Simultaneous Three-Dimensional Vascular and Tubular Imaging of Whole Mouse Kidneys With X-ray µCT.

Concurrent three-dimensional imaging of the renal vascular and tubular systems on the whole-kidney scale with capillary level resolution is labor-intensive and technically difficult. Approaches based on vascular corrosion casting and X-ray micro computed tomography (µCT), for example, suffer from vascular filling artifacts and necessitate imaging with an additional modality to acquire tubules. In this work, we report on a new sample preparation, image acquisition, and quantification protocol for simultaneous vascular and tubular µCT imaging of whole, uncorroded mouse kidneys. The protocol consists of vascular perfusion with the water-soluble, aldehyde-fixable, polymeric X-ray contrast agent XlinCA, followed by laboratory-source µCT imaging and structural analysis using the freely available Fiji/ImageJ software. We achieved consistent filling of the entire capillary bed and staining of the tubules in the cortex and outer medulla. After imaging at isotropic voxel sizes of 3.3 and 4.4 µm, we segmented vascular and tubular systems and quantified luminal volumes, surface areas, diffusion distances, and vessel path lengths. This protocol permits the analysis of vascular and tubular parameters with higher reliability than vascular corrosion casting, less labor than serial sectioning and leaves tissue intact for subsequent histological examination with light and electron microscopy.

Peritubular Capillary Rarefaction: An Underappreciated Regulator of CKD Progression.

Peritubular capillary (PTC) rarefaction is commonly detected in chronic kidney disease (CKD) such as hypertensive nephrosclerosis and diabetic nephropathy. Moreover, PTC rarefaction prominently correlates with impaired kidney function and predicts the future development of end-stage renal disease in patients with CKD. However, it is still underappreciated that PTC rarefaction is a pivotal regulator of CKD progression, primarily because the molecular mechanisms of PTC rarefaction have not been well-elucidated. In addition to the established mechanisms (reduced proangiogenic factors and increased anti-angiogenic factors), recent studies discovered significant contribution of the following elements to PTC loss: (1) prompt susceptibility of PTC to injury, (2) impaired proliferation of PTC, (3) apoptosis/senescence of PTC, and (4) pericyte detachment from PTC. Mainly based on the recent and novel findings in basic research and clinical study, this review describes the roles of the above-mentioned elements in PTC loss and focuses on the major factors regulating PTC angiogenesis, the assessment of PTC rarefaction and its surrogate markers, and an overview of the possible therapeutic agents to mitigate PTC rarefaction during CKD progression. PTC rarefaction is not only a prominent histological characteristic of CKD but also a central driving force of CKD progression.

Interleukin-24 is a novel diagnostic biomarker for the severity of acute kidney injury.

There is a clinical need for sensitive acute kidney injury (AKI) biomarkers that enable early therapeutic interventions and prediction of disease prognosis. In this study, we monitored interleukin (IL)-24 expressed in kidneys with severe AKI that progresses to atrophic kidney in a mouse model of ischemia-reperfusion injury (IRI). Therefore, we evaluated IL-24 as a potential biomarker not only for early diagnosis of AKI, but also for predicting progression to chronic kidney disease (CKD). Serum IL-24 was detected earlier than the elevation of serum creatinine levels and urinary IL-24 was detected as early as neutrophil gelatinase associated lipocalin (NGAL) in severe AKI (60 min of IRI). In addition, serum and urine IL-24 levels tended to increase in relation to ischemia duration. In such kidneys, vascular smooth muscle cells expressed IL-24 in response to the injury in the renal tubular epithelial cell and its target was the renal tubular epithelial cell itself. IL-24 may play a pivotal role in the communication between tubular epithelial cells and vascular smooth muscle cells and, in conclusion, IL-24 can be used as a sensitive biomarker for AKI.

Renal tubular injury exacerbated by vasohibin-1 deficiency in a murine cisplatin-induced acute kidney injury model.

Acute kidney injury (AKI) is frequently encountered in clinical practice, particularly secondarily to cardiovascular surgery and administration of nephrotoxic agents, and is increasingly recognized for initiating a transition to chronic kidney disease. Clarifying the pathogenesis of AKI could facilitate the development of novel preventive strategies, because the occurrence of hospital-acquired AKI is often anticipated. Vasohibin-1 (VASH1) was initially identified as an antiangiogenic factor derived from endothelial cells. VASH1 expression in endothelial cells has subsequently been reported to enhance cellular stress tolerance. Considering the importance of maintaining peritubular capillaries in preventing the progression of AKI, the present study aimed to examine whether VASH1 deletion is involved in the pathogenesis of cisplatin-induced AKI. For this, we injected male C57BL/6J wild-type (WT) and VASH1 heterozygous knockout (VASH1+/-) mice intraperitoneally with either 20 mg/kg cisplatin or vehicle solution. Seventy-two hours after cisplatin injection, increased serum creatinine concentrations and renal tubular injury accompanied by apoptosis and oxidative stress were more prominent in VASH1+/- mice than in WT mice. Cisplatin-induced peritubular capillary loss was also accelerated by VASH1 deficiency. Moreover, the increased expression of ICAM-1 in the peritubular capillaries of cisplatin-treated VASH1+/- mice was associated with a more marked infiltration of macrophages into the kidney. Taken together, VASH1 expression could have protective effects on cisplatin-induced AKI probably by maintaining the number and function of peritubular capillaries.

Matrix metalloproteinase-7 protects against acute kidney injury by priming renal tubules for survival and regeneration.

Matrix metalloproteinase-7 (MMP-7) is a secreted endopeptidase that degrades a broad range of substrates. Recent studies have identified MMP-7 as an early biomarker to predict severe acute kidney injury (AKI) and poor outcomes after cardiac surgery; however, the role of MMP-7 in the pathogenesis of AKI is unknown. In this study, we investigated the expression of MMP-7 and the impact of MMP-7 deficiency in several models of AKI. MMP-7 was induced in renal tubules following ischemia/ reperfusion injury or cisplatin administration, and in folic acid-induced AKI. MMP-7 knockout mice experienced higher mortality, elevated serum creatinine, and more severe histologic lesions after ischemic or toxic insults. Tubular apoptosis and interstitial inflammation were more prominent in MMP-7 knockout kidneys. These histologic changes were accompanied by increased expression of FasL and other components of the extrinsic apoptotic pathway, as well as increased expression of pro-inflammatory chemokines. In a rescue experiment, exogenous MMP-7 ameliorated kidney injury in MMP-7 knockout mice after ischemia/reperfusion. In vitro, MMP-7 protected tubular epithelial cells against apoptosis by directly degrading FasL. In isolated tubules ex vivo, MMP-7 promoted cell proliferation by degrading E-cadherin and thereby liberating ß-catenin, priming renal tubules for regeneration. Taken together, these results suggest that induction of MMP-7 is protective in AKI by degrading FasL and mobilizing ß-catenin, thereby priming kidney tubules for survival and regeneration.

Biglycan evokes autophagy in macrophages via a novel CD44/Toll-like receptor 4 signaling axis in ischemia/reperfusion injury.

Biglycan, a small leucine-rich proteoglycan, acts as a danger signal and is classically thought to promote macrophage recruitment via Toll-like receptors (TLR) 2 and 4. We have recently shown that biglycan signaling through TLR 2/4 and the CD14 co-receptor regulates inflammation, suggesting that TLR co-receptors may determine whether biglycan-TLR signaling is pro- or anti-inflammatory. Here, we sought to identify other co-receptors and characterize their impact on biglycan-TLR signaling. We found a marked increase in the number of autophagic macrophages in mice stably overexpressing soluble biglycan. In vitro, stimulation of murine macrophages with biglycan triggered autophagosome formation and enhanced the flux of autophagy markers. Soluble biglycan also promoted autophagy in human peripheral blood macrophages. Using macrophages from mice lacking TLR2 and/or TLR4, CD14, or CD44, we demonstrated that the pro-autophagy signal required TLR4 interaction with CD44, a receptor involved in adhesion, migration, lymphocyte activation, and angiogenesis. In vivo, transient overexpression of circulating biglycan at the onset of renal ischemia/reperfusion injury (IRI) enhanced M1 macrophage recruitment into the kidneys of Cd44+/+ and Cd44-/- mice but not Cd14-/- mice. The biglycan-CD44 interaction increased M1 autophagy and the number of renal M2 macrophages and reduced tubular damage following IRI. Thus, CD44 is a novel signaling co-receptor for biglycan, an interaction that is required for TLR4-CD44-dependent pro-autophagic activity in macrophages. Interfering with the interaction between biglycan and specific TLR co-receptors could represent a promising therapeutic intervention to curtail kidney inflammation and damage.

ß-hydroxybutyrate attenuates renal ischemia-reperfusion injury through its anti-pyroptotic effects.

Ketone bodies including ß-hydroxybutyrate (ß-OHB) have been shown to protect against ischemic tissue injury when present at low concentrations. We evaluated the impact of ß-OHB on renal ischemia/reperfusion injury (IRI). Mice were treated with a continuous infusion of ß-OHB using an osmotic mini-pump before and after IRI. We also tested the effects of increasing endogenous serum ß-OHB levels by fasting. Renal IRI was attenuated by ß-OHB treatment compared to saline control, with similar results in the fasting condition. ß-OHB treatment reduced the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells and increased expression of forkhead transcription factor O3 (FOXO3), an upstream regulator of pyroptosis. Although ß-OHB treatment did not impact markers of apoptosis, it decreased the expression of caspase-1 and proinflammatory cytokines, indicating that ß-OHB blocked pyroptosis. In a human proximal tubular cell line exposed to hypoxia and reoxygenation, ß-OHB reduced cell death in a FOXO3-dependent fashion. Histone acetylation was decreased in kidneys exposed to IRI and in proximal tubular cells exposed to hypoxia and reoxygenation, and this effect was ameliorated by ß-OHB through the inactivation of histone deacetylases. In vitro, ß-OHB treatment restored histone acetylation at the FOXO3 promoter. Consistent with epigenetic molecular effects, the renoprotective effects of ß-OHB were still observed when the continuous infusion was stopped at the time of IRI. Thus, ß-OHB attenuates renal IRI through anti-pyroptotic effects, likely mediated by an epigenetic effect on FOXO3 expression.

Caspase-3 Is a Pivotal Regulator of Microvascular Rarefaction and Renal Fibrosis after Ischemia-Reperfusion Injury.

Background Ischemia-reperfusion injury (IRI) is a major risk factor for chronic renal failure. Here, we characterize the different modes of programmed cell death in the tubular and microvascular compartments during the various stages of IRI-induced AKI, and their relative importance to renal fibrogenesis.Methods We performed unilateral renal artery clamping for 30 minutes and contralateral nephrectomy in wild-type mice (C57BL/6) or caspase-3-/- mice.Results Compared with their wild-type counterparts, caspase-3-/- mice in the early stage of AKI had high urine cystatin C levels, tubular injury scores, and serum creatinine levels. Electron microscopy revealed evidence of tubular epithelial cell necrosis in caspase-3-/- mice, and immunohistochemistry showed upregulation of the necroptosis marker receptor-interacting serine/threonine-protein kinase 3 (RIPK3) in renal cortical sections. Western blot analysis further demonstrated enhanced levels of phosphorylated RIPK3 in the kidneys of caspase-3-/- mice. In contrast, caspase-3-/- mice had less microvascular congestion and activation in the early and extension phases of AKI. In the long term (3 weeks after IRI), caspase-3-/- mice had reduced microvascular rarefaction and renal fibrosis, as well as decreased expression of α-smooth muscle actin and reduced collagen deposition within peritubular capillaries. Moreover, caspase-3-/- mice exhibited signs of reduced tubular ischemia, including lower tubular expression of hypoxia-inducible factor-1α and improved tubular injury scores.Conclusions These results establish the pivotal importance of caspase-3 in regulating microvascular endothelial cell apoptosis and renal fibrosis after IRI. These findings also demonstrate the predominant role of microvascular over tubular injury as a driver of progressive renal damage and fibrosis after IRI.

Connecting tubule glomerular feedback mediates tubuloglomerular feedback resetting after unilateral nephrectomy.

Unilaterally nephrectomized rats (UNx) have higher glomerular capillary pressure (PGC) that can cause significant glomerular injury in the remnant kidney. PGC is controlled by the ratio of afferent (Af-Art) and efferent arteriole resistance. Af-Art resistance in turn is regulated by two intrinsic feedback mechanisms: 1) tubuloglomerular feedback (TGF) that causes Af-Art constriction in response to increased NaCl in the macula densa; and 2) connecting tubule glomerular feedback (CTGF) that causes Af-Art dilatation in response to an increase in NaCl transport in the connecting tubule via the epithelial sodium channel (ENaC). Resetting of TGF post-UNx can allow systemic pressure to be transmitted to the glomerulus and cause renal damage, but the mechanism behind this resetting is unclear. Since CTGF is an Af-Art dilatory mechanism, we hypothesized that CTGF is increased after UNx and contributes to TGF resetting. To test this hypothesis, we performed UNx in Sprague-Dawley (8) rats. Twenty-four hours after surgery, we performed micropuncture of individual nephrons and measured stop-flow pressure (PSF). PSF is an indirect measurement of PGC. Maximal TGF response at 40 nl/min was 8.9 ± 1.24 mmHg in sham-UNx rats and 1.39 ± 1.02 mmHg in UNx rats, indicating TGF resetting after UNx. When CTGF was inhibited with the ENaC blocker benzamil (1 µM/l), the TGF response was 12.29 ± 2.01 mmHg in UNx rats and 13.03 ± 1.25 mmHg in sham-UNx rats, indicating restoration of the TGF responses in UNx. We conclude that enhanced CTGF contributes to TGF resetting after UNx.

Inhibition of ROMK blocks macula densa tubuloglomerular feedback yet causes renal vasoconstriction in anesthetized rats.

The Na+-K+-2Cl- cotransporter (NKCC2) on the loop of Henle is the site of action of furosemide. Because outer medullary potassium channel (ROMK) inhibitors prevent reabsorption by NKCC2, we tested the hypothesis that ROMK inhibition with a novel selective ROMK inhibitor (compound C) blocks tubuloglomerular feedback (TGF) and reduces vascular resistance. Loop perfusion of either ROMK inhibitor or furosemide caused dose-dependent blunting of TGF, but the response to furosemide was 10-fold more sensitive (IC50 = 10-6 M for furosemide and IC50 = 10-5 M for compound C). During systemic infusion, both diuretics inhibited TGF, but ROMK inhibitor was 10-fold more sensitive (compound C: 63% inhibition; furosemide: 32% inhibition). Despite blockade of TGF, 1 h of constant systemic infusion of both diuretics reduced the glomerular filtration rate (GFR) and renal blood flow (RBF) by 40-60% and increased renal vascular resistance (RVR) by 100-200%. Neither diuretic altered blood pressure or hematocrit. Proximal tubule hydrostatic pressures (PPT) increased transiently with both diuretics (compound C: 56% increase; furosemide: 70% increase) but returned to baseline. ROMK inhibitor caused more natriuresis (3,400 vs. 1,600% increase) and calciuresis (1,200 vs. 800% increase) but less kaliuresis (33 vs. 167% increase) than furosemide. In conclusion, blockade of ROMK or Na+-K+-2Cl- transport inhibits TGF yet increases renal vascular resistance. The renal vasoconstriction was independent of volume depletion, blood pressure, TGF, or PPT.

The third path of tubulointerstitial fibrosis: aberrant endothelial secretome.

The secretome, defined as a portion of proteins secreted by specific cells to the extracellular space, secures a proper microenvironmental niche not only for the donor cells, but also for the neighboring cells, thus maintaining tissue homeostasis. Communication via secretory products exists between endothelial cells and fibroblasts, and this local mechanism maintains the viability and density of each compartment. Endothelial dysfunction, apart from obvious cell-autonomous defects, leads to the aberrant secretome, which predisposes fibroblasts to acquire a myofibroblastic fibrogenic phenotype. In our recent profiling of the secretome of such dysfunctional profibrogenic renal microvascular endothelial cells, we identified unique profibrogenic signatures, among which we detected ligands of Notch and Wnt-ß-catenin pathways. Here, we stress the role of reprogramming cues in the immediate microenvironment of (myo)fibroblasts and the contribution of the endothelial secretome to the panoply of instructive signals in the vicinity of fibroblasts. We hope that this brief overview of endothelial-fibroblast communication in health and disease will lead to eventual unbiased proteomic mapping of individual secretomes of glomerular and tubular epithelial cells, pericytes, and podocytes through reductionist approaches to allow for the synthetic creation of a complex network of secretomic signals acting as reprogramming factors on individual cell types in the kidney. Knowledge of profibrogenic and antifibrogenic signatures in the secretome may garner future therapeutic efforts.

Tubulointerstitial fibrosis can sensitize the kidney to subsequent glomerular injury.

Chronic glomerular injury is associated with eventual development of tubulointerstitial fibrosis. Here we aimed to assess whether, and how, mild chronic tubulointerstitial injury affects glomeruli. For this, we generated mice expressing different toxin receptors, one on their proximal tubular epithelial cells (diphtheria toxin receptor [DTR]) and the other only on podocytes (human CD25 [IL-2R] driven by the nephrin promoter [Nep25]), allowing serial induction of tubule-specific and glomerular (podocyte)-specific injury, respectively. Six weeks after diphtheria toxin injection, mild interstitial fibrosis was found in Nep25+/DTR+, but not in Nep25+/DTR- mice. However, atubular glomeruli and neuronal nitric oxide synthase, a mediator of tubuloglomerular feedback, were higher in Nep25+/DTR+ than in DTR- mice and these atubular glomeruli had less podocyte density as assessed by WT-1 biomarker expression. Peritubular capillary density, hypoxia-inducible factor-1 and -2, and cyclooxygenase 2 expression were similar at week six in the two groups. At week seven, all mice were given the immunotoxin LMB-2, which binds to CD25 to induce podocyte injury. Ten days later, proteinuria, podocyte injury, and glomerulosclerosis were more severe in Nep25+/DTR+ than Nep25+/DTR- mice with more severe sclerosis in the tubule-connected glomeruli. This supports the concept that even mild preexisting tubulointerstitial injury sensitizes glomeruli to subsequent podocyte-specific injury. Thus, increased atubular glomeruli and abnormal tubuloglomerular feedback significantly contribute to the crosstalk between the tubulointerstitium and glomeruli.

Regardless of etiology, progressive renal disease causes ultrastructural and functional alterations of peritubular capillaries.

Progressive renal diseases are associated with rarefaction of peritubular capillaries, but the ultrastructural and functional alterations of the microvasculature are not well described. To study this, we analyzed different time points during progressive kidney damage and fibrosis in 3 murine models of different disease etiologies. These models were unilateral ureteral obstruction, unilateral ischemia-reperfusion injury, and Col4a3-deficient mice, we analyzed ultrastructural alterations in patient biopsy specimens. Compared with kidneys of healthy mice, we found a significant and progressive reduction of peritubular capillaries in all models analyzed. Ultrastructurally, compared with the kidneys of control mice, focal widening of the subendothelial space and higher numbers of endothelial vacuoles and caveolae were found in fibrotic kidneys. Quantitative analysis showed that peritubular capillary endothelial cells in fibrotic kidneys had significantly and progressively reduced numbers of fenestrations and increased thickness of the cell soma and lamina densa of the capillary basement membrane. Similar ultrastructural changes were also observed in patient's kidney biopsy specimens. Compared with healthy murine kidneys, fibrotic kidneys had significantly increased extravasation of Evans blue dye in all 3 models. The extravasation could be visualized using 2-photon microscopy in real time in living animals and was mainly localized to capillary branching points. Finally, fibrotic kidneys in all models exhibited a significantly greater degree of interstitial deposition of fibrinogen. Thus, peritubular capillaries undergo significant ultrastructural and functional alterations during experimental progressive renal diseases, independent of the underlying injury. Analyses of these alterations could provide read-outs for the evaluation of therapeutic approaches targeting the renal microvasculature.

mTORC1 maintains renal tubular homeostasis and is essential in response to ischemic stress.

Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell metabolism and autophagy. Despite widespread clinical use of mTORC1 inhibitors, the role of mTORC1 in renal tubular function and kidney homeostasis remains elusive. By using constitutive and inducible deletion of conditional Raptor alleles in renal tubular epithelial cells, we discovered that mTORC1 deficiency caused a marked concentrating defect, loss of tubular cells, and slowly progressive renal fibrosis. Transcriptional profiling revealed that mTORC1 maintains renal tubular homeostasis by controlling mitochondrial metabolism and biogenesis as well as transcellular transport processes involved in countercurrent multiplication and urine concentration. Although mTORC2 partially compensated for the loss of mTORC1, exposure to ischemia and reperfusion injury exaggerated the tubular damage in mTORC1-deficient mice and caused pronounced apoptosis, diminished proliferation rates, and delayed recovery. These findings identify mTORC1 as an important regulator of tubular energy metabolism and as a crucial component of ischemic stress responses.

Patterning the renal vascular bed.

The renal vascular bed has a stereotypic architecture that is essential for the kidney's role in excreting metabolic waste and regulating the volume and composition of body fluids. The kidney's excretory functions are dependent on the delivery of the majority of renal blood flow to the glomerular capillaries, which filter plasma removing from it metabolic waste, as well as vast quantities of solutes and fluids. The renal tubules reabsorb from the glomerular filtrate solutes and fluids required for homeostasis, while the post-glomerular capillary beds return these essential substances back into the systemic circulation. Thus, the kidney's regulatory functions are dependent on the close proximity or alignment of the post-glomerular capillary beds with the renal tubules. This review will focus on our current knowledge of the mechanisms controlling the embryonic development of the renal vasculature. An understanding of this process is critical for developing novel therapies to prevent vessel rarefaction and will be essential for engineering renal tissues suitable for restoring kidney function to the ever-increasing population of patients with end stage renal disease.

Requirement of clusterin expression for prosurvival autophagy in hypoxic kidney tubular epithelial cells.

Cellular autophagy is a prosurvival mechanism in the kidney against ischemia-reperfusion injury (IRI), but the molecular pathways that activate the autophagy in ischemic kidneys are not fully understood. Clusterin (CLU) is a chaperone-like protein, and its expression is associated with kidney resistance to IRI. The present study investigated the role of CLU in prosurvival autophagy in the kidney. Renal IRI was induced in mice by clamping renal pedicles at 32°C for 45 min. Hypoxia in renal tubular epithelial cell (TEC) cultures was induced by exposure to a 1% O2 atmosphere. Autophagy was determined by either light chain 3-BII expression with Western blot analysis or light chain 3-green fluorescent protein aggregation with confocal microscopy. Cell apoptosis was determined by flow cytometric analysis. The unfolded protein response was determined by PCR array. Here, we showed that autophagy was significantly activated by IRI in wild-type (WT) but not CLU-deficient kidneys. Similarly, autophagy was activated by hypoxia in human proximal TECs (HKC-8) and WT mouse primary TECs but was impaired in CLU-null TECs. Hypoxia-activated autophagy was CLU dependent and positively correlated with cell survival, and inhibition of autophagy significantly promoted cell death in both HKC-8 and mouse WT/CLU-expressing TECs but not in CLU-null TECs. Further experiments showed that CLU-dependent prosurvival autophagy was associated with activation of the unfolded protein response in hypoxic kidney cells. In conclusion, these data suggest that activation of prosurvival autophagy by hypoxia in kidney cells requires CLU expression and may be a key cytoprotective mechanism of CLU in the protection of the kidney from hypoxia/ischemia-mediated injury.

MiR-155 is Involved in Renal Ischemia-Reperfusion Injury via Direct Targeting of FoxO3a and Regulating Renal Tubular Cell Pyroptosis.

BACKGROUND/AIMS: Ischemia/reperfusion injury (IRI) plays a crucial role in renal transplantation and can cause renal failure associated with pyroptosis, a pro-inflammatory-induced programmed cell death. Small endogenous non-coding RNAs have been shown to be involved in renal ischemia/reperfusion injury. This study was performed to investigate which miRNAs regulate pyroptosis in response to renal ischemia/reperfusion injury and determine the mechanism underlying this regulation. METHODS: An in vivo rat model of renal IRI was established, and the serum and kidneys were harvested 24 h after reperfusion to assess renal function and histological changes. For the in vitro study, the cultured human renal proximal tubular cell line HK-2 was subjected to 24 h of hypoxia (5% CO2, 1% O2, and 94% N2) followed by 12 h of reoxygenation (5% CO2, 21% O2, and 74% N2). The mRNA expression levels were analyzed by real-time PCR, and the protein expression levels were analyzed using Western blot, immunofluorescence staining and enzyme-linked immunosorbent assay (ELISA). Bioinformatics analyses were applied to predict miR-155 targets, which were then confirmed by a luciferase reporter assay. RESULTS: We found that the levels of pyroptosis-related proteins, including caspase-1, caspase-11, IL-1ß and IL-18, were significantly increased after renal ischemia/reperfusion injury. Similarly, hypoxia-reoxygenation injury (HRI) also induced pyroptosis in HK2 cells. Furthermore, our study revealed that miR-155 expression was substantially increased in the renal tissues of IRI rats and in HRI HK2 cells. Up-regulation of miR-155 promoted HK2 cell pyroptosis in HRI; conversely, knockdown of miR-155 attenuated this process. To understand the signaling mechanisms underlying the pro-pyroptotic activity of miR-155, we found that exogenous expression of miR-155 up-regulated the expression of caspase-1 as well as the pro-inflammatory cytokines IL-1ß and IL-18. Moreover, miR-155 directly repressed FoxO3a expression and its downstream protein apoptosis repressor with caspase recruitment domain (ARC). CONCLUSIONS: Our study proposes a new signaling pathway of miR-155/FoxO3a/ARC leading to renal pyroptosis under ischemia/reperfusion injury conditions.

Kidney tubules: intertubular, vascular, and glomerular cross-talk.

PURPOSE OF REVIEW: The kidney mediates the excretion or conservation of water and electrolytes in the face of changing fluid and salt intake and losses. To ultrafilter and reabsorb the exact quantities of free water and salts to maintain euvolemia a range of endocrine, paracrine, and hormonal signaling systems have evolved linking the tubules, capillaries, glomeruli, arterioles, and other intrinsic cells of the kidney. Our understanding of these systems remains incomplete. RECENT FINDINGS: Recent work has provided new insights into the workings of the communication pathways between tubular segments and the glomeruli and vasculature, with novel therapeutic agents in development. Particular progress has also been made in the visualization of tubuloglomerular feedback. SUMMARY: The review summarizes our current understanding of pathway functions in health and disease, as well as future therapeutic options to protect the healthy and injured kidney.

Selective renal blood perfusion induces renal tubules injury in a porcine model.

OBJECTIVE: Extracorporeal circulation is routinely used in thoracoabdominal aortic aneurysm repair to preserve blood perfusion. Despite this protective measure, acute and chronic kidney disorders can develop. Therefore, the aim of this study was to establish a new large-animal model to assess the efficacy of selective renal perfusion (SRP) with extracorporeal circulation in a setting of thoracoabdominal aortic aneurysm repair. METHODS: Eighteen pigs underwent a thoracolaparotomy, during with the aorta and renal arteries were exposed. The animals were divided into three cohorts of six pigs each: cohort I--control; cohort II--thoracic aortic clamping with distal aortic perfusion (DAP) using a roller pump; and cohort III--thoracic aortic clamping with DAP plus SRP. Kidney metabolism, kidney injury, and red blood cell damage were measured by oxygen extraction ratio (O2ER), neutrophil gelatinase-associated lipocalin, a marker for acute kidney damage, and serum free hemoglobin. RESULTS: With normal mean arterial blood pressures, flow rates in the renal arteries during perfusion decreased to 75% (group II) with DAP and to 50% (group III) with SRP compared with the control animals (group I; P = .0279 for I vs II; P = .0002 for I vs III). Microcirculation, measured by microspheres, did not differ significantly among the groups. In contrast, O2ER (P = .0021 for I vs III) and neutrophil gelatinase-associated lipocalin (P = .0083 for I vs III) levels were significantly increased in group III, whereas free hemoglobin was increased in groups II and III (P = .0406 for I vs II; P = .0018 for I vs III). CONCLUSIONS: SRP with a roller pump induces kidney tubule injury. Thus, distal aortic and SRP in our model does not provide adequate kidney protection. Furthermore, the perfusion system provokes red blood cell damage with increased free hemoglobin. Hence, the SRP perfusion technique should be revised and tested.

Acute renal haemodynamic effects of glucagon-like peptide-1 receptor agonist exenatide in healthy overweight men.

AIMS: To determine the acute effect of glucagon-like peptide-1 (GLP-1) receptor agonist exenatide and the involvement of nitric oxide (NO) on renal haemodynamics and tubular function, in healthy overweight men. METHODS: Renal haemodynamics and tubular electrolyte handling were measured in 10 healthy overweight men (aged 20-27 years; BMI 26-31 kg/m(2)) during intravenous administration of placebo (saline 0.9%), exenatide, and exenatide combined with the NO-synthase inhibitor L-N(G)-monomethyl arginine (L-NMMA). Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were determined by inulin and para-aminohippurate clearance techniques, respectively, based on timed urine sampling. Glomerular hydrostatic pressure and vascular resistance of afferent and efferent renal arterioles were calculated using the Gomez formulae. Urinary electrolytes, osmolality and pH were also measured. RESULTS: GFR increased by a mean of 18 ± 20 (+20%; p = 0.021) and ERPF increased by a median (interquartile range) of 68 (26; 197) ml/min/1.73 m(2) (+14%; p = 0.015) during exenatide infusion versus placebo. During L-NMMA infusion, exenatide increased GFR by mean 8 ± 12 ml/min/1.73 m(2) (+9%; p = 0.049). Exenatide increased estimated glomerular pressure by +6% (p = 0.015) and reduced afferent renal vascular resistance by -33% (p = 0.038), whereas these effects were blunted during L-NMMA infusion. Exenatide increased absolute and fractional sodium excretion, urinary osmolality and urinary pH. The tubular effects of exenatide were not altered by concomitant L-NMMA infusion. CONCLUSIONS: Exenatide infusion in healthy overweight men acutely increases GFR, ERPF and glomerular pressure, probably by reducing afferent renal vascular resistance, and at least partially in an NO-dependent manner. As baseline renal haemodynamics in patients with type 2 diabetes differ from those in healthy individuals, clinical studies on the renal effects of GLP-1 receptor agonists are warranted.