Reduced Hepatic Synthesis of Calcidiol in Uremia

Calcidiol insufficiency is highly prevalent in chronic kidney disease (CKD), but the reasons for this are incompletely understood. CKD associates with a decrease in liver cytochrome P450 (CYP450) enzymes, and specific CYP450 isoforms mediate vitamin D₃ C-25-hydroxylation, which forms calcidiol. Abnormal levels of parathyroid hormone (PTH), which also modulates liver CYP450, could also contribute to the decrease in liver CYP450 associated with CKD. Here, we evaluated the effects of PTH and uremia on liver CYP450 isoforms involved in calcidiol synthesis in rats. Uremic rats had 52% lower concentrations of serum calcidiol than control rats (P < 0.002). Compared with controls, uremic rats produced 71% less calcidiol and 48% less calcitriol after the administration of vitamin D₃ or 1α-hydroxyvitamin D₃, respectively, suggesting impaired C-25-hydroxylation of vitamin D₃. Furthermore, uremia associated with a reduction of liver CYP2C11, 2J3, 3A2, and 27A1. Parathyroidectomy prevented the uremia-associated decreases in calcidiol and liver CYP450 isoforms. In conclusion, these data suggest that uremia decreases calcidiol synthesis secondary to a PTH-mediated reduction in liver CYP450 isoforms.It has been known for decades that chronic renal failure (CRF) is associated with low serum 1,25-dihydroxyvitamin D₃ [calcitriol, or 1,25(OH)₂D₃], the active metabolite of vitamin D₃, because of a reduction in renal 1α-hydroxylase (CYP27B1). More recently, 25-hydroxyvitamin D₃ [calcidiol, or 25(OH)D₃] deficiency has also been demonstrated in patients with stages 3 and 4 chronic kidney disease (CKD) and in patients who are on dialysis.^1–8 In fact, low serum 25(OH)D₃ is so intimately associated with CRF that in one study, only 29 and 17% of patients with stages 3 and 4 CKD, respectively, had sufficient levels [defined as a serum 25(OH)D₃ concentrations >75 nmol/L or 30 ng/ml].² A more recent study showed a prevalence of calcidiol insufficiency and deficiency as high as 98% in predialysis patients with a mean GFR of 18.3 ml/min.⁴ Prevalence of low serum 25(OH)D₃ was 78 and 89% in two large cohorts of hemodialysis patients^9,10 and 87% in a large cohort of peritoneal dialysis patients.¹¹The metabolic consequences of calcidiol deficiency are important, because low levels of 25(OH)D₃ might contribute to low levels of 1,25(OH)₂D₃ and to secondary hyperparathyroidism.^1–8 Moreover, in addition to its role in bone metabolism, there is increasing evidence that vitamin D₃ is involved in the prevention of many chronic diseases, such as type 1 diabetes, hypertension, cardiovascular diseases, and cancer.^8,9,12–14 As a consequence, according to the 2003 Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines, calcidiol levels should be measured in patients with CKD, and deficiency should be treated with ergocalciferol (vitamin D₂) or cholecalciferol (vitamin D₃)⁵; however, a paucity of information exists concerning the effect of treatment of vitamin D₃ insufficiency in CRF on the frequency and severity of secondary hyperparathyroidism among patients with decreased 25(OH)D₃ concentrations. Furthermore, the efficacy of vitamin D₃ therapy on serum calcidiol levels of patients who experience kidney failure is variable and remains poor compared with patients without CKD.^{1–7,15–18}More important, the mechanisms underlying calcidiol deficiency remain poorly understood. Lower diet intake and reduced sun exposure have been proposed but never demonstrated.^1,2,4,6,7 Vitamin D₃ is normally synthesized in the skin under the influence of sunlight or taken orally as a vitamin supplement. It is hydroxylated in the liver to 25(OH)D₃, then hydroxylated in the kidney to form 1,25(OH)₂D₃, the most bioactive form of the vitamin (Figure 1). Both calcitriol and calcidiol are degraded in part by a C-24-hydroxylation achieved by a ubiquitous 24-hydroxylase.^19,20Open in a separate windowFigure 1.Vitamin D₃ biotransformation pathway.The enzymes responsible for the C-25-hydroxylation of vitamin D₃ in rats are liver cytochrome P450 (CYP450) isoforms, namely CYP2C11, 2J3, 2R1, 3A2, and 27A1.^21–26 Several studies have shown that in rats with CRF, total hepatic CYP450 content as well as the in vitro activity and expression of several liver CYP450 isoforms (mainly CYP2C11, 3A1, and 3A2) are decreased by >50%.^27–32 More recently, we showed that this decrease in hepatic CYP450 may be explained by the presence of serum uremic factors that accumulate in CRF serum^33,34 and that parathyroid hormone (PTH) is a major mediator implicated in the downregulation of liver CYP450 and other liver drug-metabolizing enzymes.^35,36Hence, an attractive hypothesis to explain the decreased synthesis of calcidiol in CRF is that uremic toxins and, more specific, elevated PTH could downregulate liver CYP450 isoforms implicated in the C-25-hydroxylation of vitamin D₃ (Figure 1). Indirect evidence supporting such a hypothesis is that low serum levels of 25(OH)D₃ have also been reported in primary hyperparathyroidism and found to be corrected by parathyroidectomy (PTX).³⁷ The objectives of this study were to determine (1) the effect of CRF on calcidiol levels in rats, (2) the ability of CRF rats to C-25-hydroxylate vitamin D₃ after administration of vitamin D₃ or 1α-hydroxyvitamin D₃, (3) the role of liver CYP450 downregulation in calcidiol deficiency in CRF, and (4) the potential role of secondary hyperparathyroidism in calcidiol synthesis in rats with CRF.     

The Gut Microbiome,Kidney Disease,and Targeted Interventions

The human gut harbors >100 trillion microbial cells, which influence the nutrition, metabolism, physiology, and immune function of the host. Here, we review the quantitative and qualitative changes in gut microbiota of patients with CKD that lead to disturbance of this symbiotic relationship, how this may contribute to the progression of CKD, and targeted interventions to re-establish symbiosis. Endotoxin derived from gut bacteria incites a powerful inflammatory response in the host organism. Furthermore, protein fermentation by gut microbiota generates myriad toxic metabolites, including p-cresol and indoxyl sulfate. Disruption of gut barrier function in CKD allows translocation of endotoxin and bacterial metabolites to the systemic circulation, which contributes to uremic toxicity, inflammation, progression of CKD, and associated cardiovascular disease. Several targeted interventions that aim to re-establish intestinal symbiosis, neutralize bacterial endotoxins, or adsorb gut-derived uremic toxins have been developed. Indeed, animal and human studies suggest that prebiotics and probiotics may have therapeutic roles in maintaining a metabolically-balanced gut microbiota and reducing progression of CKD and uremia-associated complications. We propose that further research should focus on using this highly efficient metabolic machinery to alleviate uremic symptoms.The gut microbiota has coevolved with humans for a mutually beneficial coexistence and plays an important role in health and disease.¹ Normal gut microbiota influences the well-being of the host by contributing to its nutrition, metabolism, physiology, and immune function.^2,3 Disturbance of normal gut microbiota (dysbiosis) has been implicated in the pathogenesis of diverse illnesses, such as obesity,⁴ type 2 diabetes,⁵ inflammatory bowel disease,⁶ and cardiovascular disease.^7,8 Quantitative and qualitative alterations in gut microbiota are noted in patients with CKD and ESRD.^9–11 Preliminary evidence indicates that toxic products generated by a dysbiotic gut microbiome may contribute to progression to CKD and CKD-related complications (Figure 1).^12,13Open in a separate windowFigure 1.The human gut is host to >100 trillion bacteria with an enteric reservoir of >1 g of endotoxin. Alterations in gut microbiota and impaired intestinal barrier function in patients with CKD/ESRD have been linked to endotoxemia and accumulation of gut-derived uremic toxins leading to insulin resistance, protein energy wasting, immune dysregaulation, and atheroscleroisis. CVD, cardiovascular disease; IR, insulin resistance; PEW, protein energy wasting.     

Human Urinary Exosomes as Innate Immune Effectors

Exosomes are small extracellular vesicles, approximately 50 nm in diameter, derived from the endocytic pathway and released by a variety of cell types. Recent data indicate a spectrum of exosomal functions, including RNA transfer, antigen presentation, modulation of apoptosis, and shedding of obsolete protein. Exosomes derived from all nephron segments are also present in human urine, where their function is unknown. Although one report suggested in vitro uptake of exosomes by renal cortical collecting duct cells, most studies of human urinary exosomes have focused on biomarker discovery rather than exosome function. Here, we report results from in-depth proteomic analyses and EM showing that normal human urinary exosomes are significantly enriched for innate immune proteins that include antimicrobial proteins and peptides and bacterial and viral receptors. Urinary exosomes, but not the prevalent soluble urinary protein uromodulin (Tamm–Horsfall protein), potently inhibited growth of pathogenic and commensal Escherichia coli and induced bacterial lysis. Bacterial killing depended on exosome structural integrity and occurred optimally at the acidic pH typical of urine from omnivorous humans. Thus, exosomes are innate immune effectors that contribute to host defense within the urinary tract.Exosomes form as intraluminal vesicles of multivesicular bodies (MVBs), contain membrane and cytoplasmic proteins, have a cytoplasmic-side inward membrane orientation, and are released intact into the extracellular space (Figure 1A). First described in maturing ovine reticulocytes,¹ exosomes are released by many cell types² and have been conventionally regarded as a vehicle for shedding obsolete protein. However, emerging evidence has revealed a variety of exosomal functions, including the intercellular transfer of membrane receptors³ and RNA,^4–6 induction of immunity,⁷ antigen presentation,⁸ modulation of bone mineralization,⁹ and antiapoptotic responses.¹⁰Open in a separate windowFigure 1.Vesicles isolated from human urine are consistent with exosomes. (A) Exosomes are derived from the endocytic pathway (1–4) forming through invagination of the limiting membrane of the MVB (3). They are released into the urinary space from renal tubular epithelial cells through fusion of the MVB with the apical plasma membrane (4). (B and C) Exosomal isolation was confirmed by the identification by negative stain EM of nanovesicles (black arrows; characteristic mean 50-nm size distribution. (D) Uromodulin (white streaks in B and dark in C; open arrows in B and C) cofractionated with exosomes but was confirmed to be extraexosomal (5 nm gold-labeled; white arrows in C). (E) Western blot confirmed the presence of known exosomal constituents in vesicle preparations but did not confirm them in precipitated protein from exosome-depleted urine. (F) Immuno-EM with 5 (TSG101 and CD63) or 15 nm (AQP2) gold particle-labeled antibodies showed vesicular residency of known exosomal constituents (arrows). Vesicles were nonpermeabilized; thus, positive staining with an anti-CD63 antibody directed against an extracellular epitope indicated the cytoplasmic side inward membrane orientation characteristic of exosomes. EDUP, exosome-depleted urine protein; HKM, human kidney membrane; MW, molecular weight; TSG101, tumour susceptibility gene 101.Nanovesicles were first shown in human urine by Kanno and colleagues¹¹ and subsequently, were shown to represent exosomes.¹² Consistent with a renal tubular epithelial origin, renal tubular epithelial cells contain MVBs at the apical surface, and urine exosomes contain apical membrane proteins from every cell type along the nephron.^13,14 The array of functions ascribed to exosomes in other tissues has kindled recent interest in the functional significance of urinary exosomes. Hogan et al.¹³ suggested interaction of exosome-like vesicles with primary cilia of renal epithelial cells, and Street and coworkers¹⁵ showed in vitro uptake of exosomes by a renal cortical collecting duct cell line, leading to speculation that exosomes may provide intrarenal proximal-to-distal transapical renal tubular epithelial signaling through RNA transfer. However, most studies on urine exosomes to date have focused on biomarker discovery, resulting in the publication of several urine exosome protein compendia.^{12,13,16–21}Published reports of the urinary exosomal proteome have limited value in illuminating the potential functions of urinary exosomes for several reasons. First, protein identification by mass spectrometry (MS) has, until recently, yielded results with unacceptably low reproducibility and high false-positive rates,^22,23 and previous reports are not free of these limitations. Second, studies have often aimed to maximize the number of protein identifications and hence, biomarker candidates rather than applying or reporting rigorous protein identification thresholds. Third, most have relied on pooled samples from up to six donors and have not reported interindividual variability or reproducibility.Here, we sought, for the first time, to ascribe functionality to human urinary exosomes. We initially performed rigorous, conservative tandem MS analysis of separate human urinary exosomal samples to allow enrichment scoring²⁴ to elucidation of urine exosomal function.     

Origin of Parietal Podocytes in Atubular Glomeruli Mapped by Lineage Tracing

Parietal podocytes are fully differentiated podocytes lining Bowman’s capsule where normally only parietal epithelial cells (PECs) are found. Parietal podocytes form throughout life and are regularly observed in human biopsies, particularly in atubular glomeruli of diseased kidneys; however, the origin of parietal podocytes is unresolved. To assess the capacity of PECs to transdifferentiate into parietal podocytes, we developed and characterized a novel method for creating atubular glomeruli by electrocoagulation of the renal cortex in mice. Electrocoagulation produced multiple atubular glomeruli containing PECs as well as parietal podocytes that projected from the vascular pole and lined Bowman’s capsule. Notably, induction of cell death was evident in some PECs. In contrast, Bowman’s capsules of control animals and normal glomeruli of electrocoagulated kidneys rarely contained podocytes. PECs and podocytes were traced by inducible and irreversible genetic tagging using triple transgenic mice (PEC- or Pod-rtTA/LC1/R26R). Examination of serial cryosections indicated that visceral podocytes migrated onto Bowman’s capsule via the vascular stalk; direct transdifferentiation from PECs to podocytes was not observed. Similar results were obtained in a unilateral ureter obstruction model and in human diseased kidney biopsies, in which overlap of PEC- or podocyte-specific antibody staining indicative of gradual differentiation did not occur. These results suggest that induction of atubular glomeruli leads to ablation of PECs and subsequent migration of visceral podocytes onto Bowman’s capsule, rather than transdifferentiation from PECs to parietal podocytes.Multiple studies have reported the presence of podocytes on Bowman’s capsule in normal¹ and diseased kidneys, close to the vascular pole in particular.^2–5 These unique cells were named parietal podocytes and are defined as cells with features of differentiated podocytes lining the inner aspect of Bowman’s capsule (Figure 1A). Using an extended panel of antibodies, Bariety et al. showed that parietal podocytes expressed the same marker proteins as visceral podocytes.¹ On electron microscopic images, parietal podocytes mostly form interdigitating foot processes^3,6,7 and recruit periglomerular capillaries.⁸ In patients with membranous GN, parietal podocytes behaved similar to visceral podocytes in that they formed subepithelial deposits on the parietal basement membrane identical to those on the glomerular basement membrane (GBM).⁵Open in a separate windowFigure 1.Characterization of the early events in the coagulation model. (A) Schematic of a parietal podocyte on Bowman’s capsule forming foot processes (arrow) and recruiting periglomerular capillaries (asterisk). (B and B’) Schematic of the electrocoagulation model. Along the lateral margin of the kidney, a necrotic area is visible after coagulation (arrowheads, right kidney). (C) Immediately after coagulation, a triangular area of necrosis is observed, reaching from the renal cortex into the medulla. (D) Higher magnification of the interface between the coagulated renal tissue (arrow) and remaining parenchyma shows several necrotic tubules 3 days after coagulation (arrowheads) with inflammatory demarcation. (E) After 1 week, multiple tubules are dilated (arrowhead). (F) After 3 weeks, glomeruli with obliterated urinary poles (arrow) and atrophic tubular remnants (arrowheads) are observed. C–F show periodic acid–Schiff stainings of paraffin sections. (G) Immunohistologic double staining shows that cells obstructing the tubular outlet are SSeCKS-positive parietal cells (arrow) and that Bowman’s capsule is lined by parietal podocytes projecting from the vascular pole (arrowheads). (H–I) TUNEL assays consistently show positive nuclear staining in PECs (approximately 1–2 positive nuclei per 100 glomeruli) 1–3 weeks after electrocoagulation (G, arrow). (H) No nuclear staining is observed in healthy controls.The number of parietal podocytes on Bowman’s capsule may vary significantly. Published data indicate that their number increases throughout life.⁹ In normal kidneys, parietal podocytes are rare,¹ whereas their frequency and number is increased in transplant nephrectomies and in diseased kidneys.^2,5 In atubular glomeruli or glomerular cysts, parietal podocytes can often be found on Bowman’s capsule—in many cases, covering its entire circumference.^1,7,10–12 Atubular glomeruli and glomerular cysts are not uncommon even in “normal kidney tissue” obtained from tumor nephrectomies (up to 1% of all glomeruli).^10,13 Both are particularly common, however, in renal diseases affecting the tubulointerstitium.^14–19 Furthermore, atubular/cystic glomeruli have also been observed in other renal diseases,²⁰ including diabetic nephropathy in which on average 17% of all glomeruli were atubular,¹³ transplanted kidney affected by chronic allograft rejection (60% atubular glomeruli),¹⁰ severe renal artery stenosis (50% atubular glomeruli),²¹ GN,^5,10 pyelonephritis,¹⁴ and polycystic kidney disease.^22,23 Glomerular cysts lined by parietal podocytes have also been described in different cystic diseases in animals.^7,11,12,24In previous studies, it was proposed that parietal epithelial cells (PECs) may differentiate into podocytes acting as a potential intrinsic progenitor cell population.^25,26 Parietal podocytes provide a unique and exceptional situation to investigate whether PECs have the potential to transdifferentiate into podocytes in situ on Bowman’s capsule. In this study, we set out to resolve the origin of parietal podocytes by lineage tracing. For this purpose, a novel model to induce the generation of parietal podocytes in atubular glomeruli was established.     

Genetic Susceptibility to Obesity and Related Traits in Childhood and Adolescence: Influence of Loci Identified by Genome-Wide Association Studies

OBJECTIVE

Large-scale genome-wide association (GWA) studies have thus far identified 16 loci incontrovertibly associated with obesity-related traits in adults. We examined associations of variants in these loci with anthropometric traits in children and adolescents.

RESEARCH DESIGN AND METHODS

Seventeen variants representing 16 obesity susceptibility loci were genotyped in 1,252 children (mean ± SD age 9.7 ± 0.4 years) and 790 adolescents (15.5 ± 0.5 years) from the European Youth Heart Study (EYHS). We tested for association of individual variants and a genetic predisposition score (GPS-17), calculated by summing the number of effect alleles, with anthropometric traits. For 13 variants, summary statistics for associations with BMI were meta-analyzed with previously reported data (N_total = 13,071 children and adolescents).

RESULTS

In EYHS, 15 variants showed associations or trends with anthropometric traits that were directionally consistent with earlier reports in adults. The meta-analysis showed directionally consistent associations with BMI for all 13 variants, of which 9 were significant (0.033–0.098 SD/allele; P < 0.05). The near-TMEM18 variant had the strongest effect (0.098 SD/allele P = 8.5 × 10⁻¹¹). Effect sizes for BMI tended to be more pronounced in children and adolescents than reported earlier in adults for variants in or near SEC16B, TMEM18, and KCTD15, (0.028–0.035 SD/allele higher) and less pronounced for rs925946 in BDNF (0.028 SD/allele lower). Each additional effect allele in the GPS-17 was associated with an increase of 0.034 SD in BMI (P = 3.6 × 10⁻⁵), 0.039 SD, in sum of skinfolds (P = 1.7 × 10⁻⁷), and 0.022 SD in waist circumference (P = 1.7 × 10⁻⁴), which is comparable with reported results in adults (0.039 SD/allele for BMI and 0.033 SD/allele for waist circumference).

CONCLUSIONS

Most obesity susceptibility loci identified by GWA studies in adults are already associated with anthropometric traits in children/adolescents. Whereas the association of some variants may differ with age, the cumulative effect size is similar.Over the past three decades, the prevalence of obesity has reached epidemic proportions not only in adults, but in children and adolescents alike (1,2). A high BMI during childhood and adolescence often persists into adulthood (3–5) and has been independently associated with cardiovascular risk factors, coronary heart disease events, and all-cause mortality (2,6–9). Family and twin studies have estimated that 40–70% of the variance in obesity-related traits is due to genetic factors (10,11). Longitudinal twin studies have shown that the genetic contribution to BMI increases from childhood to adolescence (12–14), and cross-sectional twin studies suggest that the heritability of BMI is higher in adolescence than during adulthood (15,16).Six genome-wide association (GWA) studies in adults of white European descent have thus far identified 16 obesity susceptibility loci; 12 loci were consistently associated with BMI (17–22), and 4 loci were identified in GWA studies for waist circumference. Only variants in the FTO and near-MC4R loci have as of yet convincingly been associated with obesity-related traits in children and adolescents (12,18,20,23–27). Two studies have examined the effect of variants in GWA-derived loci other than FTO and MC4R in children and adolescents (20,28). However, both studies focused only on BMI and neither study examined the association of all 16 obesity susceptibility loci or their cumulative effect. Examining the association of these obesity susceptibility loci with measures of adiposity in childhood and adolescence may provide insight into their impact on obesity risk early in life. Furthermore, it has been suggested that physical activity modifies the association of genetic variation with general adiposity in adults (29–31). Thus far, this has not been demonstrated in children.In this study, we examined whether obesity susceptibility loci identified by GWA studies in adults are associated with anthropometric traits and risk of obesity in children and adolescents from the European Youth Heart Study (EYHS). To increase statistical power and to compare effect sizes in children/adolescents and adults, we additionally meta-analyzed our findings with those reported by others (20,28). Furthermore, we examined the cumulative effect of variants in the 16 loci on anthropometric traits in EYHS and tested whether the association between genetic predisposition and anthropometric traits is modified by physical activity.     

Lack of Type VIII Collagen in Mice Ameliorates Diabetic Nephropathy

OBJECTIVE

Key features of diabetic nephropathy include the accumulation of extracellular matrix proteins. In recent studies, increased expression of type VIII collagen in the glomeruli and tubulointerstitium of diabetic kidneys has been noted. The objectives of this study were to assess whether type VIII collagen affects the development of diabetic nephropathy and to determine type VIII collagen–dependent pathways in diabetic nephropathy in the mouse model of streptozotocin (STZ)-induced diabetes.

RESEARCH DESIGN AND METHODS

Diabetes was induced by STZ injections in collagen VIII–deficient or wild-type mice. Functional and histological analyses were performed 40 days after induction of diabetes. Type VIII collagen expression was assessed by Northern blots, immunohistochemistry, and real-time PCR. Proliferation of primary mesangial cells was measured by thymidine incorporation and direct cell counting. Expression of phosphorylated extracellular signal–regulated kinase (ERK1/2) and p27^Kip1 was assessed by Western blots. Finally, Col8a1 was stably overexpressed in mesangial cells.

RESULTS

Diabetic wild-type mice showed a strong renal induction of type VIII collagen. Diabetic Col8a1⁻/Col8a2⁻ animals revealed reduced mesangial expansion and cellularity and extracellular matrix expansion compared with the wild type. These were associated with less albuminuria. High-glucose medium as well as various cytokines induced Col8a1 in cultured mesangial cells. Col8a1⁻/Col8a2⁻ mesangial cells revealed decreased proliferation, less phosphorylation of Erk1/2, and increased p27^Kip1 expression. Overexpression of Col8a1 in mesangial cells induced proliferation.

CONCLUSIONS

Lack of type VIII collagen confers renoprotection in diabetic nephropathy. One possible mechanism is that type VIII collagen permits and/or fosters mesangial cell proliferation in early diabetic nephropathy.Diabetic nephropathy is the most common cause of end-stage renal failure leading to dialysis. Glomerular lesions are characterized by expansion of the mesangial matrix and thickening of peripheral glomerular basement membranes due to the synthesis and accumulation of extracellular matrix (ECM) (1,2). The degree of mesangial matrix expansion correlates with the progressive decline in the glomerular capillary surface area available for filtration and, hence, with the glomerular filtration rate (3). Early changes include a confined proliferation of mesangial cells followed by cell cycle arrest and hypertrophy (3–8). Several growth factors have been implicated in this process, among them transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor (PDGF)-BB (4,9,10). During early stages, PDGF-BB potently increases proliferation and matrix synthesis of mesangial cells and induces the expression of TGF-β1 (4,5,11). Upregulation of the PDGF-BB pathway has been shown in kidneys from patients with diabetic nephropathy as well as in experimental models of diabetic nephropathy (12,13). Further, PDGF receptor antagonists attenuate diabetic nephropathy (4). Activation of the TGF-β1 loop leads to cell cycle arrest, induction of cyclin-dependent kinase inhibitors, and further ECM synthesis (3,14).Type VIII collagen, a nonfibrillar short-chain collagen, is a structural component of many extracellular matrices (15–17). Two highly homologous polypeptides, α1(VIII) and α2(VIII), form either homotrimeric or heterotrimeric molecules (18–20). Type VIII collagen is involved in cross-talk between cells and the surrounding matrix by modulating diverse cellular responses such as proliferation, adhesion, migration, chemotaxis, and metalloproteinase synthesis (21–23). It is highly expressed by vascular smooth muscle cells in response to PDGF-BB and is thought to be a key component of vascular remodeling (24–27). In healthy kidneys, expression of type VIII collagen has been demonstrated in glomerular arterioles, larger branches of renal arteries, and in rat glomeruli and mesangial cell in vitro (28,29). Increased mRNA as well as protein expression has been noted in glomeruli and the tubulointerstitium of biopsies of kidneys from patients with diabetic nephropathy (30,31). The functional role of collagen VIII, especially in the early phase of the disease, has not been investigated and remains obscure.To address the role of type VIII collagen in the pathogenesis of diabetic nephropathy, we applied the streptozotocin (STZ) model to mice with homozygous deletions of both collagen VIII genes and compared them with wild-type mice. The objectives of this study were to assess whether collagen VIII–dependent pathways are involved in the development of diabetic nephropathy and in various cellular and molecular processes associated with this disorder.     

The Clinical Impact of Humoral Immunity in Pediatric Renal Transplantation

The development of anti-donor humoral responses after transplantation associates with higher risks for acute rejection and 1-year graft survival in adults, but the influence of humoral immunity on transplant outcomes in children is not well understood. Here, we studied the evolution of humoral immunity in low-risk pediatric patients during the first 2 years after renal transplantation. Using data from 130 pediatric renal transplant patients randomized to steroid-free (SF) or steroid-based (SB) immunosuppression in the NIH-SNSO1 trial, we correlated the presence of serum anti-HLA antibodies to donor HLA antigens (donor-specific antibodies) and serum MHC class 1-related chain A (MICA) antibody with both clinical outcomes and histology identified on protocol biopsies at 0, 6, 12, and 24 months. We detected de novo antibodies after transplant in 24% (23% of SF group and 25% of SB group), most often after the first year. Overall, 22% developed anti-HLA antibodies, of which 6% were donor-specific antibodies, and 6% developed anti-MICA antibody. Presence of these antibodies de novo associated with significantly higher risks for acute rejection (P=0.02), chronic graft injury (P=0.02), and decline in graft function (P=0.02). In summary, antibodies to HLA and MICA antigens appear in approximately 25% of unsensitized pediatric patients, placing them at greater risk for acute and chronic rejection with accelerated loss of graft function. Avoiding steroids does not seem to modify this incidence. Whether serial assessments of these antibodies after transplant could guide individual tailoring of immunosuppression requires additional study.Kidney transplantation is the modality of choice for children with ESRD. Despite an improvement in early outcomes, long-term allograft survival remains restricted^1,2; chronic graft injury is the prime limiting survival factor and largely driven by the development of antidonor humoral responses.^3,4 Anti-human leukocyte antigen (anti-HLA) antibodies (Abs) are a major cause of acute antibody-mediated rejection and also may damage the kidney in a more indolent process, leading to chronic antibody-mediated rejection and eventual allograft loss.⁵ Nevertheless, ongoing allograft rejection in HLA identical transplant recipients and patients without detectable anti-HLA Abs to donor HLA antigens (DSA) supports an additional pathogenic role for graft injury by non-HLA antigens, such as protein kinase-ζ, and MHC class 1-related chain A (MICA).^6–9In adult renal transplantation, Terasaki¹⁰ and Terasaki and Cai¹¹ have shown that preformed and de novo post-transplant production of DSAs correlates with a higher risk of acute rejection (AR) and reduced 1-year graft survival. The impact of the humoral response has not been well studied in the pediatric transplantation. We performed quantitative analysis of post-transplant de novo Abs to HLA and MICA in children undergoing kidney transplants and questioned differences in Ab profiles with steroid avoidance.^12–14 To this end, we compared the measured humoral immune responses of pediatric kidney transplant patients in a randomized, multicenter, open-labeled study for a steroid-based (SB) or steroid-free (SF) immunosuppression protocol (SNSO1).^15,16 We conducted serial monitoring of quantitative titers of circulating for MHC classes I and II and Abs to MICA in patients with stable graft function, acute graft rejection, and chronic graft injury as evaluated by matched protocol or indicated renal allograft biopsies (Figure 1). We intended to find if there were differences in the detection of these Abs with steroid avoidance, the average time for de novo Ab detection post-transplantation, and the correlation of Ab levels with graft injury and function. Correlation of the negative impact of the peripheral and intragraft humoral responses and their specificities with adverse graft outcomes in children could develop a novel means of monitoring and titrating immunosuppression in pediatric renal transplantation.Open in a separate windowFigure 1.Study outline. This study used 440 serum samples and 440 matched blinded biopsy scores for CADI, CNIT, Banff rejection grading, and C4d stains on 440 matched protocol biopsies from the SNSO1 multicenter randomized trial of SF and SB immunosuppression in pediatric renal transplantation.^15,16 Samples and biopsies were assayed at 0 (pretransplant), 6, 12, and 24 months post-transplantation. Of 130 patients in the trial, 124 patients had at least three of four sera samples collected and were included in the analysis.     

Renal Dendritic Cells Ameliorate Nephrotoxic Acute Kidney Injury

Inflammation contributes to the pathogenesis of acute kidney injury. Dendritic cells (DCs) are immune sentinels with the ability to induce immunity or tolerance, but whether they mediate acute kidney injury is unknown. Here, we studied the distribution of DCs within the kidney and the role of DCs in cisplatin-induced acute kidney injury using a mouse model in which DCs express both green fluorescence protein and the diphtheria toxin receptor. DCs were present throughout the tubulointerstitium but not in glomeruli. We used diphtheria toxin to deplete DCs to study their functional significance in cisplatin nephrotoxicity. Mice depleted of DCs before or coincident with cisplatin treatment but not at later stages experienced more severe renal dysfunction, tubular injury, neutrophil infiltration and greater mortality than nondepleted mice. We used bone marrow chimeric mice to confirm that the depletion of CD11c-expressing hematopoietic cells was responsible for the enhanced renal injury. Finally, mixed bone marrow chimeras demonstrated that the worsening of cisplatin nephrotoxicity in DC-depleted mice was not a result of the dying or dead DCs themselves. After cisplatin treatment, expression of MHC class II decreased and expression of inducible co-stimulator ligand increased on renal DCs. These data demonstrate that resident DCs reduce cisplatin nephrotoxicity and its associated inflammation.Innate immune responses are pathogenic in both ischemic and toxic acute renal failure. In response to renal injury, inflammatory chemokines and cytokines are produced both by renal parenchymal cells, such as proximal tubule epithelial cells, and resident or infiltrating leukocytes.^1–4 The elaborated chemokines and cytokines, including TNF-α, IL-18, keratinocyte-derived chemokine, and monocyte chemoattractant protein 1, subsequently recruit additional immune cells to the kidney, such as neutrophils, T cells, monocytes, and inflammatory dendritic cells (DCs), which may cause further injury through pathways that are not fully defined.^2,5–12 DCs are sentinels of the immune system and under steady-state conditions induce tolerance by various mechanisms, including production of TGF-β, IL-10, or indoleamine 2,3-dioxygenase^13–16; expression of PDL-1, PDL-2, or FcγR2B^17,18; clonal deletion of autoreactive T cells¹⁹; and induction of T regulatory cells via the inducible co-stimulator (ICOS) pathway.^20–23 In response to pathogens or products of tissue injury, DCs mature and initiate immunity or inflammatory diseases.^24,25 Monocytes recruited to inflamed tissue can also differentiate into inflammatory DCs and mediate defense against pathogens or contribute to inflammatory tissue responses.^12,26–28Although DCs represent a major population of immune cells in the kidney,²⁹ their role in renal disease is poorly defined. Liposomal clodronate has been used to study the pathophysiologic role of phagocytic cells, which include DCs and macrophages.^3,30–32 An alternative DC-specific approach uses expression of the simian diphtheria toxin receptor (DTR) driven by the CD11c promoter to target DCs for DT-mediated cell death.²⁴ This model has been used extensively to study the role of DCs in various physiologic and pathophysiologic contexts^32,33; however, its application in kidney disease has been limited to recent studies of immune complex–mediated glomerulonephritis.^12,23We have reported that inflammation plays an important role in the pathogenesis of cisplatin-induced acute kidney injury (AKI).^1,4,5,34 Given the dearth of information regarding the role of renal DCs in AKI, this study examined the renal DC population and the impact of its depletion on cisplatin nephrotoxicity. We show that DCs are the most abundant population of renal resident leukocytes and form a dense network throughout the tubulointerstitium. Renal DCs displayed surface markers that distinguished them from splenic DCs. Using a conditional DC depletion model, we determined that DC ablation markedly exacerbates cisplatin-induced renal dysfunction, structural injury, and infiltration of neutrophils.     

Confirmation of Multiple Risk Loci and Genetic Impacts by a Genome-Wide Association Study of Type 2 Diabetes in the Japanese Population

OBJECTIVE

To identify novel type 2 diabetes gene variants and confirm previously identified ones, a three-staged genome-wide association study was performed in the Japanese population.

RESEARCH DESIGN AND METHODS

In the stage 1 scan, we genotyped 519 case and 503 control subjects with 482,625 single nucleotide polymorphism (SNP) markers; in the stage 2 panel comprising 1,110 case subjects and 1,014 control subjects, we assessed 1,456 SNPs (P < 0.0025, stage 1); additionally to direct genotyping, 964 healthy control subjects formed the in silico control panel. Along with genome-wide exploration, we aimed to replicate the disease association of 17 SNPs from 16 candidate loci previously identified in Europeans. The associated and/or replicated loci (23 SNPs; P < 7 × 10^–5 for genome-wide exploration and P < 0.05 for replication) were examined in the stage 3 panel comprising 4,000 case subjects and 12,569 population-based samples, from which 4,889 nondiabetic control subjects were preselected. The 12,569 subjects were used for overall risk assessment in the general population.

RESULTS

Four loci—1 novel with suggestive evidence (PEPD on 19q13, P = 1.4 × 10^–5) and three previously reported—were identified; the association of CDKAL1, CDKN2A/CDKN2B, and KCNQ1 were confirmed (P < 10^–19). Moreover, significant associations were replicated in five other candidate loci: TCF7L2, IGF2BP2, SLC30A8, HHEX, and KCNJ11. There was substantial overlap of type 2 diabetes susceptibility genes between the two populations, whereas effect size and explained variance tended to be higher in the Japanese population.

CONCLUSIONS

The strength of association was more prominent in the Japanese population than in Europeans for more than half of the confirmed type 2 diabetes loci.The predisposition to and the course of type 2 diabetes vary according to ethnic group (1–3). In Japan, the incidence of type 2 diabetes has increased recently and is now comparable to that of other countries; this is supposedly attributable to the gradual spread of Western habits, such as consuming a high-fat diet, and the lower insulin secretory capacity of Japanese subjects (4,5). Recent technological developments have allowed the successful identification of gene regions involved in the development of type 2 diabetes in genome-wide association (GWA) studies (6–17). Several susceptibility gene loci identified by GWA studies to date have been used to obtain reproducible evidence of disease association in different populations of European descent and Asians, but not necessarily in African Americans (18–24). A number of GWA studies on type 2 diabetes have been conducted on populations of European descent (6–12). Two GWA scans in the Japanese population simultaneously reported the discovery of type 2 diabetes susceptibility gene (KCNQ1) variants in non-European populations; this result was also obtained in Scandinavian samples (25,26). Thus far, the replicated associations for a limited number of candidate genes have broadly indicated the tendency of interethnic similarity. Even though the common (or cosmopolitan) effect of type 2 diabetes risk variants is known, the extent to which the causation of this disease differs or overlaps between populations remains unknown. Here, besides comparing the genetic associations between European-descent and Japanese populations, we aimed to identify new genetic variants using a three-staged GWA study design.     

Genotype–Phenotype Correlations in Non-Finnish Congenital Nephrotic Syndrome

Mutations in NPHS1, which encodes nephrin, are the main causes of congenital nephrotic syndrome (CNS) in Finnish patients, whereas mutations in NPHS2, which encodes podocin, are typically responsible for childhood-onset steroid-resistant nephrotic syndrome in European populations. Genotype–phenotype correlations are not well understood in non-Finnish patients. We evaluated the clinical presentation, kidney histology, and disease progression in non-Finnish CNS cases by mutational screening in 107 families (117 cases) by sequencing the entire coding regions of NPHS1, NPHS2, PLCE1, WT1, LAMB2, PDSS2, COQ2, and NEPH1. We found that CNS describes a heterogeneous group of disorders in non-Finnish populations. We identified nephrin and podocin mutations in most families and only rarely found mutations in genes implicated in other hereditary forms of NS. In approximately 20% of cases, we could not identify the underlying genetic cause. Consistent with the major role of nephrin at the slit diaphragm, NPHS1 mutations associated with an earlier onset of disease and worse renal outcomes than NPHS2 mutations. Milder cases resulting from mutant NPHS1 had either two mutations in the cytoplasmic tail or two missense mutations in the extracellular domain, including at least one that preserved structure and function. In addition, we extend the spectrum of known NPHS1 mutations by describing long NPHS1 deletions. In summary, these data demonstrate that CNS is not a distinct clinical entity in non-Finnish populations but rather a clinically and genetically heterogeneous group of disorders.Congenital nephrotic syndrome (CNS) of the Finnish type (CNF; MIM# 256300) is a recessively inherited disorder characterized by massive proteinuria at birth, a large placenta, and marked edema within the first 3 months of life.^1–4 The disease is most frequent in Finland, where its incidence is 1 per 8200 newborns.⁵ Renal histology encompasses mesangial hypercellularity and matrix expansion, progressing with age to complete mesangial sclerosis and capillary obliteration.⁶ Irregular microcystic dilation of the proximal tubules (PTD) is the most typical histologic feature, observed as early as 18 to 20 weeks of gestation^7–9 and increasing in frequency with age¹⁰; however, PTD is not observed in all cases. Ultrastructural analysis of the glomerular capillary loops shows complete foot process effacement and swelling of endothelial cells.¹¹NPHS1, encoding nephrin, was identified by positional cloning more than a decade ago and is the major gene involved in CNF in Finnish populations (98% of cases).¹² The Fin_major (c.121delCT; p.L41fs) and Fin_minor (c.3325C>T; p.R1109X) mutations account for 78 and 16% of the mutated alleles, respectively, in Finnish cases¹²; however, these mutations are rarely found in other ethnic groups.¹³ NPHS1 genetic screening in patients of non-Finnish origin has shown that the frequency of NPHS1 mutations is lower than that in Finnish patients, with such mutations accounting for 39 to 55% of cases.^14,15 Indeed, more than 140 different mutations have been identified among non-Finnish cases,^14–24 including protein-truncating nonsense mutations, frameshift small insertion/deletion mutations, and splice-site changes.^14–24 In vitro functional assays have shown that most NPHS1 missense mutations lead to retention of the protein in the endoplasmic reticulum,^25,26 resulting in a complete loss of nephrin from the cell surface. This suggests that defective intracellular nephrin trafficking, presumably as a result of protein misfolding, is a common consequence of the NPHS1 missense mutations implicated in CNS.Nephrin is a transmembrane protein of the Ig superfamily characterized by eight C2-type Ig-like domains and a fibronectin type III repeat in the extracellular region, a single transmembrane domain, and a cytosolic C-terminal end.¹² The extracellular domain of nephrin forms homodimers and heterodimers with NEPH1.^27,28 Nephrin–NEPH1 interactions control nephrin signaling,²⁹ glomerular permeability,³⁰ and podocyte cell polarity.³¹ Neph1 knockout mice present massive proteinuria within the first 2 weeks of life and renal lesions resembling CNF in humans,^32,33 suggesting that recessive inactivating mutations in the NEPH1 gene may be involved in congenital human glomerular disease; however, no mutations have yet been identified in this gene.One striking finding among patients with CNS has been the detection of mutations in the NPHS2 gene,^19,34 encoding podocin, which has been implicated mainly in early-onset steroid-resistant nephrotic syndrome (SRNS).³⁵ A recent analysis showed that as many as 51% of patients who had CNS and were of European origin had mutations in the NPHS2 gene.¹⁵ In addition to mutations in the NPHS1 and NPHS2 genes, mutations in PLCE1 and WT1, which are known to cause infantile NS, have been implicated in cases of CNS and diffuse mesangial sclerosis (DMS).^36–40 Several hereditary forms of syndromic CNS have also been described. LAMB2 mutations have been implicated in Pierson syndrome, a rare autosomal recessive disorder characterized by microcoria and other complex ocular abnormalities associated with CNS.^41,42 Moreover, patients with NS and minor structural eye defects and others with isolated NS have expanded the phenotypic spectrum of mutations in the LAMB2 gene.^43–45 Finally, mutations in the PSSD2 and COQ2 genes have been found in patients with mitochondriopathies, typically presenting primary coenzyme Q10 deficiency and profound neuromuscular symptoms and occasionally developing NS.^46–51These clinical associations confirm the genetic heterogeneity of CNS in non-Finnish patients. It remains unclear whether subtle differences in phenotype between patients who have CNS and bear NPHS1 or NPHS2 mutations could be used to guide genetic screening. Distinctive extrarenal clinical features certainly help to direct mutational screening among patients with syndromic forms of CNS; however, patients with nonsyndromic CNS may have mutations in the WT1, LAMB2, COQ2, and PDSS2 genes, making genetic screening more difficult and time-consuming. Finally, the contribution of NEPH1 mutations to CNS has never been explored. We therefore carried out a comprehensive genetic analysis in the largest multiethnic cohort of patients with CNS of non-Finnish origin reported to date, with the aim of defining the epidemiologic role of mutations in the main genes implicated in CNS and elucidating potentially novel genotype–phenotype correlations.     

The C3a Anaphylatoxin Receptor Is a Key Mediator of Insulin Resistance and Functions by Modulating Adipose Tissue Macrophage Infiltration and Activation

OBJECTIVE

Significant new data suggest that metabolic disorders such as diabetes, obesity, and atherosclerosis all posses an important inflammatory component. Infiltrating macrophages contribute to both tissue-specific and systemic inflammation, which promotes insulin resistance. The complement cascade is involved in the inflammatory cascade initiated by the innate and adaptive immune response. A mouse genomic F2 cross biology was performed and identified several causal genes linked to type 2 diabetes, including the complement pathway.

RESEARCH DESIGN AND METHODS

We therefore sought to investigate the effect of a C3a receptor (C3aR) deletion on insulin resistance, obesity, and macrophage function utilizing both the normal-diet (ND) and a diet-induced obesity mouse model.

RESULTS

We demonstrate that high C3aR expression is found in white adipose tissue and increases upon high-fat diet (HFD) feeding. Both adipocytes and macrophages within the white adipose tissue express significant amounts of C3aR. C3aR^−/− mice on HFD are transiently resistant to diet-induced obesity during an 8-week period. Metabolic profiling suggests that they are also protected from HFD-induced insulin resistance and liver steatosis. C3aR^−/− mice had improved insulin sensitivity on both ND and HFD as seen by an insulin tolerance test and an oral glucose tolerance test. Adipose tissue analysis revealed a striking decrease in macrophage infiltration with a concomitant reduction in both tissue and plasma proinflammatory cytokine production. Furthermore, C3aR^−/− macrophages polarized to the M1 phenotype showed a considerable decrease in proinflammatory mediators.

CONCLUSIONS

Overall, our results suggest that the C3aR in macrophages, and potentially adipocytes, plays an important role in adipose tissue homeostasis and insulin resistance.The complement system is an integral part of both the innate and adaptive immune response involved in the defense against invading pathogens (1). Complement activation culminates in a massive amplification of the immune response leading to increased cell lysis, phagocytosis, and inflammation (1). C3 is the most abundant component of the complement cascade and the convergent point of all three major complement activation pathways. C3 is cleaved into C3a and C3b by the classical and lectin pathways, and iC3b is generated by the alternative pathway (2,3). C3a has potent anaphylatoxin activity, directly triggering degranulation of mast cells, inflammation, chemotaxis, activation of leukocytes, as well as increasing vascular permeability and smooth muscle contraction (3). C3a mediates its downstream signaling effects by binding to the C3a receptor (C3aR), a Gi-coupled G protein–coupled receptor. Several studies have demonstrated a role for C3a and C3aR in asthma, sepsis, liver regeneration, and autoimmune encephalomyelitis (1,3). Therefore, targeting C3aR may be an attractive therapeutic option for the treatment of several inflammatory diseases.Increasing literature suggests that metabolic disorders such as diabetes, obesity, and atherosclerosis also possess an important inflammatory component (4–7). Several seminal reports have demonstrated that resident macrophages can constitute as much as 40% of the cell population of adipose tissue (7–9) and can significantly affect insulin resistance (10–18). Several proinflammatory cytokines, growth factors, acute-phase proteins, and hormones are produced by the adipose tissue and implicated in insulin resistance and vascular homeostasis (4–7,19). An integrated genomics approach was performed with several mouse strains to infer causal relationships between gene expression and complex genetic diseases such as obesity/diabetes. This approach identified the C3aR gene as being causal for omental fat pad mass (20). The C3aR^−/− mice were shown to have decreased adiposity as compared with wild-type mice on a regular diet (20). Monocytes and macrophages express the C3aR (21–28). Increased C3a levels also correlate with obesity, diabetes, cholesterol, and lipid levels (29–34). We therefore sought to investigate the specific role of the C3aR in insulin resistance, obesity, and macrophage function utilizing both normal diet and the diet-induced obesity model.     

Recurrence of Lupus Nephritis after Kidney Transplantation

The frequency and outcome of recurrent lupus nephritis (RLN) among recipients of a kidney allograft vary among single-center reports. From the United Network for Organ Sharing files, we estimated the period prevalence and predictors of RLN in recipients who received a transplant between 1987 and 2006 and assessed the effects of RLN on allograft failure and recipients'' survival. Among 6850 recipients of a kidney allograft with systemic lupus erythematosus, 167 recipients had RLN, 1770 experienced rejection, and 4913 control subjects did not experience rejection. The period prevalence of RLN was 2.44%. Non-Hispanic black race, female gender, and age <33 years each independently increased the odds of RLN. Graft failure occurred in 156 (93%) of those with RLN, 1517 (86%) of those with rejection, and 923 (19%) of control subjects without rejection. Although recipients with RLN had a fourfold greater risk for graft failure compared with control subjects without rejection, only 7% of graft failure episodes were attributable to RLN compared and 43% to rejection. During follow-up, 867 (13%) recipients died: 27 (16%) in the RLN group, 313 (18%) in the rejection group, and 527 (11%) in the control group. In summary, severe RLN is uncommon in recipients of a kidney allograft, but black recipients, female recipient, and younger recipients are at increased risk. Although RLN significantly increases the risk for graft failure, it contributes far less than rejection to its overall incidence; therefore, these findings should not keep patients with lupus from seeking a kidney transplant.The frequency and clinical impact of recurrent lupus nephritis (RLN) in the kidney allograft of recipients with systemic lupus erythematosus (SLE) varies considerably in both prospective and retrospective studies.^1–25 In 1996, Mojcik and Klippel²⁶ pooled data from a total of 366 allografts transplanted in 338 recipients. In that review, histologic RLN was present in 3.8% of the grafts. Contrasting, in the studies by Goral et al.²⁷ and Nyberg et al.,¹⁰ RLN was reported in a much higher proportion: 30 and 44% of recipients, respectively.The clinical consequences of RLN on patient and allograft survival have ranged from no effect to a significant increase in the risk for graft loss and patient mortality.^24,27–31 In this case-control study, we estimated the period prevalence of RLN in kidney transplant recipients who had ESRD secondary to lupus nephritis and received a transplant between October 1987 and October 2006. We assessed the effects of RLN on graft failure and recipient survival and the risk factors leading to the development of RLN.     

Why Do SGLT2 Inhibitors Inhibit Only 30–50% of Renal Glucose Reabsorption in Humans?

Sodium glucose cotransporter 2 (SGLT2) inhibition is a novel and promising treatment for diabetes under late-stage clinical development. It generally is accepted that SGLT2 mediates 90% of renal glucose reabsorption. However, SGLT2 inhibitors in clinical development inhibit only 30–50% of the filtered glucose load. Why are they unable to inhibit 90% of glucose reabsorption in humans? We will try to provide an explanation to this puzzle in this perspective analysis of the unique pharmacokinetic and pharmacodynamic profiles of SGLT2 inhibitors in clinical trials and examine possible mechanisms and molecular properties that may be responsible.Type 2 diabetes is a serious global health issue that has reached epidemic proportions in both developed and developing countries over the last two decades (1). With currently available medicines, many diabetic patients fail to achieve optimal glycemic control (HbA_1c <6.5–7.0%). With the exception of the glucagon-like peptide 1 analogs and the thiazolidinediones (2), other antidiabetic medications lose their effectiveness to control hyperglycemia over time, partially due to the progressive decline of β-cell function (2–4). As a consequence, many patients receive multiple antidiabetic medicines and eventually require insulin therapy, which often fails to achieve the desired glycemic goal and is associated with weight gain and hypoglycemia (5,6). Failure to achieve glycemic targets is the primary factor responsible for the microvascular complications (retinopathy, neuropathy, nephropathy) and, to a lesser extent, macrovascular complications (2,7). In addition, the majority of diabetic patients are overweight or obese, and many of the current therapies are associated with weight gain, which causes insulin resistance and deterioration in glycemic control (2).Given the difficulty in achieving optimal glycemic control (8,9) for many diabetic patients using current therapies, there is an unmet medical need for new antidiabetic agents. Although it has been known for 50 years (10,11) that renal glucose reabsorption is increased in type 2 diabetic patients, only recently have the clinical therapeutic implications of this observation been recognized (2,12). Inhibition of renal tubular glucose reabsorption, leading to a reduction in blood glucose concentration through enhanced urinary glucose excretion, provides a novel insulin-independent therapy (2,12) that in animal models of diabetes has been shown to reverse glucotoxicity and improve insulin sensitivity and β-cell function (13,14). The majority (∼80–90%) of filtered plasma glucose is reabsorbed in the early proximal tubule by the high-capacity, low-affinity sodium glucose cotransporter (SGLT) 2 (15,16). The remaining 10–20% of filtered glucose is reabsorbed by the high-affinity, low-capacity SGLT1 transporter in the more distal portion of the proximal tubule. After glucose is actively reabsorbed by SGLT2 and SGLT1 into the proximal tubular cells, it is diffused out of the cells from the basolateral side into blood through facilitative GLUT 2 and 1 (15). Because the majority of glucose reabsorption occurs via the SGLT2 transporter, pharmaceutical companies have focused on the development of SGLT2 inhibitors, and multiple SGLT2 inhibitors currently are in human phase II and III clinical trials (17). This class of antidiabetic medication effectively lowers blood glucose levels and offers additional benefits, including weight loss, low propensity for causing hypoglycemia, and reduction in blood pressure. The SGLT2 inhibitors are effective as monotherapy and in combination with existing therapies (2,12,14,15,17), including insulin (18). Because of their unique mechanism of action (12,15), which is independent of the severity of insulin resistance and β-cell failure, type 2 diabetic individuals with recent-onset diabetes (<1 year) respond equally well as type 2 diabetic patients with long-standing diabetes (>10 years) (19).Dapagliflozin is the most advanced SGLT2 inhibitor in clinical trials (12,17,20). In addition, multiple other SGLT2 inhibitors are in phase II to III trials (Fig. 1) (17,21). However, none of these SGLT2 inhibitors are able to inhibit >30–50% of the filtered glucose load, despite in vitro studies indicate that 100% inhibition of the SGLT2 transporter should be achieved at the drug concentrations in humans (22,23). In this perspective, we shall examine potential explanations for this apparent paradox. Resolution of the paradox has important clinical implications with regard to the efficacy of this class of drugs and the development of more efficacious SGLT2 inhibitors.Open in a separate windowFIG. 1.SGLT2 inhibitors in late-stage clinical trials.     

Intraflagellar Transport Proteins Are Essential for Cilia Formation and for Planar Cell Polarity

The highly conserved intraflagellar transport (IFT) proteins are essential for cilia formation in multiple organisms, but surprisingly, cilia form in multiple zebrafish ift mutants. Here, we detected maternal deposition of ift gene products in zebrafish and found that ciliary assembly occurs only during early developmental stages, supporting the idea that maternal contribution of ift gene products masks the function of IFT proteins during initial development. In addition, the basal bodies in multiciliated cells of the pronephric duct in ift mutants were disorganized, with a pattern suggestive of defective planar cell polarity (PCP). Depletion of pk1, a core PCP component, similarly led to kidney cyst formation and basal body disorganization. Furthermore, we found that multiple ift genes genetically interact with pk1. Taken together, these data suggest that IFT proteins play a conserved role in cilia formation and planar cell polarity in zebrafish.The cilium is a cell surface organelle that is almost ubiquitously present on vertebrate cells. Protruding from the cell into its environment, the cilium is involved in multiple signaling pathways, including the Sonic hedgehog (Shh) pathway, the Wnt pathways, and the target of rapamycin (TOR) pathway.^1–5 Not surprisingly, defects in the cilium have been linked to a growing list of human diseases, coined “ciliopathies,” ranging from laterality defects, retinal degeneration, polycystic kidney disease (PKD), and other hepatorenal fibrocystic disorders to obesity and diabetes (for a review, see reference ⁶).Many studies have demonstrated that the formation and maintenance of the cilium depends on intraflagellar transport (IFT) particles, which are composed of at least 17 polypeptides.^7,8 These IFT particles move along microtubules in cilia and are thought to act as vehicles for transporting cargos needed for the assembly, maintenance, and function of cilia. Homologs of IFT proteins have been found in a wide spectrum of organisms including Caenorhabditis elegans, Drosophila, and mammals and have also been shown to be required for cilia formation.^3,9–12In zebrafish, mutants of ift57, ift81, ift88, and ift172 have numerous defects commonly associated with ciliary abnormalities.^13,14 However, cilia in these mutants are able to form initially but degenerate over time, giving rise to the hypothesis that IFT is essential for cilia maintenance rather than cilia assembly in zebrafish.¹⁴ Interestingly, products of many genes in zebrafish are deposited maternally. One hypothesis for the initial formation of cilia in zebrafish ift mutants is that maternal contribution of ift gene products masks the function of ift genes during early embryonic development. Accordingly, cilia formation is severely impaired in a maternal-zygotic mutant of ift88.¹⁵ In this study, we demonstrate that products of ift57 and ift172 are indeed maternally deposited. We further show that although cilia destined to form early in development show only maintenance defects in ift57^hi3417 and ift172^hi2211 mutants, cilia programmed to assemble later in development fail to form, providing further support for a conserved role of IFT in cilia formation in zebrafish.One of the most extensively studied phenotypes associated with ciliary defects is the formation of kidney cysts. Both the canonical and the noncanonical Wnt pathway, or planar cell polarity (PCP) pathway, have been implicated in kidney cyst formation.^4,16–21 However, in contrast to the well-established role of cilia in the hedgehog pathway,^2,3,22 the role of cilia in the Wnt pathways is unclear.^4,15,23,24 In this study, we demonstrate that, in the kidney duct of ift57^hi3417 and ift172^hi2211 mutants, the organization of basal bodies is impaired, a phenotype consistent with compromised PCP signaling. We further show that knockdown of prickle 1 (pk1), a core PCP player, leads to disorganization of basal bodies and kidney cyst formation. Finally, we provide evidence that ift57, ift88, and ift172 genetically interact with pk1 in convergence-extension (CE) movements during gastrulation, a process regulated by the PCP pathway. Together, these data support an intricate relationship between IFT and the PCP pathway.     

Human Recombinant ACE2 Reduces the Progression of Diabetic Nephropathy

OBJECTIVE

Diabetic nephropathy is one of the most common causes of end-stage renal failure. Inhibition of ACE2 function accelerates diabetic kidney injury, whereas renal ACE2 is downregulated in diabetic nephropathy. We examined the ability of human recombinant ACE2 (hrACE2) to slow the progression of diabetic kidney injury.

RESEARCH DESIGN AND METHODS

Male 12-week-old diabetic Akita mice (Ins2^WT/C96Y) and control C57BL/6J mice (Ins2^WT/WT) were injected daily with placebo or with rhACE2 (2 mg/kg, i.p.) for 4 weeks. Albumin excretion, gene expression, histomorphometry, NADPH oxidase activity, and peptide levels were examined. The effect of hrACE2 on high glucose and angiotensin II (ANG II)–induced changes was also examined in cultured mesangial cells.

RESULTS

Treatment with hrACE2 increased plasma ACE2 activity, normalized blood pressure, and reduced the urinary albumin excretion in Akita Ins2^WT/C96Y mice in association with a decreased glomerular mesangial matrix expansion and normalization of increased α-smooth muscle actin and collagen III expression. Human recombinant ACE2 increased ANG 1–7 levels, lowered ANG II levels, and reduced NADPH oxidase activity. mRNA levels for p47^phox and NOX2 and protein levels for protein kinase Cα (PKCα) and PKCβ1 were also normalized by treatment with hrACE2. In vitro, hrACE2 attenuated both high glucose and ANG II–induced oxidative stress and NADPH oxidase activity.

CONCLUSIONS

Treatment with hrACE2 attenuates diabetic kidney injury in the Akita mouse in association with a reduction in blood pressure and a decrease in NADPH oxidase activity. In vitro studies show that the protective effect of hrACE2 is due to reduction in ANG II and an increase in ANG 1–7 signaling.Chronic kidney disease is recognized as an increasing global public health problem due in part to the increasing prevalence of diabetes (1–3). Activation of the renin-angiotensin system (RAS) and the generation of angiotensin II (ANG II) play an important pathogenic role in diabetic nephropathy, and blockade of the RAS attenuates the development of diabetic kidney injury (4–8). The discovery of a homologue of the classical ACE, ACE2, has introduced a new enzyme in ANG peptide metabolism (9–12). Like ACE, ACE2 is membrane bound, but it is a monocarboxypeptidase that generates ANG (1–7) from the octapeptide ANG II (9,10,12,13). As such, ACE2 serves as an endogenous negative regulator of the renin-angiotensin system.In animal models of diabetes, early increases in ACE2 mRNA levels, protein expression, and ACE2 activity occurs (14,15), whereas ACE2 mRNA and protein levels have been found to decrease in older streptozotocin-induced diabetic rats (16). Loss of ACE2 is associated with age-dependent glomerulosclerosis and albuminuria (17) and exacerbation of diabetic kidney injury in Akita mice (18) and is preventable by angiotensin type 1 (AT1) receptor blockade. In patients with type 2 diabetes, glomerular and tubular ACE2 expressions are reduced in the setting of increased ACE expression (19,20). Taken together, these studies suggest that ACE2 may play an early protective role against the development of diabetic nephropathy (18,21,22). We hypothesized that treatment with human recombinant ACE2 (hrACE2) will target the diabetic glomerulus and slow progression of diabetic nephropathy in the Akita mouse (Ins2^WT/C96Y), a model of type 1 diabetes.     

Hypothalamic AMP-Activated Protein Kinase Regulates Glucose Production

OBJECTIVE

The fuel sensor AMP-activated protein kinase (AMPK) in the hypothalamus regulates energy homeostasis by sensing nutritional and hormonal signals. However, the role of hypothalamic AMPK in glucose production regulation remains to be elucidated. We hypothesize that bidirectional changes in hypothalamic AMPK activity alter glucose production.

RESEARCH DESIGN AND METHODS

To introduce bidirectional changes in hypothalamic AMPK activity in vivo, we first knocked down hypothalamic AMPK activity in male Sprague-Dawley rats by either injecting an adenovirus expressing the dominant-negative form of AMPK (Ad-DN AMPKα2 [D¹⁵⁷A]) or infusing AMPK inhibitor compound C directly into the mediobasal hypothalamus. Next, we independently activated hypothalamic AMPK by delivering either an adenovirus expressing the constitutive active form of AMPK (Ad-CA AMPKα1³¹² [T172D]) or the AMPK activator AICAR. The pancreatic (basal insulin)-euglycemic clamp technique in combination with the tracer-dilution methodology was used to assess the impact of alternations in hypothalamic AMPK activity on changes in glucose kinetics in vivo.

RESULTS

Injection of Ad-DN AMPK into the hypothalamus knocked down hypothalamic AMPK activity and led to a significant suppression of glucose production with no changes in peripheral glucose uptake during the clamps. In parallel, hypothalamic infusion of AMPK inhibitor compound C lowered glucose production as well. Conversely, molecular and pharmacological activation of hypothalamic AMPK negated the ability of hypothalamic nutrients to lower glucose production.

CONCLUSIONS

These data indicate that changes in hypothalamic AMPK activity are sufficient and necessary for hypothalamic nutrient-sensing mechanisms to alter glucose production in vivo.AMP-activated protein kinase (AMPK) is an evolutionarily conserved cellular energy sensor that regulates cellular metabolism (1). Consisting of a catalytic α subunit and two regulatory β and γ subunits, AMPK responds to an increase in intracellular AMP-to-ATP ratio and phosphorylates intracellular targets involved in cellular metabolism to promote ATP-generating processes and inhibit energy-consuming pathways. AMPK is expressed in a variety of tissues including the liver, skeletal muscles, adipose tissue, and the hypothalamus (1). AMPK phosphorylates and inhibits acetyl-CoA carboxylase (ACC) (1), which prevents the conversion of acetyl-CoA to malonyl-CoA. A decrease in malonyl-CoA relieves the inhibition of carnitine palmitoyltransferase-1 (2) and favors the transfer of long-chain fatty acyl-CoA (LCFA-CoA) into the mitochondria for β-oxidation. Conversely, direct inhibition of AMPK increases malonyl-CoA and LCFA-CoA levels (3).Studies have emerged implicating that AMPK in the hypothalamus integrates nutritional and hormonal signals to regulate food intake (4–8). In particular, direct inhibition of hypothalamic AMPK lowers food intake (8), whereas selective activation of hypothalamic AMPK negates the ability of leptin to activate hypothalamic ACC, increase hypothalamic malonyl-CoA levels, and lower food intake (9). In light of the fact that the hypothalamus integrates nutritional and hormonal signals to not only regulate energy (10–12) but also glucose (13–17) homeostasis, and that accumulation of hypothalamic malonyl-CoA and LCFA-CoA levels lowers food intake as well as hepatic glucose production (18–20), a possibility arises that direct inhibition of hypothalamic AMPK activity could alter hepatic glucose production (Fig. 1A). This working hypothesis was first tested in the current study.Open in a separate windowFIG. 1.Molecular knockdown of hypothalamic AMPK by the dominant-negative form of AMPK (DN AMPK) is sufficient to lower glucose production. A: Schematic representation of the working hypothesis: Inhibition of hypothalamic AMPK activity by DN AMPK or compound C leads to the lowering of hepatic glucose production. B: Experimental procedure and clamp protocol. A bilateral MBH catheter was implanted on day 0. Adenovirus tagged with GFP (Ad-GFP) or adenovirus-expressing DN AMPK (Ad-DN AMPK) was injected into the MBH of a group of rats immediately after MBH catheter implantation. Venous and arterial cannulations were done on day 5, and the pancreatic clamp protocol was performed on day 8. In the Ad-GFP and Ad-DN AMPK–injected rats, no MBH infusions were given during the clamp experiments. In rats with no adenovirus injection, 5% DMSO control or compound C was infused into the MBH during the clamps. C: Hypothalamic AMPK activity was significantly diminished in animals injected with Ad-DN AMPK, compared with control animals with injection of Ad-GFP (*P < 0.001). Hypothalamic injection of Ad-DN AMPK led to an increase in glucose infusion rate (D) (*P < 0.01) and a decrease in glucose production (E) (*P < 0.001) compared with the GFP control. F: Suppression of glucose production during the clamp period (180–210 min) expressed as percentage reduction from basal steady state (60–90 min) (*P < 0.01 vs. GFP control). G: Glucose uptake was not significantly different from that of GFP control. Values are shown as means ± SEM. (A high-quality color representation of this figure is available in the online issue.)Second, hypothalamus glucose metabolism to lactate, and the subsequent conversion of lactate to pyruvate and acetyl-CoA, have been reported to lower hepatic glucose production (21). However, the downstream biochemical pathways that mediate the ability of hypothalamic glucose/lactate sensing to lower glucose production remain unclear, although it was hypothesized that the formation of malonyl-CoA via the enhanced flux of acetyl-CoA could be a necessary step (3,15). Given the well-established regulatory role of AMPK on the formation of malonyl-CoA from acetyl-CoA and that hypothalamic malonyl-CoA regulates glucose production (18), we next tested the possibility that direct activation of hypothalamic AMPK negates the ability of central nervous system glucose/lactate sensing to regulate glucose production.In summary, we tested the hypothesis that molecular and pharmacological changes in hypothalamic AMPK activity are sufficient and necessary for hypothalamic nutrient-sensing mechanisms to regulate glucose production in vivo.     

Muscle Contraction, but Not Insulin, Increases Microvascular Blood Volume in the Presence of Free Fatty Acid�CInduced Insulin Resistance

OBJECTIVE

Insulin and contraction each increase muscle microvascular blood volume (MBV) and glucose uptake. Inhibiting nitric oxide synthase blocks insulin''s but not contraction''s effects. We examined whether contraction could augment the MBV increase seen with physiologic hyperinsulinemia and whether free fatty acid (FFA)-induced insulin resistance differentially affects contraction- versus insulin-mediated increases in MBV.

RESEARCH DESIGN AND METHODS

Rats were fasted overnight. Plasma FFAs were increased by intralipid/heparin infusion (3 h), insulin was increased with a euglycemic clamp (3 mU · min⁻¹ · kg⁻¹), and hindlimb muscle contraction was electrically stimulated. Muscle MBV was measured using contrast-enhanced ultrasound. Insulin transport into muscle was measured using ¹²⁵I-insulin. BQ-123 (0.4 mg/h) was used to block the endothelin-1 (ET-1) receptor A.

RESULTS

Superimposing contraction on physiologic hyperinsulinemia increased MBV within 10 min by 37 and 67% for 0.1 or 1 Hz, respectively (P < 0.01). FFA elevation alone did not affect MBV, whereas 0.1 Hz stimulation doubled MBV (P < 0.05) and increased muscle insulin uptake (P < 0.05) despite high FFA. Physiologic hyperinsulinemia during FFA elevation paradoxically decreased MBV (P < 0.05). This MBV decrease was reversed by either 0.1 Hz contraction or ET-1 receptor A antagonism, and the combination raised MBV above basal.

CONCLUSIONS

Contraction recruits microvasculature beyond that seen with physiologic hyperinsulinemia by a distinct mechanism that is not blocked by FFA-induced vascular insulin resistance. The paradoxical MBV decline seen with insulin plus FFA may result from differential inhibition of insulin-stimulated nitric oxide–dependent vasodilation relative to ET-1 vasoconstriction. Our results implicate ET-1 as a potential mediator of FFA-induced vascular insulin resistance.Insulin delivery to muscle interstitium is reported to be rate limiting for overall muscle insulin action (1,2). Insulin promotes its own access to muscle interstitium by increasing blood flow (3), by recruiting microvasculature (4,5) to expand the endothelial transporting surface available, and perhaps by also stimulating its own endothelial transport (6). Insulin''s entry to muscle interstitium is delayed in insulin-resistant states (7). This implicates insulin''s vascular actions as a significant regulator of overall insulin action in muscle.Elevated plasma concentrations of free fatty acids (FFAs), as occur with obesity and type 2 diabetes, increase cellular lipid concentrations and are associated with insulin resistance in skeletal muscle, liver, and fat (8,9). Experimentally, increased dietary fat (10–12) or acute infusion of a lipid emulsion induces insulin resistance (13–16). Increased intramyocellular lipid content in the context of obesity and type 2 diabetes could be one factor that contributes to muscle insulin resistance. Postprandially or in response to a euglycemic-insulin clamp, plasma (FFA) falls in insulin-sensitive individuals (17–19). This response is impaired in states of insulin resistance (8,17,19,20).Insulin also increases muscle blood flow and recruits microvasculature in both humans (21–24) and animals (4,25–27); both processes are inhibited by nitric oxide synthases (NOS) blockade (27). Raising plasma FFAs initiates hemodynamic effects that include decreased compliance, increased blood pressure and heart rate, and increased vascular resistance (28–31). Raising plasma (FFA) blunts insulin''s NOS-dependent effects to mediate increases in both muscle microvascular blood volume (MBV) and glucose uptake (14,32,33). Thus, FFAs exert acute vascular as well as metabolic actions.Insulin (34) and muscle contraction can each increase MBV and total flow in skeletal muscle (35–37). In addition, Wheatley et al. (38) observed that in the Zucker rat, insulin-mediated increases in MBV are blunted, but contraction-induced increases in MBV persisted. This suggests that exercise might recruit microvasculature via a mechanism that is distinct from that of insulin. Supporting this, we have recently shown that like insulin, brief low-frequency isometric contraction of the rat hindlimb (0.1 Hz, 10 min) robustly increases MBV without any observed increase in total femoral blood flow (FBF) and, unlike insulin''s effect, this process is nitric oxide (NO)-independent (39).In this study, we addressed whether 1) low-frequency contraction enhances muscle MBV and ³H-2-deoxyglucose (³H-2-DG) uptake beyond the effect of physiologic hyperinsulinemia; 2) lipid infusion differentially affects contraction- versus insulin-mediated increases in MBV; and 3) lipid infusion blunts combined insulin- and contraction-mediated effects on MBV.