traits in 5,229 nondiabetic European individuals (in whom A1C was not measured) and 1,924 diabetic patients. Glucose control–related markers other than A1C were analyzed in 18,694 nondiabetic European individuals. A type 2 diabetes case-control study included 7,447 French diabetic patients.

RESULTS

Our study confirms a strong association between the rs7072268–T allele and increased A1C (β = 0.029%; P = 2.22 × 10⁻⁷). Surprisingly, despite adequate study power, rs7072268 showed no association with any other markers of glucose control (fasting- and 2-h post-OGTT–related parameters, n = 18,694). In contrast, rs7072268–T allele decreases hemoglobin levels (n = 13,416; β = −0.054 g/dl; P = 3.74 × 10⁻⁶) and hematocrit (n = 11,492; β = −0.13%; P = 2.26 × 10⁻⁴), suggesting a proanemic effect. The T allele also increases risk for anemia (836 cases; odds ratio 1.13; P = 0.018).

CONCLUSIONS

HK1 variation, although strongly associated with A1C, does not seem to be involved in blood glucose control. Since HK1 rs7072268 is associated with reduced hemoglobin levels and favors anemia, we propose that HK1 may influence A1C levels through its anemic effect or its effect on glucose metabolism in RBCs. These findings may have implications for type 2 diabetes diagnosis and clinical management because anemia is a frequent complication of the diabetes state.Type 2 diabetes is a major source of early excess morbidity and mortality, which result from lack of adequate blood glucose control in most diabetic patients (1). In the absence of widely available continuous glucose monitoring, the A1C assay has become the most popular index to evaluate the efficiency of type 2 diabetes treatments on long-term blood glucose control (2,3). A1C, which is formed through the nonenzymatic attachment of glucose to the NH₂-terminal of the β-chain of hemoglobin, is indeed commonly considered a surrogate marker of mean blood glucose concentration over the previous 8–12 weeks (i.e., a 120-day life span of erythrocytes) (4). Furthermore, the A1C assay is often used for confirming type 2 diabetes diagnosis when fasting plasma glucose (FPG) is in the pre-diabetes range (6.1 ≤ FPG <7.0 mmol/l, defining normal glycemia and overt diabetes, respectively [2]), as postprandial or post–glucose load measurements of blood glucose are difficult to widely apply in clinical practice. However, the A1C measurement displays well-known caveats, such as genetically inherited hemoglobin defects or erythrocyte (red blood cell [RBC]) life span heterogeneity in hematologically normal people, that would oblige the use of more complex measurement of glycated serum proteins or fructosamine as a surrogate of blood glucose levels (5,6).Thus far, several genome-wide association (GWA) studies have identified 22 genes or loci, increasing the risk for type 2 diabetes or modulating FPG levels (7–19). Recently, Pare et al. (20) reported a single nucleotide polymorphism (SNP), rs7072268, at the hexokinase 1 (HK1) locus (chr10q22) that strongly associates with increased A1C in a nondiabetic population. The four isozymes of the hexokinase family (HK1, HK2, HK3, and glucokinase) contribute to commit glucose to the glycolytic pathway. The predominant HK1 isozyme is expressed in the vast majority of cells and tissues, including cells that are strictly dependent on glucose uptake for their metabolic needs (21). Importantly, while most tissues express more than one HK isozyme, RBC glucose metabolism only depends on HK1 activity (22). In humans, mutations including nonsynonymous substitutions in the active site of HK1 and intragenic deletions have been shown to cause HK1 enzymatic deficiency associated with autosomal recessive severe nonspherocytic hemolytic anemia (21,23–25). A similar phenotype has been described in the Downeast Anemia (dea) mice displaying HK1 deficiency (22).Based on these observations, we postulated that HK1 genetic variation may modulate the maintenance of the RBC pool and thus indirectly alter A1C measurements independently of the ambient blood glucose concentration. We evaluated this hypothesis by assessing the impact of HK1 rs7072268 on A1C, other glucose control-related traits, type 2 diabetes risk, and RBC-related parameters in several prospective and case-control European cohorts. Our data suggest that HK1 variation through its anemic effect impairs A1C assays, which may have important clinical implications for both type 2 diabetes diagnosis and management because anemia is commonly associated with diabetes.     

Validation of endogenous controls for quantitative gene expression analysis: application on brain cortices of human chronic alcoholics

Real-time PCR is frequently used for gene expression quantification due to its methodological sensitivity and reproducibility. The gene expression is quantified by normalization to one or more reference genes, usually beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPD) or to ribosomal RNA (18S). However, different environmental or pathological conditions might also influence the expression of normalizing genes, which could severely skew the interpretation of quantitative results. This study evaluates whether 16 genes frequently used as endogenous controls in expression studies, can serve as such for comparison of human brain tissues of chronic alcoholics and control subjects. The prefrontal and motor cortices that are affected differently by chronic alcohol consumption were analyzed. The reference genes that have no or small differences in expression in alcoholics and control subjects, were found to be specific for each region: beta-actin (ACTB) and ribosomal large P0 (RPLP0) for the prefrontal cortex while importin 8 (IPO8) and RNA polymerase II (POLR2A) for the motor cortex. Four out of sixteen analyzed genes demonstrated significant differences in expression between alcoholics and controls: phosphoglycerate kinase (PGK1), hypoxanthine phosphoribosyl transferase (HPRT1) and peptidylprolyl isomerase A (PPIA) in the motor cortex and beta-2-microglobulin (B2M) in the prefrontal cortex. Our study demonstrates the importance of validation of endogenous control genes prior to real-time PCR analysis of human brain tissues. Prescribed and non-prescribed drugs, pathological or environmental conditions along with alcohol abuse may differentially influence expression of reference genes.     

Hypertension in people with diabetes and the metabolic syndrome: Pathophysiologic insights and therapeutic update

Hypertension (HTN) and type 2 diabetes mellitus (T2DM) are emerging as epidemics of the 21st century and are important components of the metabolic syndrome (MS). Evidence demonstrates a relationship between HTN, T2DM, and several vascular and metabolic abnormalities that are components of the MS. HTN affects nearly 70 million Americans and over one billion worldwide; likewise, the MS affects 44% of the US population above the age of 60 years and is rapidly increasing. HTN associated with the MS has certain pathophysiologic characteristics that provide clinical challenges. There is growing evidence that tissue activation of the renin-angiotensin system contributes to endothelial dysfunction, microalbuminuria, insulin resistance, and subsequent increased risk for cardiovascular and chronic kidney disease. The notion that HTN is a metabolic as well as a vascular disease provides a new treatment paradigm.