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Accurate modeling of the complex three-dimensional (3D) microarchitectures of in vivo vascular structures has remained a formidable challenge for medical and biological communities. Although many platforms have been developed to mimic vascular microgeometry, difficulties associated with recreating in vivo characteristics such as permeability and tortuosity has impeded progress. To address these issues, here we present a novel strategy that combines additive manufacturing or “3D printing” with suspended microfluidics to fabricate semipermeable, tubular structures with tortuous architectures. Specifically, the approach entails: (i) using PolyJet 3D printing to create a tubular framework comprising local microcurvature and global tortuosity, and then (ii) employing a suspended microfluidics technique to coat the framework with a porous, biocompatible cellular scaffold. Fabricated proof-of-concept tubular scaffolds exhibited inner radii on the order of 100 μm. Experimental results revealed tubular rupture pressures in the range of approximately 30–65 kPa. Seeding of human umbilical vein endothelial cells (HUVECs) within the tubule suggests that the presented work could be extended to better model the micro/mesoscale architectures of vascular systems for applications including drug discovery, disease modeling, and personalized medicine.

Contains fulltext : 182590.pdf (Publisher’s version ) (Closed access) Acute kidney injury (AKI) is a common complication in the critically ill. Current standard of care mainly relies on identification of patients at risk, haemodynamic optimization, avoidance of nephrotoxicity and the use of renal replacement therapy (RRT) in established AKI. The detection of early biomarkers of renal tissue damage is a recent development that allows amending the late and insensitive diagnosis with current AKI criteria. Increasing evidence suggests that the consequences of an episode of AKI extend long beyond the acute hospitalization. Citrate has been established as the anticoagulant of choice for continuous RRT. Conflicting results have been published on the optimal timing of RRT and on the renoprotective effect of remote ischaemic preconditioning. Recent research has contradicted that acute tubular necrosis is the common pathology in AKI, that septic AKI is due to global kidney hypoperfusion, that aggressive fluid therapy benefits the kidney, that vasopressor therapy harms the kidney and that high doses of RRT improve outcome. Remaining uncertainties include the impact of aetiology and clinical context on pathophysiology, therapy and prognosis, the clinical benefit of biomarker-driven interventions, the optimal mode of RRT to improve short- and long-term patient and kidney outcomes, the contribution of AKI to failure of other organs and the optimal approach for assessing and promoting renal recovery. Based on the established gaps in current knowledge the trials that must have priority in the coming 10 years are proposed together with the definition of appropriate clinical endpoints.

Design of Phase III trials for diabetic nephropathy currently requires patients at a high risk of progression defined as within three years of a hard end point (end-stage renal disease, 40% loss of estimated glomerular filtration rate, or death). To improve the design of these trials, we used natural history data from the Joslin Kidney Studies of chronic kidney disease in patients with diabetes to develop an improved criterion to identify such patients. This included a training cohort of 279 patients with type 1 diabetes and 134 end points within three years, and a validation cohort of 221 patients with type 2 diabetes and 88 end points. Previous trials selected patients using clinical criteria for baseline urinary albumin-to-creatinine ratio and estimated glomerular filtration rate. Application of these criteria to our cohort data yielded sensitivities (detection of patients at risk) of 70-80% and prognostic values of only 52-63%. We applied classification and regression trees analysis to select from among all clinical characteristics and markers the optimal prognostic criterion that divided patients with type 1 diabetes according to risk. The optimal criterion was a serum tumor necrosis factor receptor 1 level over 4.3 ng/ml alone or 2.9-4.3 ng/ml with an albumin-to-creatinine ratio over 1900 mg/g. Remarkably, this criterion produced similar results in both type 1 and type 2 diabetic patients. Overall, sensitivity and prognostic value were high (72% and 81%, respectively). Thus, application of this criterion to enrollment in future clinical trials could reduce the sample size required to achieve adequate statistical power for detection of treatment benefits.

Early acute kidney injury (AKI) diagnosis is needed to pursue treatment trials. We evaluated cystatin C (CysC) as an early biomarker of serum creatinine (SCr)-AKI and an alternative to define AKI. We studied 160 non-cardiac children in the intensive care unit (ICU). We measured daily CysC and SCr. AKI was staged by KDIGO (Kidney Disease: Improving Global Outcomes) guidelines using SCr and CysC (CysC-AKI). We calculated area under the curve (AUC) for (1) neutrophil gelatinase-associated lipocalin (NGAL), interleukin-18 (IL-18), kidney injury molecule-1 (KIM-1) and urine CysC to diagnose SCr- and CysC-AKI; and (2) for CysC to diagnose SCr-AKI. We evaluated AKI associations with length of stay and ventilation duration. We found that 44 % of patients developed SCr-AKI; 32 % developed CysC-AKI. Early ICU NGAL was most diagnostic of CysC-AKI (AUC 0.69, 95% CI 0.54–0.84); IL-18 was most diagnostic for SCr-AKI (AUC 0.69 95% CI 0.55–0.82). Combining SCr and CysC-AKI definition led to higher biomarker diagnostic AUC’s. CysC-AKI was not more strongly associated with clinical outcomes. Early ICU CysC predicted SCr-AKI development (AUC 0.70, 95 % CI 0.53–0.89). Our findings do not support replacing SCr by CysC to define AKI. Early ICU CysC predicts SCr-AKI development and combined SCr-CysC-AKI definition leads to stronger AKI biomarker associations.

Tubular injury has a major etiological role in fibrosis. For many years, this relationship has been dominated by the perception that epithelial cells are transformed into myofibroblasts that proliferate and generate fibrotic matrix-the so-called epithelial-to-mesenchymal transition. Here we focus on mechanisms by which injury to the tubule results in fibrosis because of paracrine mechanisms. Specific injury to the proximal tubule results in inflammation, reversible injury, and adaptive repair if the insult is mild, self-limited in time, and occurs in a background of a normal kidney. Repeated injury, in contrast, leads to maladaptive repair with sustained tubule injury, chronic inflammation, proliferation of interstitial myofibroblasts, vascular rarefaction, interstitial fibrosis, and glomerular sclerosis. During the maladaptive repair process after the renal insult, many tubular cells become arrested in the G2/M phase of the cell cycle. This results in activation of the DNA repair response with the resultant synthesis and secretion of pro-fibrotic factors. Pharmacologic interventions that enhance the movement through G2/M or facilitate apoptosis of cells that otherwise would be blocked in G2/M may reduce the development of fibrosis after kidney injury and reduce the progression of chronic kidney disease.

The National Institute of Diabetes and Digestive and Kidney Diseases–supported Kidney Research National Dialogue asked the scientific community to formulate and prioritize research objectives that would improve our understanding of kidney function and disease; >1600 participants from >30 countries posted >300 ideas and >500 comments covering all areas of kidney research. Smaller groups of investigators interrogated the postings and published a series of commentaries in CJASN. Additional review of the entire series identified six cross-cutting themes: (1) increase training and team science opportunities to maintain/expand the nephrology workforce, (2) develop novel technologies to assess kidney function, (3) promote human discovery research to better understand normal and diseased kidney function, (4) establish integrative models of kidney function to inform diagnostic and treatment strategies, (5) promote interventional studies that incorporate more responsive outcomes and improved trial designs, and (6) foster translation from clinical investigation to community implementation. Together, these cross-cutting themes provide a research plan to better understand normal kidney biology and improve the prevention, diagnosis, and treatment of kidney disease, and as such, they will inform future research efforts supported by the National Institute of Diabetes and Digestive and Kidney Diseases through workshops and initiatives.

Tissue damage by oxidative stress is a key pathogenic mechanism in various diseases, including AKI and CKD. Thus, early detection of oxidative tissue damage is important. Using a tRNA-specific modified nucleoside 1-methyladenosine (m1A) antibody, we show that oxidative stress induces a direct conformational change in tRNA structure that promotes subsequent tRNA fragmentation and occurs much earlier than DNA damage. In various models of tissue damage (ischemic reperfusion, toxic injury, and irradiation), the levels of circulating tRNA derivatives increased rapidly. In humans, the levels of circulating tRNA derivatives also increased under conditions of acute renal ischemia, even before levels of other known tissue damage markers increased. Notably, the level of circulating free m1A correlated with mortality in the general population (n=1033) over a mean follow-up of 6.7 years. Compared with healthy controls, patients with CKD had higher levels of circulating free m1A, which were reduced by treatment with pitavastatin (2 mg/d; n=29). Therefore, tRNA damage reflects early oxidative stress damage, and detection of tRNA damage may be a useful tool for identifying organ damage and forming a clinical prognosis.

Acute kidney injury (AKI) leads to worsening of chronic kidney disease (CKD), and CKD predisposes to the clinical entity of AKI. The tubules of the kidney play a central role in the fibrotic response, which ultimately leads to progressive kidney disease. The cellular mechanisms responsible for the epidemiological association between AKI and CKD are complex. In order to unravel characteristics of this direct involvement of the tubules, in particular the proximal tubules, we established a model to specifically target injury to the proximal tubule using a genetic approach to express the simian diphtheria toxin (DT) receptor in the proximal tubule. A single administration of DT to the proximal tubule resulted in inflammation, reversible injury, and adaptive repair. By contrast, thrice repeated injury led to maladaptive repair with sustained tubule injury, vascular rarefaction, proliferation of interstitial myofibroblasts, interstitial fibrosis, and glomerular sclerosis. An important feature of the maladaptive repair process after severe injury is the development of cell cycle arrest in G2/M. There is a subsequent activation of the DNA repair response with activation of a secretory phenotype whereby profibrotic factors are released. This insight introduces a number of potential new targets for therapeutic intervention to prevent and/or arrest CKD progression.

Despite the increasing prevalence of CKD in the United States, there is a declining interest among United States medical graduates in nephrology as a career choice. Effective programs are needed to generate interest at early educational stages when career choices can be influenced. The Kidney Disease Screening and Awareness Program (KDSAP) is a novel program initiated at Harvard College that increases student knowledge of and interest in kidney health and disease, interest in nephrology career paths, and participation in kidney disease research. This model, built on physician mentoring, kidney screening of underserved populations, direct interactions with kidney patients, and opportunities to participate in kidney research, can be reproduced and translated to other workforce-challenged subspecialties.

Efficient clearance of apoptotic cells (efferocytosis) prevents inflammation and permits repair following tissue injury. Kidney injury molecule-1 (KIM-1) is a receptor for phosphatidylserine, an “eat-me” signal exposed on the surface of apoptotic cells that marks them for phagocytic clearance. KIM-1 is upregulated on proximal tubule epithelial cells (PTECs) during ischemic acute kidney injury (AKI), enabling efferocytosis by surviving PTECs. KIM-1 is spontaneously cleaved at its ectodomain region to generate a soluble fragment that serves a sensitive and specific biomarker for AKI, but the biological relevance of KIM-1 shedding is unknown. Here, we sought to determine how KIM-1 shedding might regulate efferocytosis. Using cells that endogenously and exogenously express KIM-1, we found that hydrogen peroxide-mediated oxidative injury or PMA treatment accelerated KIM-1 shedding in a dose-dependent manner. KIM-1 shedding was also accelerated when apoptotic cells were added. Accelerated shedding or the presence of excess soluble KIM-1 in the extracellular milieu significantly inhibited efferocytosis. We also identified that TNF-α-converting enzyme (TACE or ADAM17) mediates both the spontaneous and PMA-accelerated shedding of KIM-1. While accelerated shedding inhibited efferocytosis, we found that spontaneous KIM-1 cleavage does not affect the phagocytic efficiency of PTECs. Our results suggest that KIM-1 shedding is accelerated by worsening cellular injury, and excess soluble KIM-1 competitively inhibits efferocytosis. These findings may be important in AKI when there is severe cellular injury.

For several decades, acute kidney injury (AKI) was generally considered a reversible process leading to complete kidney recovery if the individual survived the acute illness. Recent evidence from epidemiologic studies and animal models, however, have highlighted that AKI can lead to the development of fibrosis and facilitate the progression of chronic renal failure. When kidney injury is mild and baseline function is normal, the repair process can be adaptive with few long-term consequences. When the injury is more severe, repeated, or to a kidney with underlying disease, the repair can be maladaptive and epithelial cell cycle arrest may play an important role in the development of fibrosis. Indeed, during the maladaptive repair after a renal insult, many tubular cells that are undergoing cell division spend a prolonged period in the G2/M phase of the cell cycle. These tubular cells recruit intracellular pathways leading to the synthesis and the secretion of profibrotic factors, which then act in a paracrine fashion on interstitial pericytes/fibroblasts to accelerate proliferation of these cells and production of interstitial matrix. Thus, the tubule cells assume a senescent secretory phenotype. Characteristic features of these cells may represent new biomarkers of fibrosis progression and the G2/M-arrested cells may represent a new therapeutic target to prevent, delay or arrest progression of chronic kidney disease. Here, we summarize recent advances in our understanding of the biology of the cell cycle and how cell cycle arrest links AKI to chronic kidney disease.

Objective The objective of this study was to investigate whether cytosolic phospholipase A2 (cPLA2), an important isoform of PLA2 that mediates the release of arachidonic acid, plays a role in the pathogenesis of spinal cord injury (SCI). Methods A combination of molecular, histological, immunohistochemical, and behavioral assessments were used to test whether blocking cPLA2 activation pharmacologically or genetically reduced cell death, protected spinal cord tissue, and improved behavioral recovery after a contusive SCI performed at the 10th thoracic level in adult mice. Results SCI significantly increased cPLA2 expression and activation. Activated cPLA2 was localized mainly in neurons and oligodendrocytes. Notably, the SCI-induced cPLA2 activation was mediated by the extracellular signal-regulated kinase signaling pathway. In vitro, activation of cPLA2 by ceramide-1-phosphate or A23187 induced spinal neuronal death, which was substantially reversed by arachidonyl trifluoromethyl ketone, a cPLA2 inhibitor. Remarkably, blocking cPLA2 pharmacologically at 30 minutes postinjury or genetically deleting cPLA2 in mice ameliorated motor deficits, and reduced cell loss and tissue damage after SCI. Interpretation cPLA2 may play a key role in the pathogenesis of SCI, at least in the C57BL/6 mouse, and as such could be an attractive therapeutic target for ameliorating secondary tissue damage and promoting recovery of function after SCI.

Perturbation of calcium signaling that occurs during cell injury and disease, promotes cell death. In mouse lung fibroblasts A23187 triggered mitochondrial permeability transition pore (MPTP) formation, lactate dehydrogenase (LDH) release, and necrotic cell death that were blocked by cyclosporin A (CsA) and EGTA. LDH release temporally correlated with arachidonic acid release but did not involve cytosolic phospholipase A2α (cPLA2α) or calcium-independent PLA2. Surprisingly, release of arachidonic acid and LDH from cPLA2α-deficient fibroblasts was inhibited by the cPLA2α inhibitor pyrrophenone, and another serine hydrolase inhibitor KT195, by preventing mitochondrial calcium uptake. Inhibitors of calcium/calmodulin-dependent protein kinase II, a mitochondrial Ca(2+) uniporter (MCU) regulator, also prevented MPTP formation and arachidonic acid release induced by A23187 and H2O2. Pyrrophenone blocked MCU-mediated mitochondrial calcium uptake in permeabilized fibroblasts but not in isolated mitochondria. Unlike pyrrophenone, the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol and CsA blocked cell death and arachidonic acid release not by preventing mitochondrial calcium uptake but by inhibiting MPTP formation. In fibroblasts stimulated with thapsigargin, which induces MPTP formation by a direct effect on mitochondria, LDH and arachidonic acid release were blocked by CsA and 1-oleoyl-2-acetyl-sn-glycerol but not by pyrrophenone or EGTA. Therefore serine hydrolase inhibitors prevent necrotic cell death by blocking mitochondrial calcium uptake but not the enzyme releasing fatty acids that occurs by a novel pathway during MPTP formation. This work reveals the potential for development of small molecule cell-permeable serine hydrolase inhibitors that block MCU-mediated mitochondrial calcium overload, MPTP formation, and necrotic cell death.

Patients with High Grade Serous Ovarian Cancer (HGSC) have limited therapeutic options. Immuno-oncologic (IO) agents have had limited effect. DNA damage repair gene mutations that may confer repair defects have been identified in up to 50% of HGSCs, making therapies that target repair defects, like PARP, CHK1, and ATR inhibitors, additional options. We have no means of predicting which patients will respond to any of these therapies. A model system that allows for functional assays to assess for DNA damage repair defects, prediction of response to therapies targeting such defects, and assessment of the functionality of the tumor immune infiltrate and its response to IO agents is needed. Organoids are three-dimensional structures derived from human normal or tumor tissue cells that anatomically and functionally mimic the developed human organ. Organoids mimicking the parent tumor from which they were derived have aided in the study of multiple tumor types. They are inexpensive and easily manipulated and may be an ideal model system for studying ovarian cancer. We have devised a functional assay platform to profile the DNA damage repair capacity and immune targetability of short-term patient-derived HGSC organoids. The organoids mimic the tumors from which they were derived morphologically, molecularly, and genetically. We have tested 33 organoid cultures derived from 21 HGSC patients for homologous recombination (HR) and replication fork protection capacity and compared the functional results to the tumor genomic profile. Regardless of repair gene mutational status, an HR functional defect in the organoids correlated with PARP inhibitor sensitivity. A fork protection functional defect correlated with carboplatin, and ATR and CHK1 inhibitor sensitivity. Importantly, this work has led to the discovery of potential therapeutic combinations, such as a CHK1 inhibitor plus carboplatin or gemcitabine that may be useful in treating tumors otherwise resistant to most therapies. Drugs such as carboplatin or gemcitabine can synergize with a CHK1 inhibitor by enhancing replication stress and fork deprotection. In parallel, we have immune phenotyped the parent tumors and organoid cultures from 15 patients, and shown that the organoid cultures retain lymphocytes expressing relevant IO receptors in the short term. Upon treatment with carboplatin, olaparib, and pembrolizumab as single agents or in combination, we detect changes in IO receptor expression and production of different cytokines in the cultures, suggesting an immune response induced by these agents. We have detected receptor and cytokine alterations that would create an immune suppressive environment with specific drug combinations in tumors with specific repair defects, suggesting that these may be inappropriate combinations for harnessing the immune system in tumors with specific repair capacities. Continued combined immune and DNA damage repair phenotyping analyses of the organoids will lead to a better understanding of which mechanistic defects are needed to confer sensitivity to DNA damage repair agents, what functional properties and immune milieu lead to sensitivity to IO agents, and how best to combine such therapies. In addition, through further correlation with patient responses over time, HGSC organoids may become a useful tool for rapidly predicting patient response to therapeutic agents. Citation Format: Sarah J. Hill, Brennan Decker, Emma A. Roberts, Chunyu Yang, Neil S. Horowitz, Michael G. Muto, Michael J. Worley Jr., Colleen M. Feltmate, Marisa R. Nucci, Elizabeth M. Swisher, Ryuji Morizane, Bose Kochupurakkal, Khanh T. Do, Panagiotis Konstantinopoulos, Joyce F. Liu, Joseph V. Bonventre, Ursula A. Matulonis, Geoffrey I. Shapiro, Ross S. Berkowitz, Christopher P. Crum, and Alan D. D'Andrea. REAL-TIME ASSESSMENT OF HGSC DNA DAMAGE REPAIR DEFECTS AND DEFECT-INDUCED RESPONSE TO THERAPY IN OVARIAN CANCER ORGANOIDS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP10.

Heterozygous mutations in PKD1 or PKD2, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively, cause autosomal dominant PKD (ADPKD), whereas mutations in PKHD1, which encodes fibrocystin/polyductin (FPC), cause autosomal recessive PKD (ARPKD). However, the relationship between these proteins and the pathogenesis of PKD remains unclear. To model PKD in human cells, we established induced pluripotent stem (iPS) cell lines from fibroblasts of three ADPKD and two ARPKD patients. Genetic sequencing revealed unique heterozygous mutations in PKD1 of the parental ADPKD fibroblasts but no pathogenic mutations in PKD2. Undifferentiated PKD iPS cells, control iPS cells, and embryonic stem cells elaborated primary cilia and expressed PC1, PC2, and FPC at similar levels, and PKD and control iPS cells exhibited comparable rates of proliferation, apoptosis, and ciliogenesis. However, ADPKD iPS cells as well as somatic epithelial cells and hepatoblasts/biliary precursors differentiated from these cells expressed lower levels of PC2 at the cilium. Additional sequencing confirmed the retention of PKD1 heterozygous mutations in iPS cell lines from two patients but identified possible loss of heterozygosity in iPS cell lines from one patient. Furthermore, ectopic expression of wild-type PC1 in ADPKD iPS-derived hepatoblasts rescued ciliary PC2 protein expression levels, and overexpression of PC1 but not a carboxy-terminal truncation mutant increased ciliary PC2 expression levels in mouse kidney cells. Taken together, these results suggest that PC1 regulates ciliary PC2 protein expression levels and support the use of PKD iPS cells for investigating disease pathophysiology.

Background Serum creatinine has been used as the diagnostic test for acute kidney injury (AKI) for decades despite having imperfect sensitivity and specificity. Novel tubular injury biomarkers may revolutionize the diagnosis of AKI; however, even if a novel tubular injury biomarker is 100% sensitive and 100% specific, it may appear inaccurate when using serum creatinine as the gold standard. Conclusions In general, the apparent diagnostic performance of a biomarker depends not only on its ability to detect injury but also on disease prevalence and the sensitivity and specificity of the imperfect gold standard. Apparent errors in diagnosis using a new biomarker may be a reflection of errors in the imperfect gold standard itself rather than poor performance of the biomarker.

Because of the difficulty in detecting segment-specific response in the kidney, we investigated the molecular events underlying acute kidney injury in the proximal tubules of rats with cisplatin (cis-diamminedichloroplatinum II)-induced nephrotoxicity. Microarray analysis revealed that mRNA levels of several cytokines and chemokines, such as interleukin-1beta, chemokine (C-C motif) ligand (CCL) 2, CCL20, chemokine (C-X-C motif) ligand (CXCL) 1, and CXCL10 were significantly increased after cisplatin treatment in both isolated proximal tubules and whole kidney. Interestingly, tubular CCL2 mRNA levels increased soon after cisplatin administration, whereas CCL2 mRNA levels in whole kidney first decreased and then increased. Levels of both CCL2 and kidney injury molecule-1 (KIM-1) in the whole kidney increased after cisplatin administration. Immunofluorescence analysis revealed CCL2 changes in the proximal tubular cells initially and then in the medullary interstitium. Urinary CCL2 excretion significantly increased approximately 3-fold compared with controls the day after cisplatin administration (5 mg/kg), when no changes were observed plasma creatinine and blood urea nitrogen levels. Urinary levels of KIM-1 also increased 3-fold after cisplatin administration. In addition, urinary CCL2 rather than KIM-1 increased in chronic renal failure rats after administration of low-dose cisplatin (2 mg/kg), suggesting that urinary CCL2 was selective for cisplatin-induced nephrotoxicity in renal impairment. These results indicated that the increase in cytokine and chemokine expression in renal epithelial cells might be responsible for kidney deterioration in cisplatin-induced nephrotoxicity, and that urinary CCL2 is associated with tubular injury and serves as a sensitive and noninvasive marker for the early detection of cisplatin-induced tubular injury.

Renal diseases in patients with cancer have many unique features, and often these diseases require specialized approaches. Newer cancer therapy has increased cancer cure rate and survival time, but such benefit is not fully realized, partly because of therapy-associated toxicities. Fluid and electrolyte abnormalities are very common in patients with cancer, as are acute and chronic kidney injury. With the evolving complexities of newer cancer therapies, a comprehensive team approach is becoming necessary. It is essential for nephrologists to be informed and involved in cancer care. Many nephrologists caring for patients with cancer in the United States have recently met and formed a focus group, the OncoNephrology Forum, under the American Society of Nephrology. This update addresses what is clinically unique about onconephrology, the objectives and functions of the newly formed forum, and the potential of onconephrology becoming a subspecialty in nephrology.

Background Cisplatin is a potent chemotherapeutic drug whose nephrotoxic effect is a major complication and a dose-limiting factor for antitumoral therapy. There is much evidence that inflammation contributes to the pathogenesis of cisplatin-induced nephrotoxicity. We found that cilastatin, a renal dehydropeptidase-I inhibitor, has protective effects in vitro and in vivo against cisplatin-induced renal damage by inhibiting apoptosis and oxidation. Here, we investigated the potential use of cilastatin to protect against cisplatin-induced kidney injury and inflammation in rats. Methods Male Wistar rats were divided into four groups: control, cilastatin-control, cisplatin and cilastatin-cisplatin. Nephrotoxicity was assessed 5 days after administration of cisplatin based on blood urea nitrogen, creatinine, glomerular filtration rate (GFR), kidney injury molecule (KIM)-1 and renal morphology. Inflammation was measured using the electrophoretic mobility shift assay, immunohistochemical studies and evaluation of inflammatory mediators. Results Compared with the control rats, cisplatin-administered rats were affected by significant proximal tubule damage, decreased GFR, increased production of inflammatory mediators and elevations in urea, creatinine and tissue KIM-1 levels. Cilastatin prevented these changes in renal function and ameliorated histological damage in cisplatin-administered animals. Cilastatin also reduced pro-inflammatory cytokine levels, activation of nuclear factor-κB and CD68-positive cell concentrations. Conclusions Cilastatin reduces cisplatin-induced nephrotoxicity, which is associated with decreased inflammation in vivo. Although the exact role of decreased inflammation in nephroprotection has not been fully elucidated, treatment with cilastatin could be a novel strategy for the prevention of cisplatin-induced acute kidney injury.

Rickettsial agents are sensed by pattern recognition receptors but lack pathogen-associated molecular patterns commonly observed in facultative intracellular bacteria. Due to these molecular features, the order Rickettsiales can be used to uncover broader principles of bacterial immunity. Here, we used the bacterium Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, to reveal a novel microbial surveillance system. Mechanistically, we discovered that upon A. phagocytophilum infection, cytosolic phospholipase A2 cleaves arachidonic acid from phospholipids, which is converted to the eicosanoid prostaglandin E2 (PGE2) via cyclooxygenase 2 (COX2) and the membrane associated prostaglandin E synthase-1 (mPGES-1). PGE2-EP3 receptor signaling leads to activation of the NLRC4 inflammasome and secretion of interleukin (IL)-1β and IL-18. Importantly, the receptor-interacting serine/threonine-protein kinase 2 (RIPK2) was identified as a major regulator of the immune response against A. phagocytophilum. Accordingly, mice lacking COX2 were more susceptible to A. phagocytophilum, had a defect in IL-18 secretion and exhibited splenomegaly and damage to the splenic architecture. Remarkably, Salmonella-induced NLRC4 inflammasome activation was not affected by either chemical inhibition or genetic ablation of genes associated with PGE2 biosynthesis and signaling. This divergence in immune circuitry was due to reduced levels of the PGE2-EP3 receptor during Salmonella infection when compared to A. phagocytophilum. Collectively, we reveal the existence of a functionally distinct NLRC4 inflammasome illustrated by the rickettsial agent A. phagocytophilum., Author Summary Elimination of bacteria is orchestrated by the immune system. Intracellular bacteria are generally recognized by cytosolic molecules named Nod-like receptors (NLRs). One such protein scaffold that senses needle-like structures and globular proteins, namely, the bacterial type III secretion (T3SS) and flagellin, is the NLRC4 inflammasome. The NLRC4 inflammasome induces caspase-1 autoproteolysis and secretion of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18. Here, we show that the obligate intracellular rickettsial pathogen Anaplasma phagocytophilum, which does not have a T3SS or flagellin-coding genes, induces a distinct NLRC4 inflammasome circuitry through the eicosanoid prostaglandin E2 and the EP3 receptor. Conceptually, these findings establish the existence of a distinct microbial surveillance system where the NLRC4 inflammasome senses an obligate intracellular pathogen of public health relevance. Therefore, we propose that rickettsial agents can be used to uncover broader principles of immune surveillance given their unique life style to survive inside the mammalian host and lack of pathogen associated molecular patterns commonly present in most facultative intracellular bacteria.

BACKGROUND: The primary biomarkers used to define CKD are serum creatinine and albuminuria. These biomarkers have directed focus on the filtration and barrier functions of the kidney glomerulus even though albuminuria results from tubule dysfunction as well. Given that proximal tubules make up ∼90% of kidney cortical mass, we evaluated whether a sensitive and specific marker of proximal tubule injury, urinary kidney injury molecule-1 (KIM-1), is elevated in individuals with CKD or with risk factors for CKD. METHODS: We measured urinary KIM-1 in participants of five cohort studies from the USA and Sweden. Participants had a wide range of kidney function and were racially and ethnically diverse. Multivariable linear regression models were used to test the association of urinary KIM-1 with demographic, clinical and laboratory values. RESULTS: In pooled, multivariable-adjusted analyses, log-transformed, creatinine-normalized urinary KIM-1 levels were higher in those with lower eGFR {β = -0.03 per 10 mL/min/1.73 m(2) [95% confidence interval (CI) -0.05 to -0.02]} and greater albuminuria [β = 0.16 per unit of log albumin:creatinine ratio (95% CI 0.15-0.17)]. Urinary KIM-1 levels were higher in current smokers, lower in blacks than nonblacks and lower in users versus nonusers of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers. CONCLUSION: Proximal tubule injury appears to be an integral and measurable element of multiple stages of CKD.

Few investigations have evaluated the incremental usefulness of tubular injury biomarkers for improved prediction of chronic kidney disease (CKD) progression. As such, we measured urinary kidney injury molecule-1, neutrophil gelatinase–associated lipocalin, N -acetyl-s-D-glucosaminidase and liver fatty acid binding protein under highly standardized conditions among 2466 enrollees of the prospective Chronic Renal Insufficiency Cohort Study. During 9433 person-years of follow-up, there were 581 cases of CKD progression defined as incident end-stage renal disease or halving of the estimated glomerular filtration rate. Levels of the urine injury biomarkers, normalized for urine creatinine, were strongly associated with CKD progression in unadjusted Cox proportional hazard models with hazard ratios in the range of 7 to 15 comparing the highest with the lowest quintiles. However, after controlling for the serum creatinine–based estimated glomerular filtration rate and urinary albumin/creatinine ratio, none of the normalized biomarkers was independently associated with CKD progression. None of the biomarkers improved on the high (0.89) C-statistic for the base clinical model. Thus, among patients with CKD, risk prediction with a clinical model that includes the serum creatinine–based estimated glomerular filtration rate and the urinary albumin/creatinine ratio is not improved on with the addition of renal tubular injury biomarkers.

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.

Human pluripotent stem cells (hPSCs) can self-renew extensively and differentiate into diverse tissues, including tubular organoids expressing kidney markers. However, no study has demonstrated a phenotype in these tubules relevant to kidney disease. Polycystic kidney disease (PKD) is an extremely common, life-threatening Mendelian disorder in which balloon-like cysts arise from kidney tubules. Using the CRISPR/Cas9 genome editing system, we tested the ability of hPSCs and derived kidney organoids to model PKD cystogenesis in vitro.To generate PKD hPSCs, Cas9 nuclease and guide RNAs targeting the disease-causative genes, PKD1 or PKD2, were transfected into hPSCs. hPSCs in 3D cultures were treated with specific growth factors to direct stepwise differentiation into kidney progenitor cells (SIX2+PAX2+) and subsequently proximal tubules (LTL+LRP2+). Phenotypically, PKD hPSCs exhibited self-renewal and pluripotency characteristics comparable to isogenic controls, and differentiated into tubular organoids with similar efficiencies. Interestingly, in PKD hPSC cultures, we observed formation of large, translucent, cyst-like structures alongside tubules. Confocal microscopy revealed that cyst-lining epithelia reacted strongly with LTL, were highly proliferative, and surrounded hollow interior compartments devoid of cells. Time-lapse imaging revealed that cysts arose from tubular structures. Importantly, isogenic control hPSCs plated and differentiated side-by-side did not form cysts. PKD mutations did not augment the size of cavitated spheroids of undifferentiated (epiblast-stage) hPSCs, a tissue not known to be affected in PKD patients.Our findings suggest that PKD-specific cyst formation from tubules can be reproducibly modeled in a minimal system in vitro. Cysts arise from both PKD1 and PKD2 mutants, but not in healthy controls. They are furthermore specific to the kidney lineage. These findings support the identification of hPSC-derived tubules as kidney nephron structures. Genome-modified hPSCs represent a new, pre-clinical model in which to investigate PKD pathophysiology and evaluate candidate therapeutics.

Acute kidney injury (AKI) is a global public health concern associated with high morbidity, mortality, and healthcare costs. Other than dialysis, no therapeutic interventions reliably improve survival, limit injury, or speed recovery. Despite recognized shortcomings of in vivo animal models, the underlying pathophysiology of AKI and its consequence, chronic kidney disease (CKD), is rich with biological targets. We review recent findings relating to the renal vasculature and cellular stress responses, primarily the intersection of the unfolded protein response, mitochondrial dysfunction, autophagy, and the innate immune response. Maladaptive repair mechanisms that persist following the acute phase promote inflammation and fibrosis in the chronic phase. Here macrophages, growth-arrested tubular epithelial cells, the endothelium, and surrounding pericytes are key players in the progression to chronic disease. Better understanding of these complex interacting pathophysiological mechanisms, their relative importance in humans, and the utility of biomarkers will lead to therapeutic strategies to prevent and treat AKI or impede progression to CKD or end-stage renal disease (ESRD).

Chronic kidney disease (CKD) affects millions of people in the United States, where recent studies place prevalence at greater than 13% in the adult population. In addition to a burden of excess cardiovascular risk, patients with CKD also exhibit an increased susceptibility to acute kidney injury and reduced capacity for subsequent repair after further renal insults. This chapter examines the differences between complete “adaptive” repair of the kidney and the “maladaptive” repair that is common in the context of CKD and aging. The features of CKD known or suspected to affect the ability of the damaged kidney to respond appropriately to further insult are discussed. These include changes in the control of the cell cycle, increases in numbers of senescent and G2/M arrested tubular cells, alterations in the behavior of renal pericytes, microvascular loss, and resultant chronic renal hypoxia.

Mutation of PKD1 , encoding the protein polycystin-1 (PC1), is the main cause of autosomal dominant polycystic kidney disease (ADPKD). The signaling pathways downstream of PC1 in ADPKD are still not fully understood. Here, we provide genetic evidence for the necessity of Gα12 (encoded by Gna12 , hereafter Gα12 ) for renal cystogenesis induced by Pkd1 knockout. There was no phenotype in mice with deletion of Gα12 ( Gα12 −/− ). Polyinosine-polycytosine (pI:pC)-induced deletion of Pkd1 ( Mx1Cre + Pkd1 f/f Gα12 +/+ ) in 1-week-old mice resulted in multiple kidney cysts by 9 weeks, but the mice with double knockout of Pkd1 and Gα12 ( Mx1Cre + Pkd1 f/f Gα12 −/− ) had no structural and functional abnormalities in the kidneys. These mice could survive more than one year without kidney abnormalities except multiple hepatic cysts in some mice, which indicates that the effect of Gα12 on cystogenesis is kidney specific. Furthermore, Pkd1 knockout promoted Gα12 activation, which subsequently decreased cell–matrix and cell–cell adhesion by affecting the function of focal adhesion and E-cadherin, respectively. Our results demonstrate that Gα12 is required for the development of kidney cysts induced by Pkd1 mutation in mouse ADPKD.

Care of patients with type 1 diabetes (T1D) has changed during the past 30 years. Tools to control hyperglycemia have improved and it was shown that improvement in glycemic control diminished the risk of late diabetic complications, including nephropathy. Moreover, in patients with impaired renal function, aggressive treatment of hypertension and renoprotective blockade of the renin-angiotensin system were shown to postpone end-stage renal disease (ESRD), albeit for a short while. Despite these achievements, the incidence of ESRD caused by T1D in the US population has not decreased but rather has increased over the past 20 years, although it now occurs at slightly older ages. This state of affairs is a call to action. This should begin with adopting a new model of diabetic nephropathy in human beings. In that model, instead of microalbuminuria or proteinuria, the focus should be on diagnosis and treatment of progressive renal function decline that leads to ESRD. Such a model has received significant support in clinical and epidemiologic studies. Investigation of mechanisms of such progressive renal function decline should help in the identification of new therapeutic targets and the development of new interventions. To evaluate these interventions, accurate diagnostic algorithms are needed so T1D patients will be stratified according to time to onset to ESRD. Consistent with concepts of personalized medicine, the new interventions should be tailored to and evaluated in patients predicted to have rapid, moderate, or even slow progression to ESRD.

The glomerulus has been at the center of attention as the primary site of injury in diabetic nephropathy (DN). Although there is no question that there are changes seen in the glomerulus, it is also well known that tubulointerstitial changes are a prominent component of the disease, especially in patients with type 2 diabetes. The level of albuminuria and DN disease progression best correlate with tubular degeneration and interstitial fibrosis. Nephrotoxicity studies in animals reveal that albuminuria is a highly sensitive marker of early tubular toxicity even in the absence of glomerular pathology. Urinary biomarker data in human beings support the view that proximal tubule injury contributes in a primary way, rather than in a secondary manner, to the development of early DN. I present a model in which very specific injury to the proximal tubule in vivo in the mouse results in severe inflammation, loss of blood vessels, interstitial fibrosis, and glomerulosclerosis. Increased glucose levels, free glycation adducts, reactive oxygen species, and oxidized lipids result in toxicity to tubule epithelia. This results in loss of cells with a stimulus to repair the epithelium. However, because of sublethal injury there is cell-cycle arrest in epithelial cells attempting to replace damaged cells. This leads to epithelial secretion of both profibrogenic growth factors, collagens, and factors that cause pericytes to proliferate and differentiate into myofibroblasts, leading to endothelial destabilization and capillary rarefaction. Local ischemia ensues with further injury to the tubules, more profibrogenic mediators, matrix protein deposition, fibrosis, and glomerulosclerosis.

Contrast-induced acute kidney injury (CI-AKI), or nephropathy (CIN), is frequently diagnosed in the setting of coronary angiography. The incidence varies in the literature partly related to the differences in diagnostic criteria used for CIN. In a recently published article in Circulation , Maioli et al1 used a definition of an increase of ≥0.5 mg/dL over baseline serum creatinine within 3 days of the administration of contrast medium and found an incidence of 12.1% among 1490 patients who had a baseline estimated creatinine clearance of 4-fold in patients who had mild AKI (≥0.3 mg/L or …

Chronic kidney disease (CKD) remains one of the leading causes of death in the developed world, and acute kidney injury (AKI) is now recognized as a major risk factor in its development. Understanding the factors leading to CKD after acute injury are limited by current animal models of AKI, which concurrently target various kidney cell types including epithelial, endothelial, and inflammatory cells. Here, we developed a mouse model of kidney injury using the Six2-Cre-LoxP technology to selectively activate expression of the simian diphtheria toxin (DT) receptor in renal epithelia derived from the metanephric mesenchyme. By adjusting the timing and dose of DT, a highly selective model of tubular injury was created to define the acute and chronic consequences of isolated epithelial injury. The DT-induced sublethal tubular epithelial injury was confined to the S1 and S2 segments of the proximal tubule rather than being widespread in the metanephric mesenchyme-derived epithelial lineage. Acute injury was promptly followed by inflammatory cell infiltration and robust tubular cell proliferation, leading to complete recovery after a single toxin insult. In striking contrast, three insults to renal epithelial cells at 1-week intervals resulted in maladaptive repair with interstitial capillary loss, fibrosis, and glomerulosclerosis, which was highly correlated with the degree of interstitial fibrosis. Thus, selective epithelial injury can drive the formation of interstitial fibrosis, capillary rarefaction, and potentially glomerulosclerosis, substantiating a direct role for damaged tubule epithelium in the pathogenesis of CKD.

Summary Background and objectives AKI leads to increased morbidity and mortality and progression to chronic kidney injury is a frequent consequence of AKI. Surgical treatment of mesothelioma is associated with increased risk for kidney injury. However, sustained kidney injury may limit therapeutic options for treating residual cancer. This study hypothesized that patients with significant serum creatinine (sCr) elevation within 48 hours of surgery would be at risk for sustained kidney injury. The goal was to determine the best acute sCr measure predictive of sustained kidney injury defined as a 50% increase in sCr from baseline measured 2–4 weeks after surgery. Design, setting, participants, & measurements In a prospective, observational cohort of surgical patients with mesothelioma, receiver operator characteristic curves were generated for the 24- and 48-hour absolute difference and relative sCr change over baseline in the derivation cohort (n=279). The prediction was tested in a validation cohort (n=207). The ability of various other AKI definitions to predict sustained kidney injury was evaluated. Results Sustained kidney injury occurred in 9.8% of patients in the derivation cohort. A $59% increase in sCr 48 hours after surgery was most predictive of sustained kidney injury (c statistic=0.78). Among other AKI definitions, a sCr increase of 0.3 mg/dl in 24 hours or 0.5 mg/dl increase in 48 hours (Waikar and Bonventre criteria) also reliably predicted sustained kidney injury. Conclusions Development of clinically significant sustained kidney injury can be predicted by acute postoperative sCr elevation in patients treated for mesothelioma. Clin J Am Soc Nephrol 7: 1071–1078, 2012. doi: 10.2215/CJN.12401211

Female mice lacking group IVA phospholipase A2 (Pla2g4a−/−) have a smaller litter size, which is due, in part, to defective implantation. We examined PLA2G4A dependence of the processes of ovulation and fertilization. Following induction of ovulation by equine chorionic gonadotropin (eCG)/human CG (hCG) treatment and mating, ovulation and fertilization rates were reduced significantly in Pla2g4a−/− mice as compared to wild-type littermates. Human CG triggered robust ovarian prostaglandin (PG) E2 production in the preovulatory period that was significantly attenuated in Pla2g4a−/− mice. Human CG transiently enhanced ovarian expression of PLA2G4A and prostaglandin endoperoxide synthase 2 (PTGS2) in wild-type mice. This PTGS2 induction was decreased in Pla2g4a−/− mice and also in immature rats treated with the PLA2G4A inhibitor, archidonyl trifluoromethyl ketone. A close spatiotemporal association of PLA2G4A with PTGS2 was found in mouse and rat preovulatory follicles examined by immunohistochemistry. Less association was observed with 4 other forms of PLA2. Our data strongly suggest that PLA2G4A amplifies hCG induction of PTGS2 and colocalizes with the induced PTGS2, thus contributing to robust PG production required for optimal ovulation and fertilization in rodents.—Kurusu, S., Sapirstein, A., Bonventre, J. V. Group IVA phospholipase A2 optimizes ovulation and fertilization in rodents through induction of and metabolic coupling with prostaglandin endoperoxide synthase 2.

Pre-renal acute kidney injury (AKI) is assumed to represent a physiological response to underperfusion. Its diagnosis is retrospective after a transient rise in plasma creatinine, usually associated with evidence of altered tubular transport, particularly that of sodium. In order to test whether pre-renal AKI is reversible because injury is less severe than that of sustained AKI, we measured urinary biomarkers of injury (cystatin C, neutrophil gelatinase-associated lipocalin (NGAL), γ-glutamyl transpeptidase, IL-18, and kidney injury molecule-1 (KIM-1)) at 0, 12, and 24 h following ICU admission. A total of 529 patients were stratified into groups having no AKI, AKI with recovery by 24 h, recovery by 48 h, or the composite of AKI greater than 48 h or dialysis. Pre-renal AKI was identified in 61 patients as acute injury with recovery within 48 h and a fractional sodium excretion

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.

Background: Chronic kidney disease (CKD) is a major public health problem, and despite continued research in the field, there is still a need to identify both biomarkers of risk and progression, as well as potential therapeutic targets. Structural equation modeling (SEM) is a family of statistical techniques that has been utilized in the fields of sociology and psychology for many years; however, its utilization in the biological sciences is relatively novel. SEM’s ability to investigate complex relationships in an efficient, single model could be utilized to understand the progression of CKD, as well as to develop a predictive model to assess kidney status in the patient. Methods: Fischer 344 rats were fed either an ad libitum diet or a calorically restricted diet, and a time-course study of kidney structure and function was performed. EQS, a SEM software package, was utilized to generate five CKD models of the Fisher 344 rat and identify relationships between measured variables and estimates of kidney damage and kidney function. Results: All models identified strong relationships between a biomarker for CKD, kidney injury molecule-1 (Kim-1) and kidney damage, in the Fischer 344 rat CKD model. Models also indicate a strong relationship between age and renal damage and dysfunction. Conclusion: SEM can be used to model CKD and could be useful to examine biomarkers in CKD patients.

Studies with sPLA(2) Group X, and cPLA(2) α gene-targeted mice suggest that absence of sPLA(2) Group X results in protection from ischemia/reperfusion (I/R) injury in the heart, and absence of cPLA(2) α Group IV is protective in the brain. Although latter studies might suggest a similar deleterious role for cPLA(2) α in I/R injury in the heart, the pathophysiology of stroke is intricately related to excitotoxicity and cannot necessarily be extrapolated to the heart. We report here that unlike findings in the brain, cPLA(2) α((-/-)) mice have exaggerated injury following I/R in vivo. In contrast, there is no difference in injury induced by simulated ischemia in cardiomyocytes isolated from cPLA(2) α((-/-)) versus cPLA(2) α((+/+)) mice. This suggests that cPLA(2) α does not have an important cardiomyocyte autonomous effect on ischemic injury. Prostaglandin E(2) (PGE(2) ) levels are significantly reduced in the hearts of the cPLA(2) α((-/-)) mice, and the enhanced injury is ameliorated by treatment with the PGE analog, misoprostol. We demonstrate that cPLA(2) α is cardioprotective in vivo, and this is likely via cPLA(2) α-mediated production of cardioprotective eicosanoids. These studies are the first to identify a protective role for cPLA(2) in I/R injury in any organ and raise concerns over long-term inhibition of cPLA(2).

Elevated urinary albumin excretion in patients with type 1 diabetes reverts to normoalbuminuria in a majority of patients but advances toward proteinuria in some. In order to gain valuable insights into the early pathophysiology of diabetic nephropathy we evaluated the association of kidney tubular injury biomarkers with changes in albuminuria in patients with type 1 diabetes mellitus. Urine levels of kidney injury molecule-1 (KIM-1), N-acetyl-β-D-glucosaminidase (NAG), and some inflammatory markers were determined in 38 healthy individuals and 659 patients with type 1 diabetes mellitus having varying degrees of albuminuria. Urinary interleukin-6, CXCL10/IP-10, NAG, and KIM-1 levels were very low in healthy individuals, increased in type 1 patients with normoalbuminuria, and were highest in diabetic patients that had microalbuminuria. Low baseline concentrations of urinary KIM-1 and NAG both individually and collectively were significantly associated with the regression of microalbuminuria over the subsequent 2 years; an effect independent of clinical characteristics. Progression and regression of microalbuminuria were unrelated to urinary levels of interleukins 6 and 8, CXCL10/IP-10, and monocyte chemoattractant protein-1. Thus our results show that lower urinary KIM-1 and NAG levels were associated with the regression of microalbuminuria in type 1 diabetes mellitus. Hence, tubular dysfunction is a critical component of the early course of diabetic nephropathy.

Inhibiting renal glucose transport is a potential pharmacologic approach to treat diabetes. The renal tubular sodium-glucose transporter 2 (SGLT2) reabsorbs approximately 90% of the filtered glucose load. An animal model with sglt2 dysfunction could provide information regarding the potential long-term safety and efficacy of SGLT2 inhibitors, which are currently under clinical investigation. Here, we describe Sweet Pee, a mouse model that carries a nonsense mutation in the Slc5a2 gene, which results in the loss of sglt2 protein function. The phenotype of Sweet Pee mutants was remarkably similar to patients with mutations in the Scl5a2 gene. The Sweet Pee mutants had improved glucose tolerance, higher urinary excretion of calcium and magnesium, and growth retardation. Renal physiologic studies demonstrated a prominent distal osmotic diuresis without enhanced natriuresis. Sweet Pee mutants did not exhibit increased KIM-1 or NGAL, markers of acute tubular injury. After induction of diabetes, Sweet Pee mice had better overall glycemic control than wild-type control mice, but had a higher risk for infection and an increased mortality rate (70% in homozygous mutants versus 10% in controls at 20 weeks). In summary, the Sweet Pee model allows study of the long-term benefits and risks associated with inhibition of SGLT2 for the management of diabetes. Our model suggests that inhibiting SGLT2 may improve glucose control but may confer increased risks for infection, malnutrition, volume contraction, and mortality.

Purpose of review To review the new findings about the physiological roles of kidney injury molecule-1 (KIM-1) and the rapidly expanding evidence for this molecule as a promising biomarker in preclinical kidney toxicity evaluation and various human kidney diseases. Recent findings KIM-1 has attracted increasing interest because of its possible pathophysiological role in modulating tubular damage and repair. There is rapidly accumulating evidence from both animal models and clinical studies that urinary KIM-1 is a sensitive and specific urinary biomarker for various forms of nephrotoxic injury, cardiac surgery-induced kidney injury, transplant rejection, and chronic kidney diseases. Summary KIM-1 mediates epithelial phagocytosis in the injured kidney converting the proximal epithelial cell into a phagocyte, with potentially important pathophysiological implications for modulation of the immune response and repair process after injury. KIM-1 serves as a highly sensitive and specific urinary biomarker for kidney injury and may also be a therapeutic target for various kidney diseases.

Resident tissue macrophages are activated by the fungal pathogen Candida albicans to release eicosanoids, which are important modulators of inflammation and immune responses. Our objective was to identify the macrophage receptors engaged by C. albicans that mediate activation of group IVA cytosolic phospholipase A(2) (cPLA(2)α), a regulatory enzyme that releases arachidonic acid (AA) for production of prostaglandins and leukotrienes. A comparison of peritoneal macrophages from wild type and knock-out mice demonstrates that the β-glucan receptor Dectin-1 and MyD88 regulate early release of AA and eicosanoids in response to C. albicans. However, cyclooxygenase 2 (COX2) expression and later phase eicosanoid production are defective in MyD88(-/-) but not Dectin-1(-/-) macrophages. Furthermore, C. albicans-stimulated activation of MAPK and phosphorylation of cPLA(2)α on Ser-505 are regulated by MyD88 and not Dectin-1. In contrast, Dectin-1 mediates MAPK activation, cPLA(2)α phosphorylation, and COX2 expression in response to particulate β-glucan suggesting that other receptors engaged by C. albicans preferentially mediate these responses. Results also implicate the mannan-binding receptor Dectin-2 in regulating cPLA(2)α. C. albicans-stimulated MAPK activation and AA release are blocked by d-mannose and Dectin-2-specific antibody, and overexpression of Dectin-2 in RAW264.7 macrophages enhances C. albicans-stimulated MAPK activation, AA release, and COX2 expression. In addition, calcium mobilization is enhanced in RAW264.7 macrophages overexpressing Dectin-1 or -2. The results demonstrate that C. albicans engages both β-glucan and mannan-binding receptors on macrophages that act with MyD88 to regulate the activation of cPLA(2)α and eicosanoid production.

1. Calcium is an important cation for many of the physiological and pathophysiological functions of the mesangial cell. Calcium binds to many proteins in the cell. some of these proteins seemingly serve, primarily, as intracellular buffers for calcium, whose intracellular concentration must be strictly controlled. In addition to these buffering proteins, a large number of proteins are regulated by calcium. These proteins are important for mesangial cell structural integrity, maintenance of internal ionic composition, contractility, effector responses to a variety of hormones and growth factors, and production of inflammatory mediators. 2. Calcium homeostasis in mesangial cells is complex. Cells respond to vasoactive peptides and growth factors with increases in intracellular free calcium ([Ca2+]i) due to Ca2+ release from intracellular storage sites and entry across the cell plasma membrane. 3. Individual mesangial cells respond to arginine vasopressin with repetitive [Ca2+]i spikes. Increasing the concentration of vasopressin up to 10 nmol/L increases the frequency of the repetitive Ca2+ spikes. The amplitude of the oscillations does not vary with the concentration of vasopressin used. 4. The presence of Ca2+ oscillations whose frequency varies with ligand concentration suggests that the Ca2+ signal may encode its information through a frequency-dependent mode in addition to, or rather than, an amplitude-dependent mode. Calcium can change the conformation of proteins and can change catalytic activity of enzymes directly. 5. Another important mode of regulation by calcium is demonstrated by cytosolic phospholipase A2, where calcium is important not for catalytic activity directly but for the apposition of enzyme with substrate so that the catalytic activity may be manifest.

There is a paucity of biomarkers that reliably detect nephrotoxicity. The Predictive Safety Testing Consortium (PSTC) faced several challenges in identifying novel safety biomarkers in the renal setting.