Journal of the American Society of Nephrology current issue

1. Sources of Drug Coverage among Medicare Beneficiaries with ESRD

   Despite extensive use of prescription medications in ESRD, relatively little is known about the participation of Medicare ESRD beneficiaries in the Part D program. Here, we quantitated the sources of drug coverage among ESRD beneficiaries and explored the Part D plan preferences of ESRD beneficiaries with regard to deductibles, coverage gaps, and monthly premiums. We obtained data on beneficiary sources of creditable coverage, characteristics of Part D plans, demographics, and residence from the Centers for Medicare and Medicaid Chronic Condition Data Warehouse and identified beneficiaries with ESRD from the US Renal Data System. We found that a substantial proportion (17.0%) of ESRD beneficiaries lacked a known source of creditable drug coverage in 2007 and 64.3% were enrolled in Part D. Of those enrolled, 72% received the Medicare Part D low-income subsidy. ESRD beneficiaries who enrolled in standalone Part D plans without the assistance of the low-income subsidy tended to prefer more comprehensive coverage options. In conclusion, more outreach is needed to ensure that beneficiaries who lack coverage obtain the coverage they need and that ESRD beneficiaries join the best plans for managing their disease and accompanying comorbid conditions.

2. Molecular Phenotypes of Acute Kidney Injury in Kidney Transplants

   Little is known regarding the molecular phenotype of kidneys with AKI because biopsies are performed infrequently. However, all kidney transplants experience acute injury, making early kidney transplants an excellent model of acute injury, provided the absence of rejection, because donor kidneys should not have CKD, post-transplant biopsies occur relatively frequently, and follow-up is excellent typically. Here, we used histopathology and microarrays to compare indication biopsies from 26 transplants with acute injury with 11 pristine protocol biopsies of stable transplants. Kidneys with acute injury showed increased expression of 394 transcripts associated with the repair response to injury, including many epithelium-like injury molecules tissue, remodeling molecules, and inflammation molecules. Many other genes also predicted the phenotype, including the acute injury biomarkers HAVCR1 and IL18. Pathway analysis of the injury-repair transcripts revealed similarities to cancer, development, and cell movement. The injury-repair transcript score in kidneys with acute injury correlated with reduced graft function, future renal recovery, brain death, and need for dialysis, but not with future graft loss. In contrast, histologic features of acute tubular injury did not correlate with function or with the molecular changes. Thus, the transcripts associated with repair of injury suggest a massive coordinated response of the kidney parenchyma to acute injury, providing both an objective measure for assessing the severity of injury in kidney biopsies and validation for many biomarkers of AKI.

3. Serum Amyloid A in Uremic HDL Promotes Inflammation

   Uremia impairs the atheroprotective properties of HDL, but the mechanisms underlying why this occurs are unknown. Here, we observed that HDL isolated from healthy individuals inhibited the production of inflammatory cytokines by peripheral monocytes stimulated with a Toll-like receptor 2 agonist. In contrast, HDL isolated from the majority of patients with ESRD did not show this anti-inflammatory property; many HDL samples even promoted the production of inflammatory cytokines. To investigate this difference, we used shotgun proteomics to identify 49 HDL-associated proteins in a uremia-specific pattern. Proteins enriched in HDL from patients with ESRD (ESRD-HDL) included surfactant protein B (SP-B), apolipoprotein C-II, serum amyloid A (SAA), and α-1-microglobulin/bikunin precursor. In addition, we detected some ESRD-enriched proteins in earlier stages of CKD. We did not detect a difference in oxidation status between HDL isolated from uremic and healthy patients. Regarding function of these uremia-specific proteins, only SAA mimicked ESRD-HDL by promoting inflammatory cytokine production. Furthermore, SAA levels in ESRD-HDL inversely correlated with its anti-inflammatory potency. In conclusion, HDL has anti-inflammatory activities that are defective in uremic patients as a result of specific changes in its molecular composition. These data suggest a potential link between the high levels of inflammation and cardiovascular mortality in uremia.

4. Identification of Gene Mutations in Autosomal Dominant Polycystic Kidney Disease through Targeted Resequencing

   Mutations in two large multi-exon genes, PKD1 and PKD2, cause autosomal dominant polycystic kidney disease (ADPKD). The duplication of PKD1 exons 1–32 as six pseudogenes on chromosome 16, the high level of allelic heterogeneity, and the cost of Sanger sequencing complicate mutation analysis, which can aid diagnostics of ADPKD. We developed and validated a strategy to analyze both the PKD1 and PKD2 genes using next-generation sequencing by pooling long-range PCR amplicons and multiplexing bar-coded libraries. We used this approach to characterize a cohort of 230 patients with ADPKD. This process detected definitely and likely pathogenic variants in 115 (63%) of 183 patients with typical ADPKD. In addition, we identified atypical mutations, a gene conversion, and one missed mutation resulting from allele dropout, and we characterized the pattern of deep intronic variation for both genes. In summary, this strategy involving next-generation sequencing is a model for future genetic characterization of large ADPKD populations.

5. Biomarkers Predict Progression of Acute Kidney Injury after Cardiac Surgery

   Being able to predict whether AKI will progress could improve monitoring and care, guide patient counseling, and assist with enrollment into trials of AKI treatment. Using samples from the Translational Research Investigating Biomarker Endpoints in AKI study (TRIBE-AKI), we evaluated whether kidney injury biomarkers measured at the time of first clinical diagnosis of early AKI after cardiac surgery can forecast AKI severity. Biomarkers included urinary IL-18, urinary albumin to creatinine ratio (ACR), and urinary and plasma neutrophil gelatinase-associated lipocalin (NGAL); each measurement was on the day of AKI diagnosis in 380 patients who developed at least AKI Network (AKIN) stage 1 AKI. The primary end point (progression of AKI defined by worsening AKIN stage) occurred in 45 (11.8%) patients. Using multivariable logistic regression, we determined the risk of AKI progression. After adjustment for clinical predictors, compared with biomarker values in the lowest two quintiles, the highest quintiles of three biomarkers remained associated with AKI progression: IL-18 (odds ratio=3.0, 95% confidence interval=1.3–7.3), ACR (odds ratio=3.4, 95% confidence interval=1.3–9.1), and plasma NGAL (odds ratio=7.7, 95% confidence interval=2.6–22.5). Each biomarker improved risk classification compared with the clinical model alone, with plasma NGAL performing the best (category-free net reclassification improvement of 0.69, P<0.0001). In conclusion, biomarkers measured on the day of AKI diagnosis improve risk stratification and identify patients at higher risk for progression of AKI and worse patient outcomes.

6. Survival in Daily Home Hemodialysis and Matched Thrice-Weekly In-Center Hemodialysis Patients

   Frequent hemodialysis improves cardiovascular surrogates and quality-of-life indicators, but its effect on survival remains unclear. We used a matched-cohort design to assess relative mortality in daily home hemodialysis and thrice-weekly in-center hemodialysis patients between 2005 and 2008. We matched 1873 home hemodialysis patients with 9365 in-center patients (i.e., 1:5 ratio) selected from the prevalent population in the US Renal Data System database. Matching variables included first date of follow-up, demographic characteristics, and measures of disease severity. The cumulative incidence of death was 19.2% and 21.7% in the home hemodialysis and in-center patients, respectively. In the intention-to-treat analysis, home hemodialysis associated with a 13% lower risk for all-cause mortality than in-center hemodialysis (hazard ratio [HR], 0.87; 95% confidence interval [95% CI], 0.78–0.97). Cause-specific mortality HRs were 0.92 (95% CI, 0.78–1.09) for cardiovascular disease, 1.13 (95% CI, 0.84–1.53) for infection, 0.63 (95% CI, 0.41–0.95) for cachexia/dialysis withdrawal, 1.06 (95% CI, 0.81–1.37) for other specified cause, and 0.59 (95% CI, 0.44–0.79) for unknown cause. Findings were similar using as-treated analyses. We did not detect statistically significant evidence of heterogeneity of treatment effects in subgroup analyses. In summary, these data suggest that relative to thrice-weekly in-center hemodialysis, daily home hemodialysis associates with modest improvements in survival. Continued surveillance should strengthen inference about causes of mortality and determine whether treatment effects are homogeneous throughout the dialysis population.

7. The Impact of Renal Function on Outcomes of Bariatric Surgery

   The effect of CKD on the risks of bariatric surgery is not well understood. Using the American College of Surgeons National Surgical Quality Improvement Program Participant Use File, we analyzed 27,736 patients who underwent bariatric surgery from 2006 through 2008. Before surgery, 34 (0.12%) patients were undergoing long-term dialysis. Among those not undergoing dialysis, 20,806 patients (75.0%) had a normal estimated GFR or stage 1 CKD, 5011 (18.07%) had stage 2 CKD, 1734 (6.25%) had stage 3 CKD, 94 (0.34%) had stage 4 CKD, and 91 (0.33%) had stage 5 CKD. In an unadjusted analysis, CKD stage was directly associated with complication rate, ranging from 4.6% for those with stage 1 CKD or normal estimated GFR to 9.9% for those with stage 5 CKD (test for trend, P<0.001). Multivariable logistic regression demonstrated that CKD stage predicts higher complication rates (odds ratio for each higher CKD stage, 1.30) after adjustment for diabetes and hypertension. Although patients with higher CKD stage had higher complication rates, the absolute incidence of complications remained <10%. In conclusion, these data demonstrate higher risks of bariatric surgery among patients with worse renal function, but whether the potential benefits outweigh the risks in this population requires further study.

8. Pericyte TIMP3 and ADAMTS1 Modulate Vascular Stability after Kidney Injury

   Kidney pericytes are progenitors of scar-forming interstitial myofibroblasts that appear after injury. The function of kidney pericytes as microvascular cells and how these cells detach from peritubular capillaries and migrate to the interstitial space, however, are poorly understood. Here, we used an unbiased approach to identify genes in kidney pericytes relevant to detachment and differentiation in response to injury in vivo, with a particular focus on genes regulating proteolytic activity and angiogenesis. Kidney pericytes rapidly activated expression of a disintegrin and metalloprotease with thrombospondin motifs-1 (ADAMTS1) and downregulated its inhibitor, tissue inhibitor of metalloproteinase 3 (TIMP3) in response to injury. Similarly to brain pericytes, kidney pericytes bound to and stabilized capillary tube networks in three-dimensional gels and inhibited metalloproteolytic activity and angiogenic signaling in endothelial cells. In contrast, myofibroblasts did not have these vascular stabilizing functions despite their derivation from kidney pericytes. Pericyte-derived TIMP3 stabilized and ADAMTS1 destabilized the capillary tubular networks. Furthermore, mice deficient in Timp3 had a spontaneous microvascular phenotype in the kidney resulting from overactivated pericytes and were more susceptible to injury-stimulated microvascular rarefaction with an exuberant fibrotic response. Taken together, these data support functions for kidney pericytes in microvascular stability, highlight central roles for regulators of extracellular proteolytic activity in capillary homoeostasis, and identify ADAMTS1 as a marker of activation of kidney pericytes.

9. Genetic or Pharmacologic Blockade of EGFR Inhibits Renal Fibrosis

   Although enhanced activation of the EGF receptor (EGFR) associates with the development and progression of renal fibrosis, the mechanisms linking these observations are not completely understood. Here, after unilateral ureteral obstruction (UUO), wild-type mice exhibited sustained EGFR phosphorylation in the kidney and developed renal fibrosis that was more severe than the renal fibrosis observed in waved-2 mice, which have reduced EGFR tyrosine kinase activity. Waved-2 mice also showed fewer renal tubular cells arrested at G2/M, reduced expression of α-smooth muscle actin (α-SMA), downregulation of multiple genes encoding profibrogenic cytokines, including TGF-β1, and dephosphorylation of Smad3, STAT3, and ERK1/2. Administration of the specific EGFR inhibitor gefitinib recapitulated this phenotype in wild-type mice after UUO. Furthermore, inactivation of either EGFR or STAT3 reduced UUO-induced expression of lipocalin–2, a molecule associated with the pathogenesis of CKD. In cultured renal interstitial fibroblasts, inhibition of EGFR also abrogated TGF-β1– or serum-induced phosphorylation of EGFR, STAT3, ERK1/2, and Smad3 as well as expression of α-SMA and extracelluar matrix proteins. Taken together, these data suggest that EGFR may mediate renal fibrogenesis by promoting transition of renal epithelial cells to a profibrotic phenotype, increased production of inflammatory factors, and activation of renal interstitial fibroblasts. Inhibition of EGFR may have therapeutic potential for fibrotic kidney disease.

10. Expression of Heme Oxygenase-1 in Thick Ascending Loop of Henle Attenuates Angiotensin II-Dependent Hypertension

    Kidney-specific induction of heme oxygenase-1 (HO-1) attenuates the development of angiotensin II (Ang II) -dependent hypertension, but the relative contribution of vascular versus tubular induction of HO-1 is unknown. To determine the specific contribution of thick ascending loop of Henle (TALH) -derived HO-1, we generated a transgenic mouse in which the uromodulin promoter controlled expression of human HO-1. Quantitative RT-PCR and confocal microscopy confirmed successful localization of the HO-1 transgene to TALH tubule segments. Medullary HO activity, but not cortical HO activity, was significantly higher in transgenic mice than control mice. Enhanced TALH HO-1 attenuated the hypertension induced by Ang II delivered by an osmotic minipump for 10 days (139±3 versus 153±2 mmHg in the transgenic and control mice, respectively; P<0.05). The lower blood pressure in transgenic mice associated with a 60% decrease in medullary NKCC2 transporter expression determined by Western blot. Transgenic mice also exhibited a 36% decrease in ouabain-sensitive sodium reabsorption and a significantly attenuated response to furosemide in isolated TALH segments. In summary, these results show that increased levels of HO-1 in the TALH can lower blood pressure by a mechanism that may include alterations in NKCC2-dependent sodium reabsorption.

11. Sonic Hedgehog Signaling Mediates Epithelial-Mesenchymal Communication and Promotes Renal Fibrosis

    Sonic hedgehog (Shh) signaling is a developmental signal cascade that plays an essential role in regulating embryogenesis and tissue homeostasis. Here, we investigated the potential role of Shh signaling in renal interstitial fibrogenesis. Ureteral obstruction induced Shh, predominantly in the renal tubular epithelium of the fibrotic kidneys. Using Gli1^lacZ knock-in mice, we identified renal interstitial fibroblasts as Shh-responding cells. In cultured renal fibroblasts, recombinant Shh protein activated Gli1 and induced α-smooth muscle actin (α-SMA), desmin, fibronectin, and collagen I expression, suggesting that Shh signaling promotes myofibroblast activation and matrix production. Blockade of Shh signaling with cyclopamine abolished the Shh-mediated induction of Gli1, Snail1, α-SMA, fibronectin, and collagen I. In vivo, the kidneys of Gli1-deficient mice were protected against the development of interstitial fibrosis after obstructive injury. In wild-type mice, cyclopamine did not affect renal Shh expression but did inhibit induction of Gli1, Snail1, and α-SMA. In addition, cyclopamine reduced matrix expression and mitigated fibrotic lesions. These results suggest that tubule-derived Shh mediates epithelial–mesenchymal communication by targeting interstitial fibroblasts after kidney injury. We conclude that Shh/Gli1 signaling plays a critical role in promoting fibroblast activation, production of extracellular matrix, and development of renal interstitial fibrosis.

12. Folate Receptor-Targeted Antioxidant Therapy Ameliorates Renal Ischemia-Reperfusion Injury

    Antioxidant therapy can protect against ischemic injury, but the inability to selectively target the kidney would require extremely high doses to achieve effective local concentrations of drug. Here, we developed a directed therapeutic that specifically targets an antioxidant to renal proximal tubule cells via the folate receptor. Because a local increase in superoxide contributes to renal ischemic injury, we created the folate-antioxidant conjugate 4-hydroxy-Tempo (tempol)-folate to target folate receptors, which are highly expressed in the proximal tubule. Dihydroethidium high-performance liquid chromatography demonstrated that conjugated tempol retained its efficacy to scavenge superoxide in proximal tubule cells. In a mouse model of renal ischemia-reperfusion injury, tempol-folate reduced renal superoxide levels more effectively than tempol alone. Furthermore, electron spin resonance revealed the successful targeting of the tempol-folate conjugate to the kidney and other tissues expressing folate receptors. Administration of tempol-folate protected the renal function of mice after ischemia-reperfusion injury and inhibited infiltration of macrophages. In conclusion, kidney-specific targeting of an antioxidant has therapeutic potential to prevent renal ischemic injury. Conjugation of other pharmaceuticals to folate may also facilitate the development of treatments for other kidney diseases.

13. Imaging of Podocyte Foot Processes by Fluorescence Microscopy

    Visualizing podocyte foot processes requires electron microscopy, a technique that depends on special equipment, requires immunogold for colabeling, and does not take advantage of the growing number of in vivo fluorophores available. To address these limitations, we developed a genetic strategy to allow detailed visualization of single podocytes and their foot processes by conventional fluorescence microscopy. We generated a transgenic mouse line expressing a GFP-Cre-ERT2 fusion protein under the control of the collagen α1(I) promoter with strong podocyte expression. Administration of submaximal tamoxifen allowed genetic labeling of single podocytes when crossed with a Cre-reporter line. Of three different reporter systems that we evaluated for the ability to reveal fine structural details of podocytes, bigenic Coll1α1GCE;Gt(ROSA)26Sor^{tm9(CAG-tdTomato)} mice allowed podocyte labeling with a strong and homogeneous reporter signal that was easily observed by epifluorescence. We could easily detect anatomic features of podocytes down to tertiary foot processes, and we were able to visualize and quantitate ultrastructural changes to foot processes after podocyte injury. In summary, using this method of genetic labeling and conventional fluorescence microscopy to visualize podocyte foot processes will complement electron microscopy and facilitate the analysis of podocytes and their precursors in vivo.

14. Dose-Dependent Effects of Sirolimus on mTOR Signaling and Polycystic Kidney Disease

    Inhibition of the mammalian target of rapamycin (mTOR) shows beneficial effects in animal models of polycystic kidney disease (PKD); however, two clinical trials in patients with autosomal dominant PKD failed to demonstrate a short-term benefit in either the early or progressive stages of disease. The stage of disease during treatment and the dose of mTOR inhibitors may account for these differing results. Here, we studied the effects of a conventional low dose and a higher dose of sirolimus (blood levels of 3 ng/ml and 30–60 ng/ml, respectively) on mTOR activity and renal cystic disease in two Pkd1-mutant mouse models at different stages of the disease. When initiated at early but not late stages of disease, high-dose treatment strongly reduced mTOR signaling in renal tissues, inhibited cystogenesis, accelerated cyst regression, and abrogated fibrosis and the infiltration of immune cells. In contrast, low-dose treatment did not significantly reduce renal cystic disease. Levels of p-S6Rp^Ser240/244, which marks mTOR activity, varied between kidneys; severity of the renal cystic phenotype correlated with the level of mTOR activity. Taken together, these data suggest that long-term treatment with conventional doses of sirolimus is insufficient to inhibit mTOR activity in renal cystic tissue. Mechanisms to increase bioavailability or to target mTOR inhibitors more specifically to kidneys, alone or in combination with other compounds, may improve the potential for these therapies in PKD.

15. Vasculature and Kidney Complications in Sickle Cell Disease

    Recent developments in sickle cell disease include the concept of a vasculopathic state and the classification of sickle cell disease into a hemolysis-endothelial dysfunction phenotype or a viscosity-vasoocclusion phenotype. The hemolysis-endothelial dysfunction phenotype largely reflects deficiency of or resistance to nitric oxide. In addition to discussing these areas, we suggest that the hemolysis-endothelial dysfunction phenotype also reflects the instability of sickle hemoglobin, the release of heme, and the induction of heme oxygenase-1. From these perspectives the renal complications of sickle cell disease are discussed and classified.

16. Molecular Mechanisms of Acid-Base Sensing by the Kidney

    A major function of the kidney is to collaborate with the respiratory system to maintain systemic acid-base status within limits compatible with normal cell and organ function. It achieves this by regulating the excretion and recovery of bicarbonate (mainly in the proximal tubule) and the secretion of buffered protons (mainly in the distal tubule and collecting duct). How proximal tubular cells and distal professional proton transporting (intercalated) cells sense and respond to changes in pH, bicarbonate, and CO₂ status is a question that has intrigued many generations of renal physiologists. Over the past few years, however, some candidate molecular pH sensors have been identified, including acid/alkali-sensing receptors (GPR4, InsR-RR), kinases (Pyk2, ErbB1/2), pH-sensitive ion channels (ASICs, TASK, ROMK), and the bicarbonate-stimulated adenylyl cyclase (sAC). Some acid-sensing mechanisms in other tissues, such as CAII-PDK2L1 in taste buds, might also have similar roles to play in the kidney. Finally, the function of a variety of additional membrane channels and transporters is altered by pH variations both within and outside the cell, and the expression of several metabolic enzymes are altered by acid-base status in parts of the nephron. Thus, it is possible that a master pH sensor will never be identified. Rather, the kidney seems equipped with a battery of molecules that scan the epithelial cell environment to mount a coordinated physiologic response that maintains acid-base homeostasis. This review collates current knowledge on renal acid-base sensing in the context of a whole organ sensing and response process.

17. New Observational Data Demonstrate that Mortality Is Lower in Patients Receiving More Frequent Dialysis
18. The Impact of Renal Function on Outcomes of Bariatric Surgery
19. Activated Pericytes and the Inhibition of Renal Vascular Stability: Obstacles for Kidney Repair
20. microRNA-Induced IgA Nephropathy
21. This Month's Highlights
22. Abnormal miR-148b Expression Promotes Aberrant Glycosylation of IgA1 in IgA Nephropathy

    Aberrant O-glycosylation in the hinge region of IgA1 characterizes IgA nephropathy. The mechanisms underlying this abnormal glycosylation are not well understood, but reduced expression of the enzyme core 1, β1,3-galactosyltransferase 1 (C1GALT1) may contribute. In this study, high-throughput microRNA (miRNA) profiling identified 37 miRNAs differentially expressed in PBMCs of patients with IgA nephropathy compared with healthy persons. Among them, we observed upregulation of miR-148b, which potentially targets C1GALT1. Patients with IgA nephropathy exhibited lower C1GALT1 expression, which negatively correlated with miR-148b expression. Transfection of PBMCs from healthy persons with a miR-148b mimic reduced endogenous C1GALT1 mRNA levels threefold. Conversely, loss of miR-148b function in PBMCs of patients with IgA nephropathy increased C1GALT1 mRNA and protein levels to those observed in healthy persons. Moreover, we found that upregulation of miR-148b directly correlated with levels of galactose-deficient IgA1. In vitro, we used an IgA1-producing cell line to confirm that miR-148b modulates IgA1 O-glycosylation and the levels of secreted galactose-deficient IgA1. Taken together, these data suggest a role for miRNAs in the pathogenesis of IgA nephropathy. Abnormal expression of miR-148b may explain the aberrant glycosylation of IgA1, providing a potential pharmacologic target for IgA nephropathy.

23. A 4.1-Mb Congenic Region of Rf-4 Contributes to Glomerular Permeability

    The combined transfer of two renal function quantitative trait loci (QTLs), Rf-1 (rat chromosome 1) and Rf-4 (rat chromosome 14), from the Fawn-hooded hypertensive rat onto the August Copenhagen Irish genetic background significantly increases proteinuria and demonstrates an interaction between these QTLs. Because the original Rf-4 congenic region is 61.9 Mbp, it is necessary to reduce this interval to feasibly search for variants responsible for renal susceptibility in this region. Here, we generated a minimal congenic line (Rf-1a+4_a) to identify a 4.1-Mb region of the Rf-4 QTL that significantly contributes to the severity of proteinuria in the Fawn-hooded hypertensive rat. Rf-1a+4_a animals have an increased glomerular permeability to albumin without significant changes in BP, indicating that at least one genetic element in this refined region directly affects renal function. Sequence analysis revealed no variants predicted to damage protein function, implying that regulatory elements are responsible for the Rf-4 phenotype. Multiple human studies, including recent genome-wide association studies, link the homologous human region with susceptibility to renal disease, suggesting that this congenic line is an important model for studying pathways that contribute to the progression of kidney disease.