Danegaptide Prevents TGFß1-Induced Damage in Human Proximal Tubule Epithelial Cells of the Kidney.

Chronic kidney disease (CKD) is a global health problem associated with a number of comorbidities. Recent evidence implicates increased hemichannel-mediated release of adenosine triphosphate (ATP) in the progression of tubulointerstitial fibrosis, the main underlying pathology of CKD. Here, we evaluate the effect of danegaptide on blocking hemichannel-mediated changes in the expression and function of proteins associated with disease progression in tubular epithelial kidney cells. Primary human proximal tubule epithelial cells (hPTECs) were treated with the beta1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFß1) ± danegaptide. qRT-PCR and immunoblotting confirmed mRNA and protein expression, whilst a cytokine antibody array assessed the expression/secretion of proinflammatory and profibrotic cytokines. Carboxyfluorescein dye uptake and ATP biosensing measured hemichannel activity and ATP release, whilst transepithelial electrical resistance was used to assess paracellular permeability. Danegaptide negated carboxyfluorescein dye uptake and ATP release and protected against protein changes associated with tubular injury. Blocking Cx43-mediated ATP release was paralleled by partial restoration of the expression of cell cycle inhibitors, adherens and tight junction proteins and decreased paracellular permeability. Furthermore, danegaptide inhibited TGFß1-induced changes in the expression and secretion of key adipokines, cytokines, chemokines, growth factors and interleukins. The data suggest that as a gap junction modulator and hemichannel blocker, danegaptide has potential in the future treatment of CKD.

Serum D-serine accumulation after proximal renal tubular damage involves neutral amino acid transporter Asc-1.

Chiral separation has revealed enantio-specific changes in blood and urinary levels of amino acids in kidney diseases. Blood D-/L-serine ratio has been identified to have a correlation with creatinine-based kidney function. However, the mechanism of distinctive behavior in serine enantiomers is not well understood. This study was performed to investigate the role of renal tubules in derangement of serine enantiomers using a mouse model of cisplatin-induced tubular injury. Cisplatin treatment resulted in tubular damage histologically restricted to the proximal tubules and showed a significant increase of serum D-/L-serine ratio with positive correlations to serum creatinine and blood urine nitrogen (BUN). The increased D-/L-serine ratio did not associate with activity of a D-serine degrading enzyme, D-amino acid oxidase, in the kidney. Screening transcriptions of neutral amino acid transporters revealed that Asc-1, found in renal tubules and collecting ducts, was significantly increased after cisplatin-treatment, which correlates with serum D-serine increase. In vitro study using a kidney cell line showed that Asc-1 is induced by cisplatin and mediated influx of D-serine preferably to L-serine. Collectively, these results suggest that cisplatin-induced damage of proximal tubules accompanies Asc-1 induction in tubules and collecting ducts and leads to serum D-serine accumulation.

Lithium ameliorates tubule-interstitial injury through activation of the mTORC2/protein kinase B pathway.

Tubule-interstitial injury (TII) is a critical step in the progression of renal disease. It has been proposed that changes in proximal tubule (PT) albumin endocytosis plays an important role in the development of TII. Some reports have shown protective effects of lithium on kidney injury animal models that was correlated to proteinuria. We tested the hypothesis that lithium treatment ameliorates the development of TII due to changes in albumin endocytosis. Two experimental models were used: (1) TII induced by albumin overload in an animal model; (2) LLC-PK1 cells, a PT cell line. Lithium treatment ameliorates TII induced by albumin overload measured by (1) proteinuria; (2) collagen deposition; (3) area of tubule-interstitial space, and (4) macrophage infiltration. Lithium treatment increased mTORC2 activity leading to the phosphorylation of protein kinase B (PKB) at Ser473 and its activation. This mechanism enhanced albumin endocytosis in PT cells, which decreased the proteinuria observed in TII induced by albumin overload. This effect did not involve changes in the expression of megalin, a PT albumin receptor. In addition, activation of this pathway decreased apoptosis in LLC-PK1 cells, a PT cell line, induced by higher albumin concentration, similar to that found in pathophysiologic conditions. Our results indicate that the protective role of lithium treatment on TII induced by albumin overload involves an increase in PT albumin endocytosis due to activation of the mTORC2/PKB pathway. These results open new possibilities in understanding the effects of lithium on the progression of renal disease.

Alpha-lipoic acid attenuates p-cresyl sulfate-induced renal tubular injury through suppression of apoptosis and autophagy in human proximal tubular epithelial cells.

The p-cresyl sulfate accumulates in kidney disease and may be involved in renal injury. α-Lipoic acid (α-LA) acts as an antioxidant in cell injury. We investigated the effects of α-LA treatment on p-cresyl sulfate-induced renal tubular injury. p-Cresyl sulfate induced cell death, and increased Bax/Bcl-2, cleaved caspase-3, Beclin-1, and LC3BII/LC3BI in human renal proximal tubular epithelial (HK-2) cells, which was counteracted by α-LA treatment. p-Cresyl sulfate-induced apoptosis was reduced by autophagy inhibitor 3-methyladenine, and p-cresyl sulfate induced autophagy was reduced by pan-caspase inhibitor Z-VAD-FMK. Moreover, p-cresyl sulfate treatment increased the expression of ER stress proteins and decreased the expression of baculoviral IAP repeat-containing proteins 6; these effects were prevented by α-LA treatment. Apoptosis and autophagy were associated with the phosphorylation of mitogen-activated protein kinase and nuclear translocation of the nuclear factor-κB p65 subunit. Pretreatment inhibitors of p38 and JNK, and knockdown of ATF4 gene reduced apoptosis- and autophagy-related protein expressions in p-cresyl sulfate treated HK-2 cells. These results demonstrate that α-lipoic acid attenuated p-cresyl sulfate-induced cell death by suppression of apoptosis and autophagy via regulation of ER stress in HK-2 cells.

Renal Interstitial Platelet-Derived Growth Factor Receptor-ß Cells Support Proximal Tubular Regeneration.

BACKGROUND: The kidney is considered to be a structurally stable organ with limited baseline cellular turnover. Nevertheless, single cells must be constantly replaced to conserve the functional integrity of the organ. PDGF chain B (PDGF-BB) signaling through fibroblast PDGF receptor-ß (PDGFRß) contributes to interstitial-epithelial cell communication and facilitates regenerative functions in several organs. However, the potential role of interstitial cells in renal tubular regeneration has not been examined. METHODS: In mice with fluorescent protein expression in renal tubular cells and PDGFRß-positive interstitial cells, we ablated single tubular cells by high laser exposure. We then used serial intravital multiphoton microscopy with subsequent three-dimensional reconstruction and ex vivo histology to evaluate the cellular and molecular processes involved in tubular regeneration. RESULTS: Single-tubular cell ablation caused the migration and division of dedifferentiated tubular epithelial cells that preceded tubular regeneration. Moreover, tubular cell ablation caused immediate calcium responses in adjacent PDGFRß-positive interstitial cells and the rapid migration thereof toward the injury. These PDGFRß-positive cells enclosed the injured epithelium before the onset of tubular cell dedifferentiation, and the later withdrawal of these PDGFRß-positive cells correlated with signs of tubular cell redifferentiation. Intraperitoneal administration of trapidil to block PDGFRß impeded PDGFRß-positive cell migration to the tubular injury site and compromised the recovery of tubular function. CONCLUSIONS: Ablated tubular cells are exclusively replaced by resident tubular cell proliferation in a process dependent on PDGFRß-mediated communication between the renal interstitium and the tubular system.

Kidney injury molecule-1 staining in renal allograft biopsies 10 days after transplantation is inversely correlated with functioning proximal tubular epithelial cells.

BACKGROUND: Kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) are promising biomarkers for monitoring delayed graft function (DGF) after kidney transplantation. Here we investigated localization and distribution of KIM-1 and NGAL staining in renal allograft biopsies and studied their association with histological features, functional DGF (fDGF) and the tubular function slope (TFS), a functioning proximal tubular epithelial cell (PTEC) marker. METHODS: Day 10 protocol biopsies of 64 donation after circulatory death recipients were stained for KIM-1 and NGAL and the positive area was quantified using ImageJ software. Biopsies were scored according to Banff and acute tubular necrosis (ATN) criteria. A 99mtechnetium-mercaptoacetyltriglycine (99mTc-MAG3)-renography was performed to calculate TFS. RESULTS: KIM-1 staining was located on the brush border of tubular epithelial cells (TECs) and correlated with denudation, while NGAL was present more focally in a cytoplasmic distribution. KIM-1 and NGAL staining were not correlated and no co-localization was observed. Quantitative stainings were not associated with fDGF, but KIM-1 tended to be higher in patients with prolonged fDGF (≥21 days; P = 0.062). No correlation was observed between the quantitative tissue stainings and urinary KIM-1 or NGAL. Quantitative KIM-1 staining was inversely correlated with the TFS (Spearman's ρ = -0.53; P < 0.001), whereas NGAL was not. The latter finding might be because cortical NGAL staining is dependent on filtration and subsequent reabsorption by functioning PTECs. Staining of NGAL was indeed restricted to PTECs, as shown by co-localization with a PTEC-specific lectin. CONCLUSIONS: KIM-1 and NGAL staining showed different localization and distribution. Quantitative KIM-1 staining was inversely correlated with functioning PTECs.

Nicotine Exposure Augments Renal Toxicity of 5-aza-cytidine Through p66shc: Prevention by Resveratrol.

BACKGROUND/AIM: We have shown that either chronic nicotine (NIC) exposure or 5-aza-cytidine (AZA) augments oxidative stress-dependent injury through stimulating p66shc in renal cells. Hence, NIC could exacerbate adverse effects of AZA while antioxidants such as resveratrol (RES) could prevent it. MATERIALS AND METHODS: Renal proximal tubule cells (NRK52E) were treated with 20 µM RES prior to 200 µM NIC plus 100 nM AZA and cell injury (LDH release) was determined. Reporter luciferase assays determined p66shc activation and RES-induced antioxidant responses. Genetic manipulations identified the mechanism of RES action. RESULTS: NIC exacerbated AZA-dependent injury via augmenting p66shc transcription. While RES suppressed NIC+AZA-mediated injury, -surprisingly-it further enhanced activity of the p66shc promoter. RES protected cells via the cytoplasmic p66shc/Nrf2/heme oxygenase-1 (HO-1) axis. CONCLUSION: RES can protect the kidney from adverse effects of NIC in patients undergoing anticancer therapy.

Resveratrol Attenuates Nicotine-mediated Oxidative Injury by Inducing Manganese Superoxide Dismutase in Renal Proximal Tubule Cells.

BACKGROUND/AIM: Nicotine (NIC) exposure - via smoking and the increasingly popular E-cigarettes- increases oxidative stress and hence, renal risk in smokers. Resveratrol (RES) may help ameliorate this risk by mounting anti-oxidant responses in the kidney. MATERIALS AND METHODS: Renal proximal tubule cells (NRK52E) were treated with vehicle or 20 µM RES prior to treatment with 200 µM NIC and generation of reactive oxygen species (ROS) as well as cell viability was determined. RES-induced antioxidant responses were determined in reporter luciferase assays. Gene silencing was used to determine mechanism of RES action. RESULTS: RES protected NRK52E cells from NIC-induced oxidative injury. RES activated the promoter of the anti-oxidant manganese superoxide dismutase (MnSOD) gene via activation of the forkhead box O (FoxO3a) transcription factor. Silencing of MnSOD abolished the protective effects of RES on NIC-associated oxidative injury. CONCLUSION: RES may provide protection to the kidney from the adverse effects of NIC in smokers.

Hyponatremia with Persistent Elevated Urinary Fractional Uric Acid Excretion: Evidence for Proximal Tubular Injury?

BACKGROUND/AIMS: Hyponatremia associated with high urinary fractional excretion of uric acid which persists after serum sodium is corrected is the cardinal feature of salt losing nephropathy (SLN). We hypothesize that low grade proximal tubular injury is present in SLN because the proximal tubule is the main site of uric acid and sodium transport. METHODS: Five subjects with SLN were compared to four subjects with recurrent hyponatremia and three healthy individuals. Urinary NGAL (neutrophil gelatinase associated lipocalin, a marker of tubular injury) and fasting urinary fructose levels (a marker of proximal tubular injury) were measured. RESULTS: Subjects with SLN (n=5) showed elevated fractional uric acid excretion (22 ± 6 vs 4 ± 2 percent, p<0.0001), elevated urinary NGAL levels (62 ± 37 vs 9 ± 7 ng/mg creatinine, p=0.001) and fasting urinary fructose levels compared with the 7 controls (383 ± 465 vs 60 ± 34µmole/µg creatinine, p <0.001). A strong correlation between urinary NGAL levels and urinary fructose levels was observed (r =0.87, p<0.001). CONCLUSION: High urinary fractional excretion of uric acid in SLN is associated with elevated NGAL and fasting urinary fructose levels suggesting that SLN may involve tubular injury.

Renoprotective effect of DPP-4 inhibitors against free fatty acid-bound albumin-induced renal proximal tubular cell injury.

Dipeptidyl peptidase (DPP)-4 inhibitors, a new class of antidiabetic agent, have recently been suggested to exert pleiotropic effects beyond glucose lowering. Renal prognosis in patients with diabetic nephropathy depends on the severity of tubulointerstitial injury induced by massive proteinuria. We thus examined the renoprotective effect of DPP-4 inhibitors on inflammation in cultured mouse proximal tubular cells stimulated with free fatty acid (FFA)-bound albumin. Linagliptin and higher concentrations of sitagliptin, vildagliptin, and alogliptin all inhibited FFA-bound albumin-induced increases in mRNA expression of MCP-1 in cultured mouse proximal tubular cells. Furthermore, linagliptin significantly inhibited tubulointerstitial injury induced by peritoneal injection of FFA-bound albumin, such as inflammation, fibrosis, and apoptosis, in mice without altering systemic characteristics including body weight, fasting blood glucose, and food intake. These results indicate that DPP-4 inhibitors pleiotropically exert a direct renoprotective effect, and may serve as an additional therapeutic strategy to protect proximal tubular cells against proteinuria in patients with diabetic nephropathy.

Evaluation of cystatin C as an early biomarker of cadmium nephrotoxicity in the rat.

Cadmium (Cd) is a nephrotoxic environmental pollutant that causes insidious injury to the proximal tubule that results in severe polyuria and proteinuria. Cystatin C is a low molecular weight protein that is being evaluated as a serum and urinary biomarker for various types of ischemic and nephrotoxic renal injury. The objective of the present study was to determine if cystatin C might be a useful early biomarker of Cd nephrotoxicity. Male Sprague-Dawley rats were given daily injections of Cd for up to 12 weeks. At 3, 6, 9 and 12 weeks, urine samples were analyzed for cystatin C, protein, creatinine, ß2 microglobulin and kidney injury molecule-1. The results showed that Cd caused a significant increase in the urinary excretion of cystatin C that occurred 3-4 weeks before the onset of polyuria and proteinuria. Serum levels of cystatin C were not altered by Cd. Immunolabeling studies showed that Cd caused the relocalization of cystatin C from the cytoplasm to the apical surface of the epithelial cells of the proximal tubule. The Cd-induced changes in cystatin C labelling paralleled those of the brush border transport protein, megalin, which has been implicated as a mediator of cystatin C uptake in the proximal tubule. These results indicate that Cd increases the urinary excretion of cystatin C, and they suggest that this effect may involve disruption of megalin-mediated uptake of cystatin C by epithelial cells of the proximal tubule.

Complement activation and kidney injury molecule-1-associated proximal tubule injury in severe preeclampsia.

Kidney injury with proteinuria is a characteristic feature of preeclampsia, yet the nature of injury in specific regions of the nephron is incompletely understood. Our study aimed to use existing urinary biomarkers to describe the pattern of kidney injury and proteinuria in pregnancies affected by severe preeclampsia. We performed a case-control study of pregnant women from Brigham and Women's Hospital from 2012 to 2013. We matched cases of severe preeclampsia (n=25) 1:1 by parity and gestational age to 2 control groups with and without chronic hypertension. Urinary levels of kidney injury molecule-1 and complement components (C3a, C5a, and C5b-9) were measured by enzyme-linked immunosorbent assay, and other markers (albumin, ß2 microglobulin, cystatin C, epithelial growth factor, neutrophil gelatinase-associated lipocalin, osteopontin, and uromodulin) were measured simultaneously with a multiplex electrochemiluminescence assay. Median values between groups were compared with the Wilcoxon signed-rank test and correlations with Spearman correlation coefficient. Analysis of urinary markers revealed higher excretion of albumin and kidney injury molecule-1 and lower excretion of neutrophil gelatinase-associated lipocalin and epithelial growth factor in severe preeclampsia compared with chronic hypertension and healthy controls. Among subjects with severe preeclampsia, urinary excretion of complement activation products correlated most closely with kidney injury molecule-1, a specific marker of proximal tubule injury (C5a: r=0.60; P=0.001; and C5b-9: r=0.75; P<0.0001). Taken together, we describe a pattern of kidney injury in severe preeclampsia that is characterized by glomerular impairment and complement-mediated inflammation and injury, possibly localized to the proximal tubule in association with kidney injury molecule-1.

Adrenomedullin-RAMP2 system suppresses ER stress-induced tubule cell death and is involved in kidney protection.

Various bioactive peptides have been implicated in the homeostasis of organs and tissues. Adrenomedullin (AM) is a peptide with various bioactivities. AM-receptor, calcitonin-receptor-like receptor (CLR) associates with one of the subtypes of the accessory proteins, RAMPs. Among the RAMP subisoforms, only RAMP2 knockout mice ⁻/⁻ reproduce the phenotype of embryonic lethality of AM⁻/⁻, illustrating the importance of the AM-RAMP2-signaling system. Although AM and RAMP2 are abundantly expressed in kidney, their function there remains largely unknown. We used genetically modified mice to assess the pathophysiological functions of the AM-RAMP2 system. RAMP2⁺/⁻ mice and their wild-type littermates were used in a streptozotocin (STZ)-induced renal injury model. The effect of STZ on glomeruli did not differ between the 2 types of mice. On the other hand, damage to the proximal urinary tubules was greater in RAMP2⁺/⁻. Tubular injury in RAMP2⁺/⁻ was resistant to correction of blood glucose by insulin administration. We examined the effect of STZ on human renal proximal tubule epithelial cells (RPTECs), which express glucose transporter 2 (GLUT2), the glucose transporter that specifically takes up STZ. STZ activated the endoplasmic reticulum (ER) stress sensor protein kinase RNA-like endoplasmic reticulum kinase (PERK). AM suppressed PERK activation, its downstream signaling, and CCAAT/enhancer-binding homologous protein (CHOP)-induced cell death. We confirmed that the tubular damage was caused by ER stress-induced cell death using tunicamycin (TUN), which directly evokes ER stress. In RAMP2⁺/⁻ kidneys, TUN caused severe injury with enhanced ER stress. In wild-type mice, TUN-induced tubular damage was reversed by AM administration. On the other hand, in RAMP2⁺/⁻, the rescue effect of exogenous AM was lost. These results indicate that the AM-RAMP2 system suppresses ER stress-induced tubule cell death, thereby exerting a protective effect on kidney. The AM-RAMP2 system thus has the potential to serve as a therapeutic target in kidney disease.

Responses of proximal tubular cells to injury in congenital renal disease: fight or flight.

Most chronic kidney disease in children results from congenital or inherited disorders, which can be studied in mouse models. Following 2 weeks of unilateral ureteral obstruction (UUO) in the adult mouse, nephron loss is due to proximal tubular mitochondrial injury and cell death. In neonatal mice, proximal tubular cell death is delayed beyond 2 weeks of complete UUO, and release of partial UUO allows remodeling of remaining nephrons. Progressive cyst expansion develops in polycystic kidney disease (PKD), a common inherited renal disorder. The polycystic kidney and fibrosis (pcy)-mutant mouse (which develops late-onset PKD) develops thinning of the glomerulotubular junction in parallel with growth of cysts in adulthood. Renal insufficiency in nephropathic cystinosis, a rare inherited renal disorder, results from progressive tubular cystine accumulation. In the Ctns knockout mouse (a model of cystinosis), proximal tubular cells become flattened, with loss of mitochondria and thickening of tubular basement membrane. In each model, persistent obstructive or metabolic stress leads ultimately to the formation of atubular glomeruli. The initial "fight" response (proximal tubular survival) switches to a "flight" response (proximal tubular cell death) with ongoing oxidative injury and mitochondrial damage. Therapies should be directed at reducing proximal tubular mitochondrial oxidative injury to enhance repair and regeneration.

Differentiated kidney epithelial cells repair injured proximal tubule.

Whether kidney proximal tubule harbors a scattered population of epithelial stem cells is a major unsolved question. Lineage-tracing studies, histologic characterization, and ex vivo functional analysis results conflict. To address this controversy, we analyzed the lineage and clonal behavior of fully differentiated proximal tubule epithelial cells after injury. A CreER(T2) cassette was knocked into the sodium-dependent inorganic phosphate transporter SLC34a1 locus, which is expressed only in differentiated proximal tubule. Tamoxifen-dependent recombination was absolutely specific to proximal tubule. Clonal analysis after injury and repair showed that the bulk of labeled cells proliferate after injury with increased clone size after severe compared with mild injury. Injury to labeled proximal tubule epithelia induced expression of CD24, CD133, vimentin, and kidney-injury molecule-1, markers of putative epithelial stem cells in the human kidney. Similar results were observed in cultured proximal tubules, in which labeled clones proliferated and expressed dedifferentiation and injury markers. When mice with completely labeled kidneys were subject to injury and repair there was no dilution of fate marker despite substantial proliferation, indicating that unlabeled progenitors do not contribute to kidney repair. During nephrogenesis and early kidney growth, single proximal tubule clones expanded, suggesting that differentiated cells also contribute to tubule elongation. These findings provide no evidence for an intratubular stem-cell population, but rather indicate that terminally differentiated epithelia reexpress apparent stem-cell markers during injury-induced dedifferentiation and repair.

Ang II induce kidney damage by recruiting inflammatory cells and up regulates PPAR gamma and Renin 1 gene: effect of ß carotene on chronic renal damage.

Antioxidants are widely used for prevention of diseases associated with oxidative stress and ischemic disorder. We investigated the hypothesis of antioxidants (α-tocopherol and ß-carotene) can suppress the renal disorder in apo E-/-mice. Renal damage induced by chronic infusion of Angiotensin II (Ang II) into 4 month old male apo E-/-mice. After that the mice were treated with diet enriched α tocopherol and ß carotene (800 mg/kg) for 150 days. Ang II treated kidney showed polycystic appearance with accumulation of clear fluid and constriction of renal artery and renal vein was noticed. Vacuolar/cystic degeneration as well as inflammatory reactions was noticed in the tubules/glomerulus of Ang II treated mice. ß carotene treated mice showed enormous numbers of regenerated tubules in the kidney and over expression of ICAM proteins in the regenerated tubules. CD 45.2, MAC 3 proteins were over expressed in the inflammatory cells infiltrated into the tubular region of Ang II treated kidney. Gene expression studies revealed up regulation of Renin 1 (Ren 1) and PPARγ genes in the kidney of Ang II treated animals, but the ß carotene treatment controlled the expression of these genes in the regenerated kidneys. ß carotene may have protective effective on chronic renal disorder. It may repress the inflammatory genes (Ren 1, PPARγ) to achieve the protective effect on Ang II induced renal damage.

Protective effects of L-type fatty acid-binding protein (L-FABP) in proximal tubular cells against glomerular injury in anti-GBM antibody-mediated glomerulonephritis.

BACKGROUND: In glomerulonephritis (GN), an overload of free fatty acids (FFA) bound to albumin in urinary protein may induce oxidative stress in the proximal tubules. Human liver-type fatty acid-binding protein (hL-FABP) expressed in human proximal tubules, but not rodents, participates in intracellular FFA metabolism and exerts anti-oxidative effects on the progression of tubulointerstitial damage. We examined whether tubular enhancement of this anti-oxidative action modulates the progression of glomerular damage in immune-mediated GN in hL-FABP chromosomal gene transgenic (Tg) mice. METHODS: Anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM GN) was induced in Tg and wild-type mice (WT). Proteinuria, histopathology, polymorphonuclear (PMN) influx, expression of tubulointerstitial markers for oxidative stress 4-hydroxy-2-Nonenal (HNE) and fibrosis (α-smooth muscle actin), proximal tubular damage (Kim-1), Peroxisome Proliferator-Activated Receptor γ (PPAR γ) and inflammatory cytokines [Monocyte Chemotactic Protein-1, tumor necrosis factor-alpha (TNF-α) and Transforming growth factor beta (TGF-ß)] were analyzed. The mice were also treated with an angiotensin type II receptor blocker (ARB). RESULTS: The urinary protein level in Tg mice decreased significantly during the acute phase (~Day 5). Tg mice survived for a significantly longer time than WT mice, with an attenuation of tubulointerstitial damage score and expression of each tubulointerstitial damage marker observed at Day 7. Expression of inflammatory cytokines on Day 7 was higher in WT mice than Tg mice and correlated strongly with PPARγ expression in WT mice, but not in Tg mice. Interestingly, Tg mice showed insufficient PMN influx at 3 and 6 h, with simultaneous elevation of urinary L-FABP and reduction in HNE expression. The two strains of mice showed different types of glomerular damage, with mild mesangial proliferation in Tg mice and severe endothelial swelling with vascular thrombosis in WT mice. The glomerular damage in Tg mice was improved by administration of an ARB. CONCLUSIONS: The present experimental model suggests that tubular enhancement of L-FABP may protect mice with anti-GBM GN from progression of both tubulointerstitial and glomerular injury.

Annexin 1 mimetic peptide protects against renal ischemia/reperfusion injury in rats.

Inflammation is currently recognized as a key mechanism in the pathogenesis of renal ischemia-reperfusion (I/R) injury. The importance of infiltrating neutrophil, lymphocytes, and macrophage in this kind of injury has been assessed with conflicting results. Annexin 1 is a protein with potent neutrophil anti-migratory activity. In order to evaluate the effects of annexin A1 on renal I/R injury, uninephrectomized rats received annexin A1 mimetic peptide Ac2-26 (100 µg) or vehicle before 30 min of renal artery clamping and were compared to sham surgery animals. Annexin A1 mimetic peptide granted a remarkable protection against I/R injury, preventing glomerular filtration rate and urinary osmolality decreases and acute tubular necrosis development. Annexin A1 infusion aborted neutrophil extravasation and attenuated macrophage infiltration but did not prevent tissue lymphocyte traffic. I/R increased annexin A1 expression (assessed by transmission electron microscopy) in renal epithelial cells, which was attenuated by exogenous annexin A1 infusion. Additionally, annexin A1 reduced I/R injury in isolated proximal tubules suspension. Annexin A1 protein afforded striking functional and structural protection against renal I/R. These results point to an important role of annexin A1 in the epithelial cells defense against I/R injury and indicate that neutrophils are key mediators for the development of tissue injury after renal I/R. If these results were confirmed in clinical studies, annexin A1 might emerge as an important tool to protect against I/R injury in renal transplantation and in vascular surgery.

Kidney-specific overexpression of Sirt1 protects against acute kidney injury by retaining peroxisome function.

Sirt1, a NAD-dependent protein deacetylase, is reported to regulate intracellular metabolism and attenuate reactive oxidative species (ROS)-induced apoptosis leading to longevity and acute stress resistance. We created transgenic (TG) mice with kidney-specific overexpression of Sirt1 using the promoter sodium-phosphate cotransporter IIa (Npt2) driven specifically in proximal tubules and investigated the kidney-specific role of Sirt1 in the protection against acute kidney injury (AKI). We also elucidated the role of number or function of peroxisome and mitochondria in mediating the mechanisms for renal protective effects of Sirt1 in AKI. Cisplatin-induced AKI decreased the number and function of peroxisomes as well as mitochondria and led to increased local levels of ROS production and renal tubular apoptotic cells. TG mice treated with cisplatin mitigated AKI, local ROS, and renal tubular apoptotic tubular cells. Consistent with these results, TG mice treated with cisplatin also exhibited recovery of peroxisome number and function, as well as rescued mitochondrial function; however, mitochondrial number was not recovered. Immunoelectron microscopic findings consistently demonstrated that the decrease in peroxisome number by cisplatin in wild type mice was restored in transgenic mice. In HK-2 cells, a cultured proximal tubule cell line, overexpression of Sirt1 rescued the cisplatin-induced cell apoptosis through the restoration of peroxisome number, although the mitochondria number was not restored. These results indicate that Sirt1 overexpression in proximal tubules rescues cisplatin-induced AKI by maintaining peroxisomes number and function, concomitant up-regulation of catalase, and elimination of renal ROS levels. Renal Sirt1 can be a potential therapeutic target for the treatment of AKI.

siRNA targeted to p53 attenuates ischemic and cisplatin-induced acute kidney injury.

Proximal tubule cells (PTCs), which are the primary site of kidney injury associated with ischemia or nephrotoxicity, are the site of oligonucleotide reabsorption within the kidney. We exploited this property to test the efficacy of siRNA targeted to p53, a pivotal protein in the apoptotic pathway, to prevent kidney injury. Naked synthetic siRNA to p53 injected intravenously 4 h after ischemic injury maximally protected both PTCs and kidney function. PTCs were the primary site for siRNA uptake within the kidney and body. Following glomerular filtration, endocytic uptake of Cy3-siRNA by PTCs was rapid and extensive, and significantly reduced ischemia-induced p53 upregulation. The duration of the siRNA effect in PTCs was 24 to 48 h, determined by levels of p53 mRNA and protein expression. Both Cy3 fluorescence and in situ hybridization of siRNA corroborated a short t(1/2) for siRNA. The extent of renoprotection, decrease in cellular p53 and attenuation of p53-mediated apoptosis by siRNA were dose- and time-dependent. Analysis of renal histology and apoptosis revealed improved injury scores in both cortical and corticomedullary regions. siRNA to p53 was also effective in a model of cisplatin-induced kidney injury. Taken together, these data indicate that rapid delivery of siRNA to proximal tubule cells follows intravenous administration. Targeting siRNA to p53 leads to a dose-dependent attenuation of apoptotic signaling, suggesting potential therapeutic benefit for ischemic and nephrotoxic kidney injury.