A kidney resident macrophage subset is a candidate biomarker for renal cystic disease in preclinical models.

Although renal macrophages have been shown to contribute to cyst development in polycystic kidney disease (PKD) animal models, it remains unclear whether there is a specific macrophage subpopulation involved. Here, we analyzed changes in macrophage populations during renal maturation in association with cystogenesis rates in conditional Pkd2 mutant mice. We observed that CD206+ resident macrophages were minimal in a normal adult kidney but accumulated in cystic areas in adult-induced Pkd2 mutants. Using Cx3cr1 null mice, we reduced macrophage number, including CD206+ macrophages, and showed that this significantly reduced cyst severity in adult-induced Pkd2 mutant kidneys. We also found that the number of CD206+ resident macrophage-like cells increased in kidneys and in the urine from autosomal-dominant PKD (ADPKD) patients relative to the rate of renal functional decline. These data indicate a direct correlation between CD206+ resident macrophages and cyst formation, and reveal that the CD206+ resident macrophages in urine could serve as a biomarker for renal cystic disease activity in preclinical models and ADPKD patients. This article has an associated First Person interview with the first author of the paper.

Urinary T cells correlate with rate of renal function loss in autosomal dominant polycystic kidney disease.

Several innate immune response components were recognized as outcome predictors in autosomal dominant polycystic kidney disease (ADPKD) and their causative role in disease pathogenesis was confirmed in animal models. In contrast, the role of adaptive immunity in ADPKD remains relatively unexplored. Therefore, we evaluated T cell populations in kidney and urine of ADPKD patients using flow cytometry and confocal immunofluorescence microscopy approaches. These analyses revealed ADPKD-associated overall increases in the number of intrarenal CD4 and CD8 T cells that were associated with a loss of polarity in distribution between the cortex and medulla (higher in medulla vs. cortex in controls). Also, the urinary T cell-based index correlated moderately with renal function decline in a small cohort of ADPKD patients. Together, these data suggest that similar to innate immune responses, T cells participate in ADPKD pathogenesis. They also point to urinary T cells as a novel candidate marker of the disease activity in ADPKD.

Heterozygous Pkhd1C642* mice develop cystic liver disease and proximal tubule ectasia that mimics radiographic signs of medullary sponge kidney.

Heterozygosity for human polycystic kidney and hepatic disease 1 ( PKHD1) mutations was recently associated with cystic liver disease and radiographic findings resembling medullary sponge kidney (MSK). However, the relevance of these associations has been tempered by a lack of cystic liver or renal disease in heterozygous mice carrying Pkhd1 gene trap or exon deletions. To determine whether heterozygosity for a smaller Pkhd1 defect can trigger cystic renal disease in mice, we generated and characterized mice with the predicted truncating Pkhd1C642* mutation in a region corresponding to the middle of exon 20 cluster of five truncating human mutations (between PKHD1G617fs and PKHD1G644*). Mouse heterozygotes or homozygotes for the Pkhd1C642* mutation did not have noticeable liver or renal abnormalities on magnetic resonance images during their first weeks of life. However, when aged to ~1.5 yr, the Pkhd1C642* heterozygotes developed prominent cystic liver changes; tissue analyses revealed biliary cysts and increased number of bile ducts without signs of congenital hepatic fibrosis-like portal field inflammation and fibrosis that was seen in Pkhd1C642* homozygotes. Interestingly, aged female Pkhd1C642* heterozygotes, as well as homozygotes, developed radiographic changes resembling MSK. However, these changes correspond to proximal tubule ectasia, not an MSK-associated collecting duct ectasia. In summary, by demonstrating that cystic liver and kidney abnormalities are triggered by heterozygosity for the Pkhd1C642* mutation, we provide important validation for relevant human association studies. Together, these investigations indicate that PKHD1 mutation heterozygosity (predicted frequency 1 in 70 individuals) is an important underlying cause of cystic liver disorders and MSK-like manifestations in a human population.

Truncating PKHD1 and PKD2 mutations alter energy metabolism.

Deficiency in polycystin 1 triggers specific changes in energy metabolism. To determine whether defects in other human cystoproteins have similar effects, we studied extracellular acidification and glucose metabolism in human embryonic kidney (HEK-293) cell lines with polycystic kidney and hepatic disease 1 ( PKHD1) and polycystic kidney disease (PKD) 2 ( PKD2) truncating defects along multiple sites of truncating mutations found in patients with autosomal recessive and dominant PKDs. While neither the PKHD1 or PKD2 gene mutations nor their position enhanced cell proliferation rate in our cell line models, truncating mutations in these genes progressively increased overall extracellular acidification over time ( P < 0.001 for PKHD1 and PKD2 mutations). PKHD1 mutations increased nonglycolytic acidification rate (1.19 vs. 1.03, P = 0.002), consistent with an increase in tricarboxylic acid cycle activity or breakdown of intracellular glycogen. In addition, they increased basal and ATP-linked oxygen consumption rates [7.59 vs. 5.42 ( P = 0.015) and 4.55 vs. 2.98 ( P = 0.004)]. The PKHD1 and PKD2 mutations also altered mitochondrial morphology, resembling the effects of polycystin 1 deficiency. Together, these data suggest that defects in major PKD genes trigger changes in mitochondrial energy metabolism. After validation in in vivo models, these initial observations would indicate potential benefits of targeting energy metabolism in the treatment of PKDs.

Transcription factor avian erythroblastosis virus E26 oncogen homolog-1 is a novel mediator of renal injury in salt-sensitive hypertension.

Transcription factor E26 transformation-specific sequence-1 (ETS-1) is a transcription factor that regulates the expression of a variety of genes, including growth factors, chemokines, and adhesion molecules. We recently demonstrated that angiotensin II increases the glomerular expression of ETS-1 and that blockade of ETS-1 ameliorates the profibrotic and proinflammatory effects of angiotensin II. The Dahl salt-sensitive rat is a paradigm of salt-sensitive hypertension associated with local activation of the renin-angiotensin system. In these studies, we determined whether: (1) salt-sensitive hypertension is associated with renal expression of ETS-1 and (2) ETS-1 participates in the development of end-organ injury in salt-sensitive hypertension. Dahl salt-sensitive rats were fed a normal-salt diet (0.5% NaCl diet) or a high-salt diet (4% NaCl) for 4 weeks. Separate groups on high-salt diet received an ETS-1 dominant-negative peptide (10 mg/kg/d), an inactive ETS-1 mutant peptide (10 mg/kg/d), the angiotensin II type 1 receptor blocker candesartan (10 mg/kg/d), or the combination high-salt diet/dominant-negative peptide/angiotensin II type 1 receptor blocker for 4 weeks. High-salt diet rats had a significant increase in the glomerular expression of the phosphorylated ETS-1 that was prevented by angiotensin II type 1 receptor blocker. ETS-1 blockade reduced proteinuria, glomerular injury score, fibronectin expression, urinary transforming growth factor-ß excretion, and macrophage infiltration. Angiotensin II type 1 receptor blocker reduced proteinuria, glomerular injury score, and macrophage infiltration, whereas concomitant ETS-1 blockade and angiotensin II type 1 receptor blocker had additive effects and reduced interstitial fibrosis. Our studies demonstrated that salt-sensitive hypertension results in increased glomerular expression of phosphorylated ETS-1 and suggested that ETS-1 plays an important role in the pathogenesis of end-organ injury in salt-sensitive hypertension.

The transcription factor E26 transformation-specific sequence-1 mediates neointima formation in arteriovenous fistula.

Hemodialysis vascular access dysfunction contributes to increased morbidity and mortality in hemodialysis patients. Arteriovenous fistula (AVF) is the preferred type of vascular access for hemodialysis but has high rates of dysfunction, in part because of excessive neointima formation. The transcription factor E26 transformation-specific sequence-1 (ETS-1) is a mediator of proinflammatory responses in hypertension and endovascular injury. We examined the role of ETS-1 in the formation of neointima in AVF. Right carotid artery to internal jugular vein fistulas were created in C57BL/6 mice and assigned to treatment with an ETS-1-dominant negative peptide (ETS-DN), an inactive mutant peptide (ETS-MU), or vehicle (n=6 per group). After 7 and 21 days, AVFs or contralateral internal jugular veins were processed for PCR, immunofluorescence, immunohistochemistry, and morphometry. In AVFs, ETS-1 mRNA increased 2.5-fold at 7 days and 4-fold at 21 days. By immunofluorescence, we confirmed increased expression of ETS-1 predominantly in the neointima and overlying endothelium. Similarly, ETS-1 expression increased in human AVFs compared with normal veins. In mice, ETS-DN, but not ETS-MU, reduced neointima formation at days 7 and 21 and reduced the expression of nitric oxide synthase 2, NADPH oxidase (NOX) 2, NOX4, E-selectin, and monocyte chemotactic protein-1. Shear stress increased ETS-1 phosphorylation in human umbilical vein cells in a NOX-dependent manner, demonstrating a role for reactive oxygen species in ETS-1 activation. These results unveil the role of ETS-1 as a mediator of neointima formation in AVF and may result in the development of novel strategies for the treatment of AVF dysfunction.

Dasatinib-induced nephrotic-range proteinuria.

Since the introduction of imatinib, tyrosine kinase inhibition has been a mainstay in the treatment of many malignancies. The number of these medications is growing, as are the number of targeted tyrosine kinases. Off-target effects of these medications can have beneficial or adverse effects on the kidney. The onus of knowing the implications of these medications on kidney function, and appropriate treatment when such adverse effects occur, is on the nephrologist. We present a patient with chronic myelogenous leukemia who developed nephrotic-range proteinuria after initiation on dasatinib therapy that resolved after changing therapy to imatinib. The mechanism of kidney injury caused by dasatinib has not been described previously in the literature. We provide a review of vascular endothelial growth factor and its pharmacologic inhibition as it pertains to kidney pathology and propose possible mechanisms by which dasatinib induces kidney injury.

Role of the transcription factor erythroblastosis virus E26 oncogen homolog-1 (ETS-1) as mediator of the renal proinflammatory and profibrotic effects of angiotensin II.

Angiotensin II (Ang II) plays a major role in the pathogenesis of end-organ injury in hypertension via its diverse hemodynamic and nonhemodynamic effects. Erythroblastosis virus E26 oncogen homolog-1 (ETS-1) is an important transcription factor recently recognized as an important mediator of cell proliferation, inflammation, and fibrosis. In the present studies, we tested the hypothesis that ETS-1 is a common mediator of the renal proinflammatory and profibrotic effects of Ang II. C57BL6 mice (n=6 per group) were infused with vehicle (control), Ang II (1.4 mg/kg per day), Ang II and an ETS-1 dominant-negative peptide (10 mg/kg per day), or Ang II and an ETS-1 mutant peptide (10 mg/kg per day) via osmotic minipump for 2 or 4 weeks. The infusion of Ang II resulted in significant increases in blood pressure and left ventricular hypertrophy, which were not modified by ETS-1 blockade. The administration of ETS-1 dominant-negative peptide significantly attenuated Ang II-induced renal injury as assessed by urinary protein excretion, mesangial matrix expansion, and cell proliferation. Furthermore, ETS-1 dominant-negative peptide but not ETS-1 mutant peptide significantly reduced Ang II-mediated upregulation of transforming growth factor-ß, connective tissue growth factor, and α-smooth muscle actin. In addition, ETS-1 blockade reduced several proinflammatory effects of Ang II, including macrophage infiltration, nitrotyrosine expression, and NOX4 mRNA expression. Our studies suggest that ETS-1 is a common mediator of the proinflammatory and profibrotic effects of Ang II-induced hypertensive renal damage and may result in the development of novel strategies in the treatment and prevention of end-organ injury in hypertension.

Nicotine exposure and the progression of chronic kidney disease: role of the α7-nicotinic acetylcholine receptor.

Clinical studies have established the role of cigarette smoking as a risk factor in the progression of chronic kidney disease (CKD). We have shown that nicotine promotes mesangial cell proliferation and hypertrophy via nonneuronal nicotinic acetylcholine receptors (nAChRs). The α7-nAChR is one of the most important subunits of the nAChRs. These studies were designed to test the hypothesis that nicotine worsens renal injury in rats with 5/6 nephrectomy (5/6Nx) and that the α7-nAChR subunit is required for these effects. We studied five different groups: Sham, 5/6Nx, 5/6Nx + nicotine (Nic; 100 µg/ml dry wt), 5/6Nx + Nic + α7-nAChR blocker methyllicaconitine (MLA; 3 mg·kg(-1)·day(-1) sq), and Sham + Nic. Blood pressure was measured by the tail-cuff method, and urine was collected for proteinuria. After 12 wk, the rats were euthanized and kidneys were collected. We observed expression of the α7-nAChR in the proximal and distal tubules. The administration of nicotine induced a small increase in blood pressure and resulted in cotinine levels similar to those found in the plasma of smokers. In 5/6Nx rats, the administration of nicotine significantly increased urinary protein excretion (onefold), worsened the glomerular injury score and increased fibronectin (∼ 50%), NADPH oxidase 4 (NOX4; ∼100%), and transforming growth factor-ß expression (∼200%). The administration of nicotine to sham rats increased total proteinuria but not albuminuria, suggesting direct effects on tubular protein reabsorption. These effects were prevented by MLA, demonstrating a critical role for the α7-nAChR as a mediator of the effects of nicotine in the progression of CKD.

Sodium nitrite protects against kidney injury induced by brain death and improves post-transplant function.

Renal injury induced by brain death is characterized by ischemia and inflammation, and limiting it is a therapeutic goal that could improve outcomes in kidney transplantation. Brain death resulted in decreased circulating nitrite levels and increased infiltrating inflammatory cell infiltration into the kidney. Since nitrite stimulates nitric oxide signaling in ischemic tissues, we tested whether nitrite therapy was beneficial in a rat model of brain death followed by kidney transplantation. Nitrite, administered over 2 h of brain death, blunted the increased inflammation without affecting brain death-induced alterations in hemodynamics. Kidneys were transplanted after 2 h of brain death and renal function followed over 7 days. Allografts collected from nitrite-treated brain-dead rats showed significant improvement in function over the first 2 to 4 days after transplantation compared with untreated brain-dead animals. Gene microarray analysis after 2 h of brain death without or with nitrite therapy showed that the latter significantly altered the expression of about 400 genes. Ingenuity Pathway Analysis indicated that multiple signaling pathways were affected by nitrite, including those related to hypoxia, transcription, and genes related to humoral immune responses. Thus, nitrite therapy attenuates brain death-induced renal injury by regulating responses to ischemia and inflammation, ultimately leading to better post-transplant kidney function.

The transcription factor ETS-1 regulates angiotensin II-stimulated fibronectin production in mesangial cells.

Angiotensin II (ANG II) produced as result of activation of the renin-angiotensin system (RAS) plays a critical role in the pathogenesis of chronic kidney disease via its hemodynamic effects on the renal microcirculation as well as by its nonhemodynamic actions including the production of extracellular matrix proteins such as fibronectin, a multifunctional extracellular matrix protein that plays a major role in cell adhesion and migration as well as in the development of glomerulosclerosis. ETS-1 is an important transcription factor essential for normal kidney development and glomerular integrity. We previously showed that ANG II increases ETS-1 expression and is required for fibronectin production in mesangial cells. In these studies, we determined that ANG II induces phosphorylation of ETS-1 via activation of the type 1 ANG II receptor and that Erk1/2 and Akt/PKB phosphorylation are required for these effects. In addition, we characterized the role of ETS-1 on the transcriptional activation of fibronectin production in mesangial cells. We determined that ETS-1 directly activates the fibronectin promoter and by utilizing gel shift assays and chromatin immunoprecipitation assays identified two different ETS-1 binding sites that promote the transcriptional activation of fibronectin in response to ANG II. In addition, we identified the essential role of CREB and its coactivator p300 on the transcriptional activation of fibronectin by ETS-1. These studies unveil novel mechanisms involved in RAS-induced production of the extracellular matrix protein fibronectin in mesangial cells and establish the role of the transcription factor ETS-1 as a direct mediator of these effects.

Downregulation of claudin-2 expression in renal epithelial cells by metabolic acidosis.

Chronic metabolic acidosis (CMA) is associated with an inhibition of fluid reabsorption in the renal proximal tubule. The effects of CMA on paracellular transport across the renal epithelial tight junction (TJ) is unknown. Claudin-2 is a transmembrane TJ-associated protein which confers TJ paracellular permeability to Na(+). We examined the effects of CMA on the expression of TJ transport proteins using both in vivo and in vitro models of CMA. The results showed downregulation of claudin-2 mRNA and protein expression in the cortex of rats subjected to the NH(4)Cl loading model of CMA. Madin-Darby canine kidney (MDCK) and HK-2 cells are models of renal epithelial cells and express claudin-2 protein in their TJ. We examined the effects of acidic pH exposure on the expression of claudin-2 in MDCK and HK-2 renal epithelial cells. Exposure of MDCK cells to pH 6.96 medium caused a significant and reversible decrease in claudin-2 protein abundance. A dose-response analysis of acidic medium exposure of MDCK and HK-2 cells demonstrated a downregulation of claudin-2 protein. The downregulation effect of acidic pH is specific to claudin-2 expression as the expression of other TJ-associated proteins (i.e., claudin-1, -3, -4, and -7, occludin, and zonula occludens-1) remained unchanged compared with control pH (7.40). Collectively, these data demonstrate that CMA downregulates the expression of claudin-2 likely through a direct effect of acidic pH. Potential physiological significance of these changes is discussed.