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.     

Epac Regulates UT-A1 to Increase Urea Transport in Inner Medullary Collecting Ducts

Urea plays a critical role in the concentration of urine, thereby regulating water balance. Vasopressin, acting through cAMP, stimulates urea transport across rat terminal inner medullary collecting ducts (IMCD) by increasing the phosphorylation and accumulation at the apical plasma membrane of UT-A1. In addition to acting through protein kinase A (PKA), cAMP also activates Epac (exchange protein activated by cAMP). In this study, we tested whether the regulation of urea transport and UT-A1 transporter activity involve Epac in rat IMCD. Functional analysis showed that an Epac activator significantly increased urea permeability in isolated, perfused rat terminal IMCD. Similarly, stimulating Epac by adding forskolin and an inhibitor of PKA significantly increased urea permeability. Incubation of rat IMCD suspensions with the Epac activator significantly increased UT-A1 phosphorylation and its accumulation in the plasma membrane. Furthermore, forskolin-stimulated cAMP significantly increased ERK 1/2 phosphorylation, which was not prevented by inhibiting PKA, indicating that Epac mediated this phosphorylation of ERK 1/2. Inhibition of MEK 1/2 phosphorylation decreased the forskolin-stimulated UT-A1 phosphorylation. Taken together, activation of Epac increases urea transport, accumulation of UT-A1 at the plasma membrane, and UT-A1 phosphorylation, the latter of which is mediated by the MEK–ERK pathway.Urea plays a crucial role in the urinary concentrating mechanism, and therefore, in the regulation of water balance. Urea''s importance to the generation of a concentrated urine has been appreciated since at least 1934.^1,2 Several studies have shown that maximal urine concentrating ability is decreased in protein-deprived mammals and is restored by urea infusion.³ More recently, a UT-A1/UT-A3 knock-out mouse,^4,5 a UT-A2 knock-out mouse,⁶ and a UT-B knock-out mouse^7–9 were each shown to have urine concentrating defects. Thus, any hypothesis regarding the mechanism by which the kidney concentrates urine needs to include some effect derived from urea.The UT-A1 urea transporter is expressed in the terminal inner medullary collecting duct (IMCD).¹⁰ Vasopressin stimulates urea transport across perfused rat terminal IMCDs by increasing UT-A1 phosphorylation and apical plasma membrane accumulation.^11–15 Vasopressin acts by binding to V₂ receptors in the basolateral plasma membrane, stimulating adenylyl cyclase, increasing cAMP production, and increasing urea transport.^11,16–18 Forskolin, which directly activates adenylyl cyclase,¹⁹ also increases urea transport in perfused rat terminal IMCDs.²⁰cAMP is traditionally thought to act through protein kinase A (PKA). However, when we stimulate the PKA activity by increasing cAMP with forskolin in MDCK cells that are stably transfected with UT-A1 (UT-A1-MDCK cells), only 50% of the forskolin-stimulated urea flux is inhibited by H-89, a PKA inhibitor.²¹ Vasopressin and forskolin work in a similar manner to increase the cAMP levels, so this partial inhibition by H-89 suggests that vasopressin may signal through two cAMP-dependent pathways: one involving PKA and one that is independent of PKA. Because the UT-A1-MDCK cells reproduce many of the properties of native rat IMCDs,^13,21,22 these findings raise the possibility that vasopressin may signal through a second cAMP-dependent, but non–PKA-dependent, pathway in rat IMCDs.In addition to PKA, cAMP can activate Epac (exchange protein activated by cAMP), which signals by activating Rap1, a Ras-related small molecular weight G protein, which in turn signals through mitogen-activated protein kinase kinase (MEK) and extracellular signal-related kinase (ERK)^23,24 (Figure 1). There are two closely related exchange proteins activated by cAMP (Epac) proteins, Epac1 and Epac2, and both have been detected in rat IMCDs, although one or the other predominates in different studies.^25–28 The purpose of this study was to determine whether activation of the Epac pathway resulted in a functional change in urea transport in perfused rat terminal IMCDs.Open in a separate windowFigure 1.Vasopressin signaling diagram.     

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.     

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.     

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.     

Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice

OBJECTIVE

Oxyntomodulin (OXM) is a glucagon-like peptide 1 (GLP-1) receptor (GLP1R)/glucagon receptor (GCGR) dual agonist peptide that reduces body weight in obese subjects through increased energy expenditure and decreased energy intake. The metabolic effects of OXM have been attributed primarily to GLP1R agonism. We examined whether a long acting GLP1R/GCGR dual agonist peptide exerts metabolic effects in diet-induced obese mice that are distinct from those obtained with a GLP1R-selective agonist.

RESEARCH DESIGN AND METHODS

We developed a protease-resistant dual GLP1R/GCGR agonist, DualAG, and a corresponding GLP1R-selective agonist, GLPAG, matched for GLP1R agonist potency and pharmacokinetics. The metabolic effects of these two peptides with respect to weight loss, caloric reduction, glucose control, and lipid lowering, were compared upon chronic dosing in diet-induced obese (DIO) mice. Acute studies in DIO mice revealed metabolic pathways that were modulated independent of weight loss. Studies in Glp1r^−/− and Gcgr^−/− mice enabled delineation of the contribution of GLP1R versus GCGR activation to the pharmacology of DualAG.

RESULTS

Peptide DualAG exhibits superior weight loss, lipid-lowering activity, and antihyperglycemic efficacy comparable to GLPAG. Improvements in plasma metabolic parameters including insulin, leptin, and adiponectin were more pronounced upon chronic treatment with DualAG than with GLPAG. Dual receptor agonism also increased fatty acid oxidation and reduced hepatic steatosis in DIO mice. The antiobesity effects of DualAG require activation of both GLP1R and GCGR.

CONCLUSIONS

Sustained GLP1R/GCGR dual agonism reverses obesity in DIO mice and is a novel therapeutic approach to the treatment of obesity.Obesity is an important risk factor for type 2 diabetes, and ∼90% of patients with type 2 diabetes are overweight or obese (1). Among new therapies for type 2 diabetes, peptidyl mimetics of the gut-derived incretin hormone glucagon-like peptide 1 (GLP-1) stimulate insulin biosynthesis and secretion in a glucose-dependent manner (2,3) and cause modest weight loss in type 2 diabetic patients. The glucose-lowering and antiobesity effects of incretin-based therapies for type 2 diabetes have prompted evaluation of the therapeutic potential of other glucagon-family peptides, in particular oxyntomodulin (OXM). The OXM peptide is generated by post-translational processing of preproglucagon in the gut and is secreted postprandially from l-cells of the jejuno-ileum together with other preproglucagon-derived peptides including GLP-1 (4,5). In rodents, OXM reduces food intake and body weight, increases energy expenditure, and improves glucose metabolism (6–8). A 4-week clinical study in obese subjects demonstrated that repeated subcutaneous administration of OXM was well tolerated and caused significant weight loss with a concomitant reduction in food intake (9). An increase in activity-related energy expenditure was also noted in a separate study involving short-term treatment with the peptide (10).OXM activates both, the GLP-1 receptor (GLP1R) and glucagon receptor (GCGR) in vitro, albeit with 10- to 100-fold reduced potency compared with the cognate ligands GLP-1 and glucagon, respectively (11–13). It has been proposed that OXM modulates glucose and energy homeostasis solely by GLP1R agonism, because its acute metabolic effects in rodents are abolished by coadministration of the GLP1R antagonist exendin(9–39) and are not observed in Glp1r^−/− mice (7,8,14,15). Other aspects of OXM pharmacology, however, such as protective effects on murine islets and inhibition of gastric acid secretion appear to be independent of GLP1R signaling (14). In addition, pharmacological activation of GCGR by glucagon, a master regulator of fasting metabolism (16), decreases food intake in rodents and humans (17–19), suggesting a potential role for GCGR signaling in the pharmacology of OXM. Because both OXM and GLP-1 are labile in vivo (T_1/2 ∼12 min and 2–3 min, respectively) (20,21) and are substrates for the cell surface protease dipeptidyl peptidase 4 (DPP-4) (22), we developed two long-acting DPP-4–resistant OXM analogs as pharmacological agents to better investigate the differential pharmacology and therapeutic potential of dual GLP1R/GCGR agonism versus GLP1R-selective agonism. Peptide DualAG exhibits in vitro GLP1R and GCGR agonist potency comparable to that of native OXM and is conjugated to cholesterol via a Cys sidechain at the C-terminus for improved pharmacokinetics. Peptide GLPAG differs from DualAG by only one residue (Gln³→Glu) and is an equipotent GLP1R agonist, but has no significant GCGR agonist or antagonist activity in vitro. The objective of this study was to leverage the matched GLP1R agonist potencies and pharmacokinetics of peptides DualAG and GLPAG in comparing the metabolic effects and therapeutic potential of a dual GLP1R/GCGR agonist with a GLP1R-selective agonist in a mouse model of obesity.     

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.     

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.     

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.     

Pregnancy and Maternal Outcomes Among Kidney Transplant Recipients

Fertility rates, pregnancy, and maternal outcomes are not well described among women with a functioning kidney transplant. Using data from the Australian and New Zealand Dialysis and Transplant Registry, we analyzed 40 yr of pregnancy-related outcomes for transplant recipients. This analysis included 444 live births reported from 577 pregnancies; the absolute but not relative fertility rate fell during these four decades. Of pregnancies achieved, 97% were beyond the first year after transplantation. The mean age at the time of pregnancy was 29 ± 5 yr. Compared with previous decades, the mean age during the last decade increased significantly to 32 yr (P < 0.001). The proportion of live births doubled during the last decade, whereas surgical terminations declined (P < 0.001). The fertility rate (or live-birth rate) for this cohort of women was 0.19 (95% confidence interval 0.17 to 0.21) relative to the Australian background population. We also matched 120 parous with 120 nulliparous women by year of transplantation, duration of transplant, age at transplantation ±5 yr, and predelivery creatinine for parous women or serum creatinine for nulliparous women; a first live birth was not associated with a poorer 20-yr graft or patient survival. Maternal complications included preeclampsia in 27% and gestational diabetes in 1%. Taken together, these data confirm that a live birth in women with a functioning graft does not have an adverse impact on graft and patient survival.One of the many perceived benefits of kidney transplantation has been restoration of pituitary-ovarian function and fertility in women of reproductive age. Prenatal advice for women with a functioning kidney transplant has been primarily based on data derived from observational research,^1–13 and the reported live-birth rates achieved in such women range from 43.2¹⁴ to 82%.¹⁵Although an increased pregnancy event number has been reported for women with a functioning kidney transplant,¹⁶ little is actually known about “pregnancy rate changes” during the past 40 yr. More importantly, long-term graft and maternal survival analyses, referred to when advising women who have undergone transplantation and are considering a pregnancy, have been mostly performed without adequate matching,¹² or, alternatively, matching has been used but outcomes followed up for only brief intervals^14,17,18 and in small cohorts.^19–22 Published graft matching studies to date suggest no adverse impact 10 yr after a live birth.¹⁴In most instances, pregnancies in women with a kidney graft have been encouraged. Historically, renal function,^8,15,17,18 baseline proteinuria,²³ intercurrent hypertension,^1,24 and time from transplantation^{1,3,5,8,14,15,18,24,25} have been used to predict adverse event risks to the mother, kidney, and offspring. To this are added the often unquantifiable inherent risks for genetically transmitted diseases or the problems associated with prematurity.^26,27 More recently, epidemiologic evidence suggests low birth weight may be associated with the development of hypertension,²⁸ cardiovascular disease,²⁹ insulin resistance,³⁰ and end-stage renal failure.³¹ Moreover, low birth weight is associated with an increased risk for hypertension, independent of genetic and shared environmental factors.³²Series published to date have not captured all pregnancy events or their outcomes. Limitations of some of the published studies include short duration of follow-up and studies with no adequate or long-term matching for decade and renal function.We examined fertility rates, pregnancy rates, and pregnancy outcomes over 40 yr in an at-risk population, defined as women who were aged between 15 and 49 and had a functioning kidney transplant, using ANZDATA registry data. In addition, maternal and graft outcomes were analyzed, and, uniquely, a matched cohort analysis of 120 nulliparous and 120 parous women who had undergone transplantation enabled analysis of outcomes at 20 yr.     

Association of 18 Confirmed Susceptibility Loci for Type 2 Diabetes With Indices of Insulin Release, Proinsulin Conversion, and Insulin Sensitivity in 5,327 Nondiabetic Finnish Men

OBJECTIVE

We investigated the effects of 18 confirmed type 2 diabetes risk single nucleotide polymorphisms (SNPs) on insulin sensitivity, insulin secretion, and conversion of proinsulin to insulin.

RESEARCH DESIGN AND METHODS

A total of 5,327 nondiabetic men (age 58 ± 7 years, BMI 27.0 ± 3.8 kg/m²) from a large population-based cohort were included. Oral glucose tolerance tests and genotyping of SNPs in or near PPARG, KCNJ11, TCF7L2, SLC30A8, HHEX, LOC387761, CDKN2B, IGF2BP2, CDKAL1, HNF1B, WFS1, JAZF1, CDC123, TSPAN8, THADA, ADAMTS9, NOTCH2, KCNQ1, and MTNR1B were performed. HNF1B rs757210 was excluded because of failure to achieve Hardy-Weinberg equilibrium.

RESULTS

Six SNPs (TCF7L2, SLC30A8, HHEX, CDKN2B, CDKAL1, and MTNR1B) were significantly (P < 6.9 × 10⁻⁴) and two SNPs (KCNJ11 and IGF2BP2) were nominally (P < 0.05) associated with early-phase insulin release (InsAUC_0–30/GluAUC_0–30), adjusted for age, BMI, and insulin sensitivity (Matsuda ISI). Combined effects of these eight SNPs reached −32% reduction in InsAUC_0–30/GluAUC_0–30 in carriers of ≥11 vs. ≤3 weighted risk alleles. Four SNPs (SLC30A8, HHEX, CDKAL1, and TCF7L2) were significantly or nominally associated with indexes of proinsulin conversion. Three SNPs (KCNJ11, HHEX, and TSPAN8) were nominally associated with Matsuda ISI (adjusted for age and BMI). The effect of HHEX on Matsuda ISI became significant after additional adjustment for InsAUC_0–30/GluAUC_0–30. Nine SNPs did not show any associations with examined traits.

CONCLUSIONS

Eight type 2 diabetes–related loci were significantly or nominally associated with impaired early-phase insulin release. Effects of SLC30A8, HHEX, CDKAL1, and TCF7L2 on insulin release could be partially explained by impaired proinsulin conversion. HHEX might influence both insulin release and insulin sensitivity.Impaired insulin secretion and insulin resistance, two main pathophysiological mechanisms leading to type 2 diabetes, have a significant genetic component (1). Recent studies have confirmed 20 genetic loci reproducibly associated with type 2 diabetes (2–13). Three were previously known (PPARG, KCNJ11, and TCF7L2), whereas 17 loci were recently discovered either by genome-wide association studies (SLC30A8, HHEX-IDE, LOC387761, CDKN2A/2B, IGF2BP2, CDKAL1, FTO, JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, THADA, ADAMTS9, NOTCH2, KCNQ1, and MTNR1B), or candidate gene approach (WFS1 and HNF1B). The mechanisms by which these genes contribute to the development of type 2 diabetes are not fully understood.PPARG is the only gene from the 20 confirmed loci previously associated with insulin sensitivity (14,15). Association with impaired β-cell function has been reported for 14 loci (KCNJ11, SLC30A8, HHEX-IDE, CDKN2A/2B, IGF2BP2, CDKAL1, TCF7L2, WFS1, HNF1B, JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, KCNQ1, and MTNR1B) (6,12,13,16–38). Although associations of variants in HHEX (16–22), CDKAL1 (6,21–26), TCF7L2 (22,27–30), and MTNR1B (13,31,32) with impaired insulin secretion seem to be consistent across different studies, information concerning other genes is limited (12,18–25,27,33–38). The mechanisms by which variants in these genes affect insulin secretion are unknown. However, a few recent studies suggested that variants in TCF7L2 (22,39–42), SLC30A8 (22), CDKAL1 (22), and MTNR1B (31) might influence insulin secretion by affecting the conversion of proinsulin to insulin. Variants of FTO have been shown to confer risk for type 2 diabetes through their association with obesity (7,16) and therefore were not included in this study.Large population-based studies can help to elucidate the underlying mechanisms by which single nucleotide polymorphisms (SNPs) of different risk genes predispose to type 2 diabetes. Therefore, we investigated confirmed type 2 diabetes–related loci for their associations with insulin sensitivity, insulin secretion, and conversion of proinsulin to insulin in a population-based sample of 5,327 nondiabetic Finnish men.     

Albuminuria and Estimated Glomerular Filtration Rate Independently Associate with Acute Kidney Injury

Acute kidney injury (AKI) is increasingly common and a significant contributor to excess death in hospitalized patients. CKD is an established risk factor for AKI; however, the independent graded association of urine albumin excretion with AKI is unknown. We analyzed a prospective cohort of 11,200 participants in the Atherosclerosis Risk in Communities (ARIC) study for the association between baseline urine albumin-to-creatinine ratio and estimated GFR (eGFR) with hospitalizations or death with AKI. The incidence of AKI events was 4.0 per 1000 person-years of follow-up. Using participants with urine albumin-to-creatinine ratios <10 mg/g as a reference, the relative hazards of AKI, adjusted for age, gender, race, cardiovascular risk factors, and categories of eGFR were 1.9 (95% CI, 1.4 to 2.6), 2.2 (95% CI, 1.6 to 3.0), and 4.8 (95% CI, 3.2 to 7.2) for urine albumin-to-creatinine ratio groups of 11 to 29 mg/g, 30 to 299 mg/g, and ≥300 mg/g, respectively. Similarly, the overall adjusted relative hazard of AKI increased with decreasing eGFR. Patterns persisted within subgroups of age, race, and gender. In summary, albuminuria and eGFR have strong, independent associations with incident AKI.It has long been recognized that an episode of acute kidney injury (AKI) can have serious health consequences.^1–4 Even a relatively small degree of renal injury increases a patient''s risk of a prolonged hospital stay, chronic kidney disease (CKD), ESRD, and death.^2,5–10 Over the last 2 decades, the incidence of hospitalized AKI has increased dramatically.^11–14 Precise estimations vary depending on population and method of case identification, but a recent community-based study of AKI estimated the incidence of nondialysis requiring AKI at 522 per 100,000 population per year and dialysis-requiring AKI at 30 per 100,000,¹³ which is well over that of ESRD.¹⁴ This increase in the burden of disease, taken with the associated poor long-term outcomes, has established AKI as a major public health issue.¹⁴Beyond routine supportive care, there exists little established medical therapy for AKI.¹⁵ Many current lines of research are focused on the prevention of AKI. However, few prospective, population-based studies have evaluated the development of AKI.^3,13,16 Hsu et al.,^13,17 along with multiple observational series in various clinical settings, have clearly established older age and CKD as risk factors for AKI.^18–24 Other observed associations with AKI include black race and male gender.^11,18,25 Proteinuria, an established risk factor in the development of cardiovascular disease,^26,27 ESRD,²⁸ and death,²⁹ is less studied in its role in the development of AKI. Hsu and colleagues demonstrated the prospective association of proteinuria with dialysis-requiring AKI; however, the proteinuria classification was binary and based on dipstick measurement.¹⁷ To our knowledge, no study has quantified the independent dose response of albuminuria with AKI hospitalization, including less severe AKI. Our study''s objective was thus to characterize prospectively the association between baseline urine albumin-to-creatinine ratio (UACR) and hospitalizations for AKI, controlling for established and potential risk factors such as CKD, age, and cardiovascular comorbidities.     

Kallikrein 5-Mediated Inflammation in Rosacea: Clinically Relevant Correlations with Acute and Chronic Manifestations in Rosacea and How Individual Treatments May Provide Therapeutic Benefit

Rosacea is a chronic inflammatory condition of facial skin estimated to affect more than 16 million Americans. Although the pathogenesis of rosacea is not fully understood, recent evidence in vitro as well as in vivo has supported the role of increased levels of the trypsin-like serine protease, kallikrein 5, in initiating an augmented inflammatory response in rosacea. The increase in the quantity and magnitude of biological activity of kallikrein 5 leads to production of greater quantities of cathelicidin (LL-37), an antimicrobial peptide associated with increases in innate cutaneous inflammation, vasodilation, and vascular proliferation, all of which are characteristic features of rosacea. In this article, the authors review the literature supporting the role of kallikrein 5 in the pathophysiology of rosacea, including how therapeutic interventions modulate the effects of kallikrein 5, thus providing further support for this pathophysiological model that at least partially explains many of the clinical features of cutaneous rosacea.Cutaneous rosacea (rosacea) is a chronic inflammatory facial skin disorder noted most commonly in individuals of northern European descent, although people of any ethnicity or skin color may be affected.1-4 The visible manifestations with central facial predominance are characteristic of rosacea, including erythema, papules, pustules, telangiectasias, and phymatous changes.1-4 However, persistent (nontransient) erythema involving the central face that intensifies during flares and the presence of telangiectasias, which are also accentuated mostly on the central face, are the core clinical features that support a diagnosis of rosacea.1-9 Papules and pustules are not consistently present in rosacea, characterizing only those individuals with rosacea who exhibit the papulopustular subtype of the disease.3-6 In fact, papulopustular lesions never emerge in many individuals affected by rosacesa, and phymatous changes affect only a relatively small number of the rosacea-affected population; however, central facial erythema is present to some extent in essentially all people with rosacea.1-8Why do some people get rosacea and others do not? Although the entire explanation that would fully answer this question remains elusive, current evidence suggests that individuals affected by rosacea exhibit rosacea-prone skin, which inherently displays dysregulation of two main systems present within skin—the neurovascular/neuroimmune system and the immune detection/response system (innate immunity).3,5-8,1–9 Both of these systems normally serve physiological functions related to how skin responds to exogenous changes or insults (i.e., changes in temperature, exposure to microbial pathogens). However, in rosacea, both the cutaneous neurovascular/neuroimmune system and the immune detection/response system are dysregulated, with both demonstrating augmented responses that correlate with clinical manifestations commonly seen in patients with cutaneous rosacea.Neurovascular/neuroimmune dysregulation, which includes both anatomic and physiochemical differences present in rosacea-prone skin as compared to healthy facial skin, appears to be a major contributor that exacerbates the vasodilation of facial skin vasculature with increased facial blood flow that occurs during a rosacea flare.3,5-7,17,18,20 This increased vasodilation in rosacea-affected skin, which can be acute or subacute in onset, is commonly referred to as flushing.1,3,4,6,7,17,20 Neurosensory symptoms (i.e., stinging, burning) are often associated with or exacerbated during a rosacea flare.1,3-8 Exogenous factors that are commonly recognized by patients as triggers, which seem to induce a flare, include increased ambient heat/warmth and certain spices (i.e., capsaicin), all of which can induce signaling of neurogenic inflammation via specific receptor channels (transient receptor potential vanilloid [TRPV] subfamily) shown to be increased in rosacea-prone skin.3,6,17,18 The immune detection/response dysregulation of rosacea is evidenced by the upregulation of the pattern recognition receptor, toll-like receptor 2 (TLR2) and the cathelicidin innate immunity pathway.3,5-17,19,21,22 Ultraviolet light (UV) exposure, another recognized trigger factor associated with flares of rosacea, produces changes that induce ligand-binding of TLR2, which signals innate inflammation.3,5-16,19,21 Lastly, upregulated production of several matrix metalloproteases (MMPs) has been demonstrated in rosacea, further contributing to cascades of inflammation and degradation of the dermal matrix.1,3,5-7,19 Accentuated immune detection/response as a major component of the pathophysiology of rosacea has been discussed extensively in the literature and is addressed in more detail as a major subject of this article.5-7,10-16,19,21,22Although the pathophysiology of rosacea is not completely understood, dysregulation of the innate immune detection/response system plays a significant role in the inflammatory and vascular responses seen in this condition.5-7,10-16,19,21,22 As a known inducer of innate and cellular inflammation, increased vascularity, and angiogenesis, cathelicidin (LL-37), an antimicrobial peptide that physiologically provides near-immediate innate defense against several microbial organisms, has been investigated to determine its potential role in the pathophysiology of rosacea.10,11,23,24 Results have shown that patients with rosacea express elevated levels of LL-37 in facial skin, with this increased expression attributed to abnormally high levels of the trypsin-like serine protease enzyme, kallikrein 5 (KLK5), which selectively cleaves an inactive precursor protein (hCAP18) to form the biologically active antimicrobial peptide (LL-37).10,22 Investigations of the mechanism of action of two agents proven to be effective in reducing papulopustular lesions and perilesional erythema in rosacea, topical azelaic acid (AzA) and oral doxycycline, demonstrated direct and indirect inhibition of KLK5, respectively.25-29 In one study with AzA 15% gel, the reduction in KLK5 activity correlated with clinical improvement of rosacea.29 In this review, the authors further describe the role of KLK5 in the pathophysiology of rosacea, including the inflammatory cascades that result from increased KLK5 expression, as well as a more detailed discussion of different therapies shown to inhibit the progression of this cascade.     

Cutaneous Collagenous Vasculopathy

Cutaneous collagenous vasculopathy is a rare microangiopathy of dermal blood vessels. Clinically indistinguishable from generalized essential telangiectasia, this condition is diagnosed by its unique histological appearance. In contrast to other primary telangiectatic processes, cutaneous collagenous vasculopathy has dilated vascular structures that contain deposits of eosinophilic hyaline material within the vessel walls. To date, cutaneous collagenous vasculopathy has been described in a total of 19 cases in the medical literature. The first several cases were described exclusively in middle-aged to elderly men. Though it has now been described in both men and women, cutaneous collagenous vasculopathy is still most often described in middle-aged to older adults. No particular disease or medication has been linked to the development of cutaneous collagenous vasculopathy, and the etiology remains unknown. In this case series, the authors present three additional patients diagnosed with cutaneous collagenous vasculopathy and discuss their clinical and histopathologic features.Cutaneous collagenous vasculopathy (CCV) is a rare, idiopathic microangiopathy first reported in 2000 by Salama and Rosenthal.1 CCV has characteristic microscopic findings, including dilated capillaries and post-capillary venules with marked collagen deposition,2 which are features essential to diagnosis. Clinically, CCV presents as blanchable, non-urticating macules that typically begin on the lower extremities and then spread to the trunk and upper extremities. 1,3-6 Due to its clinical similarity to generalized essential telangiectasia (GET), dermatologists may not biopsy these patients, potentially causing CCV to be underdiagnosed and under-reported. 4,5,7,8 To date, CCV has been described in approximately equal numbers in men and women of Caucasian race with patients ranging from 16 to 83 years of age.4,9 The majority of cases have been diagnosed in patients with other concomitant diseases, most commonly hypertension and cardiovascular disease,3,5,6,9,10 but also in patients with autoimmune conditions2,5,8,9 and diabetes mellitus.5,10 Additionally, in most described cases, patients were taking at least one medication on an intermittent or ongoing basis. 1,2,4-7,9,1° Despite this observation, specific medical conditions or medications are yet to be linked to the development of CCV. In this article, the authors present three female patients who have been diagnosed with CCV along with their clinical and histopathological features.     

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.     

Connexin 30 Deficiency Impairs Renal Tubular ATP Release and Pressure Natriuresis

In the renal tubule, ATP is an important regulator of salt and water reabsorption, but the mechanism of ATP release is unknown. Several connexin (Cx) isoforms form mechanosensitive, ATP-permeable hemichannels. We localized Cx30 to the nonjunctional apical membrane of cells in the distal nephron and tested whether Cx30 participates in physiologically important release of ATP. We dissected, partially split open, and microperfused cortical collecting ducts from wild-type and Cx30-deficient mice in vitro. We used PC12 cells as ATP biosensors by loading them with Fluo-4/Fura Red to measure cytosolic calcium and positioning them in direct contact with the apical surface of either intercalated or principal cells. ATP biosensor responses, triggered by increased tubular flow or by bath hypotonicity, were approximately three-fold greater when positioned next to intercalated cells than next to principal cells. In addition, these responses did not occur in preparations from Cx30-deficient mice or with purinergic receptor blockade. After inducing step increases in mean arterial pressure by ligating the distal aorta followed by the mesenteric and celiac arteries, urine output increased 4.2-fold in wild-type mice compared with 2.6-fold in Cx30-deficient mice, and urinary Na⁺ excretion increased 5.2-fold in wild-type mice compared with 2.8-fold in Cx30-deficient mice. Furthermore, Cx30-deficient mice developed endothelial sodium channel–dependent, salt-sensitive elevations in mean arterial pressure. Taken together, we suggest that mechanosensitive Cx30 hemichannels have an integral role in pressure natriuresis by releasing ATP into the tubular fluid, which inhibits salt and water reabsorption.It is well established that renal tubular epithelial cells release ATP,^1–4 which then binds to purinergic receptors along the nephron and collecting ducts to regulate salt and water reabsorption^5–12; however, the molecular mechanism of ATP release and the identity of ATP channels are less clear. The possible mechanisms include vesicular release,^1,2 pannexin or connexin hemichannels,^13–16 ATP-permeable anion channels such as the cystic fibrosis transmembrane conductance regulator,¹⁷ or the maxi anion channel.¹⁸ Previous work also suggested that the release of ATP in epithelia may be triggered by mechanical stimulation,^19–21 including elevations in tubular fluid flow rates. For example, tubular flow–dependent ATP release resulting in purinergic calcium signaling has been well characterized in the cortical collecting duct (CCD)^21–23; however, the ATP release mechanism has not been identified.Connexin 30 (Cx30) is a member of the connexin (Cx) family of transmembrane proteins (more than 20 isoforms). Various Cx proteins can form large pores in the nonjunctional plasma membrane of cells (gap junction hemichannels) before the assembly of two Cx hemichannels into complete gap junction channels between adjacent cells.¹⁴ Cx hemichannels are large, mechanosensitive ion channels that allow the passage of a variety of small molecules and metabolites, including ATP.^14–16 Our laboratory localized Cx30 in the nonjunctional apical plasma membrane of cells in the distal nephron,²⁴ suggesting that Cx30 may function as an ATP-releasing, luminal membrane hemichannel in this nephron segment. To address this hypothesis, we applied a recently developed ATP biosensor technique¹⁸ combined with a Cx30 knockout mouse model²⁵ to show tubular flow–induced ATP release through luminal Cx30 hemichannels in the renal collecting duct. The physiologic significance of this mechanically induced Cx30-mediated luminal ATP release was further studied by assessing its involvement in pressure natriuresis, a multifactorial, multimechanistic intrarenal phenomenon that causes diuresis and natriuresis in response to elevations in systemic BP.²⁶ Pressure natriuresis involves proximal and distal nephron components; hemodynamic factors such as medullary blood flow and renal interstitial hydrostatic pressure; and renal autocoids such as nitric oxide, prostaglandins, kinins, and angiotensin II.^26–32 Because pressure natriuresis is related to mechanical factors and Cx hemichannels are mechanosensitive, we hypothesized that Cx30-mediated ATP release is involved, at least in part, in pressure natriuresis, a critically important phenomenon in the maintenance of body fluid balance and BP.     

Racial Composition of Residential Areas Associates with Access to Pre-ESRD Nephrology Care

Referral to a nephrologist before initiation of chronic dialysis occurs less frequently for blacks than whites, but the reasons for this disparity are incompletely understood. Here, we examined the contribution of racial composition by zip code on access and quality of nephrology care before initiation of renal replacement therapy (RRT). We retrospectively studied a cohort study of 92,000 white and black adults who initiated RRT in the United States between June 1, 2005, and October 5, 2006. The percentage of patients without pre-ESRD nephrology care ranged from 30% among those who lived in zip codes with <5% black residents to 41% among those who lived in areas with >50% black residents. In adjusted analyses, as the percentage of blacks in residential areas increased, the likelihood of not receiving pre-ESRD nephrology care increased. Among patients who received nephrology care, the quality of care (timing of care and proportion of patients who received a pre-emptive renal transplant, who initiated therapy with peritoneal dialysis, or who had a permanent hemodialysis access) did not differ by the racial composition of their residential area. In conclusion, racial composition of residential areas associates with access to nephrology care but not with quality of the nephrology care received.Clinical practice guidelines for chronic kidney disease emphasize the importance of timely referral to a nephrologist for patients expected to require renal replacement therapy.^1–3 Nevertheless, approximately 33% of end-stage renal disease (ESRD) patients in the United States do not see a nephrologist before initiation of chronic dialysis.^4,5 Lack of timely access to nephrology care is associated with several adverse outcomes after initiation of dialysis including higher mortality rates,^6–9 higher rates of hospitalization,¹⁰ lower rates of renal transplantation,^11,12 delayed creation of arteriovenous fistulae,¹³ lower rates of achievement of dialysis treatment targets,^14,15 and a lower likelihood of receiving home-based dialysis therapies such as peritoneal dialysis and home hemodialysis.^3,16–18In the United States, black dialysis patients are less likely than white patients to have received nephrology care before onset of ESRD.^9,19–21 They are also less likely to receive a pre-emptive kidney transplant,^22,23 select peritoneal dialysis over hemodialysis,²⁴ and have a vascular access in place at onset of hemodialysis.^25,26 Several factors may contribute to these disparities including differences in insurance status, level of education, physician knowledge or biases, and patient preferences.^9,27–29 In addition, geographic factors such as proximity to dialysis facilities and degree of urbanization also affect access to nephrology care.^30–33A substantial proportion of black patients are also more likely to live in areas where most other residents are black. A recent study demonstrated that both black and white dialysis patients living in predominantly black zip codes were less likely to receive a kidney transplant than patients living in other areas.³⁴ Although levels of income, wealth and education tended to be lower among residents of predominantly black zip codes, lower transplant rates among dialysis patients living in these areas were not completely explained by these measures. Patients who live in predominantly black zip codes may face unique barriers to care that are not explained by measured socioeconomic characteristics of those zip codes. We therefore hypothesized that dialysis patients living in zip codes with a greater proportion of black residents would be less likely to have received pre-ESRD care and less likely to have received high-quality nephrology care than patients living in other zip codes. We also hypothesized that these relationships would not be completely explained by differences in zip code socioeconomic status or patient race.