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1.  C-kit+ cells isolated from developing kidneys are a novel population of stem cells with regenerative potential 
Stem cells (Dayton, Ohio)  2013;31(8):1644-1656.
The presence of tissue specific precursor cells is an emerging concept in organ formation and tissue homeostasis. Several progenitors are described in the kidneys. However, their identity as a true stem cell remains elusive. Here, we identify a neonatal kidney-derived c-kit+ cell population that fulfills all of the criteria as a stem cell. These cells were found in the thick ascending limb of Henle's loop and exhibited clonogenicity, self-renewal, and multipotentiality with differentiation capacity into mesoderm and ectoderm progeny. Additionally, c-kit+ cells formed spheres in nonadherent conditions when plated at clonal density and expressed markers of stem cells, progenitors, and differentiated cells. Ex-vivo expanded c-kit+ cells integrated into several compartments of the kidney, including tubules, vessels, and glomeruli, and contributed to functional and morphological improvement of the kidney following acute ischemia-reperfusion injury in rats. Together these findings document a novel neonatal rat kidney c-kit+ stem cell population that can be isolated, expanded, cloned, differentiated, and employed for kidney repair following acute kidney injury. These cells have important biological and therapeutic implications.
PMCID: PMC3804254  PMID: 23733311
C-kit; kidney stem cells; clonogenicity; self-renewal; multipotentiality; regenerative potential
2.  Circulating urokinase receptor as a cause of focal segmental glomerulosclerosis 
Nature medicine  2011;17(8):952-960.
Focal segmental glomerulosclerosis (FSGS) is a cause of proteinuric kidney disease, compromising both native and transplanted kidneys. Treatment is limited because of a complex pathogenesis, including unknown serum factors. Here we report that serum soluble urokinase receptor (suPAR) is elevated in two-thirds of subjects with primary FSGS, but not in people with other glomerular diseases. We further find that a higher concentration of suPAR before transplantation underlies an increased risk for recurrence of FSGS after transplantation. Using three mouse models, we explore the effects of suPAR on kidney function and morphology. We show that circulating suPAR activates podocyte β3 integrin in both native and grafted kidneys, causing foot process effacement, proteinuria and FSGS-like glomerulopathy. Our findings suggest that the renal disease only develops when suPAR sufficiently activates podocyte β3 integrin. Thus, the disease can be abrogated by lowering serum suPAR concentrations through plasmapheresis, or by interfering with the suPAR–β3 integrin interaction through antibodies and small molecules targeting either uPAR or β3 integrin. Our study identifies serum suPAR as a circulating factor that may cause FSGS.
PMCID: PMC4089394  PMID: 21804539
3.  Type 1 angiotensin receptors on macrophages ameliorate IL-1 receptor–mediated kidney fibrosis 
The Journal of Clinical Investigation  2014;124(5):2198-2203.
In a wide array of kidney diseases, type 1 angiotensin (AT1) receptors are present on the immune cells that infiltrate the renal interstitium. Here, we examined the actions of AT1 receptors on macrophages in progressive renal fibrosis and found that macrophage-specific AT1 receptor deficiency exacerbates kidney fibrosis induced by unilateral ureteral obstruction (UUO). Macrophages isolated from obstructed kidneys of mice lacking AT1 receptors solely on macrophages had heightened expression of proinflammatory M1 cytokines, including IL-1. Evaluation of isolated AT1 receptor–deficient macrophages confirmed the propensity of these cells to produce exaggerated levels of M1 cytokines, which led to more severe renal epithelial cell damage via IL-1 receptor activation in coculture compared with WT macrophages. A murine kidney crosstransplantation concomitant with UUO model revealed that augmentation of renal fibrosis instigated by AT1 receptor–deficient macrophages is mediated by IL-1 receptor stimulation in the kidney. This study indicates that a key role of AT1 receptors on macrophages is to protect the kidney from fibrosis by limiting activation of IL-1 receptors in the kidney.
PMCID: PMC4001526  PMID: 24743144
Transplantation proceedings  2009;41(1):238-239.
Laser scanning cytometry (iCys; CompuCyte) is recently developed methodology that utilizes fluorescence-based quantitative measurements on tissue sections or other cellular preparations at a single-cell level. The purpose of this study was to develop objective quantitative immunoprofiling of regulatory T cells (T regs) on formalin-fixed/paraffin embedded (FFPE) biopsy sample from transplanted allograft using iCys. We analyzed the population of CD4 (+) Foxp3 (+) T regs among the entire CD4 (+) T cells and the entire T cells (the total of CD4 (+) and CD8 (+)) in human intestinal allograft biopsy samples to evaluate the usefulness of iCys. Primary antibodies (Foxp3 and CD4) were incubated on one section. Foxp3 and CD4 were labeled by Alexa Fluoro 488 (Alexa488) and 647 (Alexa647) using polymer HRP and catalyzed signal amplification. On the other section, CD8 and CD4 were labeled by Alexa488 and Alexa647 using the same protocol. Data acquisition was performed using iCys. The signal intensity of Alexa488 and Alexa647 was sufficient to analyze by iCys. Distribution of the integrals of Alexa488 and Alexa647 visualize each cell population and enable to calculate the population of T reg among CD4 (+) T cell, CD4 (+) T cell among the total T cells and T reg among the entire T cells. iCys and signal amplified immunofluorescent staining allowed objective quantitative immunoprofiling of in situ T reg populations, with precise quantitative analysis at a single-cell level on FFPE section. This objective method can be applied on the biopsy sample from various transplant organs.
PMCID: PMC3777540  PMID: 19249524
5.  Quantitative In Situ Analysis of FoxP3+ T Regulatory Cells on Transplant Tissue Using Laser Scanning Cytometry 
Cell transplantation  2011;21(1):113-125.
There is abundant evidence that immune cells infiltrating into a transplanted organ play a critical role for destructive inflammatory or regulatory immune reactions. Quantitative in situ analysis (i.e. in tissue sections) of immune cells remains challenging due to a lack of objective methodology. Laser scanning cytometry (LSC/iCys) is a recently developed methodology that utilizes fluorescence-based quantitative measurements on tissue sections or other cellular preparations at a single-cell level. In this study, we have developed a novel objective method for analysis of immune cells, including Foxp3+ T regulatory cell (T reg), on formalin-fixed / paraffin embedded (FFPE) transplant biopsy sections using LSC/iCys.
The development of multiple immunofluorescent staining was established using FFPE human tonsil sample. The CD4/CD8 ratio and the population of T reg among CD4+ cells were analyzed using LSC/iCys and compared with the results from conventional flow cytometry analysis (FCM).
Our multiple immunofluorescent staining techniques allow obtaining clear staining on FFPE sections. The CD4/CD8 ratio analyzed by LSC/iCys was concordant with those obtained by FCM. This method was also applicable for liver, small intestine, kidney, pancreas and heart transplant biopsy sections and provide an objective quantification of T regs within the grafts.
PMCID: PMC3777543  PMID: 21929847
T regulatory cell; Foxp3; laser scanning cytometry; transplant biopsy; quantification
6.  B Lymphocytes Differentially Influence Acute and Chronic Allograft Rejection in Mice1 
The relative contributions of B lymphocytes and plasma cells during allograft rejection remain unclear. Therefore, the effects of B cell depletion on acute cardiac rejection, chronic renal rejection, and skin graft rejection were compared using CD20 or CD19 mAbs. Both CD20 and CD19 mAbs effectively depleted mature B cells, while CD19 mAb treatment depleted plasmablasts and some plasma cells. B cell depletion did not affect acute cardiac allograft rejection, although CD19 mAb treatment prevented allograft-specific IgG production. Strikingly, CD19 mAb treatment significantly reduced renal allograft rejection and abrogated allograft-specific IgG development, while CD20 mAb treatment did not. By contrast, B cell depletion exacerbated skin allograft rejection and augmented the proliferation of adoptively transferred alloantigen-specific CD4+ T cells, demonstrating that B cells can also negatively regulate allograft rejection. Thereby, B cells can either positively or negatively regulate allograft rejection depending on the nature of the allograft and the intensity of the rejection response. Moreover, CD19 mAb may represent a new approach for depleting both B cells and plasma cells to concomitantly impair T cell activation, inhibit the generation of new allograft-specific Abs, or reduce preexisting allograft-specific Ab levels in transplant patients.
PMCID: PMC3734565  PMID: 21248259
7.  A novel role for type 1 angiotensin receptors on T lymphocytes to limit target organ damage in hypertension 
Circulation Research  2012;110(12):1604-1617.
Human clinical trials using type 1 angiotensin (AT1) receptor antagonists indicate that angiotensin II is a critical mediator of cardiovascular and renal disease. However, recent studies have suggested that individual tissue pools of AT1 receptors may have divergent effects on target organ damage in hypertension.
We examined the role of AT1 receptors on T lymphocytes in the pathogenesis of hypertension and its complications.
Methods and Results
Deficiency of AT1 receptors on T cells potentiated kidney injury during hypertension with exaggerated renal expression of chemokines and enhanced accumulation of T cells in the kidney. Kidneys and purified CD4+ T cells from “T cell knockout” mice lacking AT1 receptors on T lymphocytes had augmented expression of Th1-associated cytokines including IFN-γ and TNF-α. Within T lymphocytes, the transcription factors T-bet and GATA-3 promote differentiation toward the Th1 and Th2 lineages, respectively, and AT1 receptor-deficient CD4+ T cells had enhanced T-bet / GATA-3 expression ratios favoring induction of the Th1 response. Inversely, mice that were unable to mount a Th1 response due to T-bet deficiency were protected from kidney injury in our hypertension model.
The current studies identify an unexpected role for AT1 receptors on T lymphocytes to protect the kidney in the setting of hypertension by favorably modulating CD4+ T helper cell differentiation.
PMCID: PMC3393036  PMID: 22534490
Hypertension; Kidney disease; T lymphocytes; Inflammation
8.  A Role for Angiotensin II Type 1 Receptors on Bone Marrow-Derived Cells in the Pathogenesis of AngiotensinII–Dependent Hypertension 
Hypertension  2009;55(1):99-108.
Activation of type 1 angiotensin (AT1) receptors causes hypertension, leading to progressive kidney injury. AT1 receptors are expressed on immune cells, and previous studies have identified a role for immune cells in angiotensin II–dependent hypertension. We, therefore, examined the role of AT1 receptors on immune cells in the pathogenesis of hypertension by generating bone marrow chimeras with wild-type donors or donors lacking AT1A receptors (BMKO). The 2 groups had virtually identical blood pressures at baseline, suggesting that AT1 receptors on immune cells do not make a unique contribution to the determination of baseline blood pressure. By contrast, in response to chronic angiotensin II infusion, the BMKOs had an augmented hypertensive response, suggesting a protective effect of AT1 receptors on immune cells with respect to blood pressure elevation. The BMKOs had 50% more albuminuria after 4 weeks of angiotensin II–dependent hypertension. Angiotensin II–induced pathological injury to the kidney was similar in the experimental groups. However, there was exaggerated renal expression of the macrophage chemokine monocyte chemoattractant protein 1 in the BMKO group, leading to persistent accumulation of macrophages in the kidney. This enhanced mononuclear cell infiltration into the BMKO kidneys was associated with exaggerated renal expression of the vasoactive mediators interleukin-1β and interleukin-6. Thus, in angiotensin II-induced hypertension, bone marrow-derived AT1 receptors limited mononuclear cell accumulation in the kidney and mitigated the chronic hypertensive response, possibly through the regulation of vasoactive cytokines.
PMCID: PMC3676183  PMID: 19996062
angiotensin II; hypertension; inflammation; kidney diseases; lymphocytes
9.  Endothelial Nitric Oxide Synthase Reduces Crescentic and Necrotic Glomerular Lesions, Reactive Oxygen Production, and MCP1 Production in Murine Lupus Nephritis 
PLoS ONE  2013;8(5):e64650.
Systemic lupus erythematosus, in both animal models and in humans, is characterized by autoantibody production followed by immune complex deposition in target tissues. Ensuing target organ damage is modulated by reactive intermediates, including reactive nitrogen and oxygen species, through as of now incompletely understood mechanisms. Endothelial nitric oxide synthase is known to impact vascular reactivity; however its impact on reactive intermediate production and inflammatory renal disease is less well defined. In this study, we assessed the impact of endothelial nitric oxide synthase (eNOS) on disease in lupus prone MRL/lpr mice. Mice lacking eNOS developed earlier more severe disease with decreased survival. eNOS deficient mice died sooner and developed significantly more glomerular crescents, necrosis, inflammatory infiltrates and vasculitis, indicating a role for eNOS in modulating these renal lesions. Immune complex deposition was similar between groups, indicating the impact of eNOS is distal to antibody/complement glomerular deposition. Urinary nitric oxide production was decreased in the eNOS deficient mice, while proteinuria was increased. Urinary monocyte chemotactic protein-1 was also increased in the knockout mice. CD4+ T cells from MRL/lpr mice demonstrated mitochondrial hyperpolarization, increased nitric oxide and superoxide production and increased calcium flux compared to B6 control mice. Deficiency of eNOS resulted in decreased nitric oxide and mitochondrial calcium levels but had no effect on mitochondrial hyperpolarization. Renal cortices from MRL/lpr mice that are eNOS deficient demonstrated increased superoxide production, which was blocked by both nitric oxide synthase and NADPH oxidase inhibitors. These studies thus demonstrate a key role for eNOS in modulating renal disease in lupus prone MRL/lpr mice. The impact appears to be mediated by effects on superoxide production in the kidney, impacting downstream mediators such as monocyte chemotactic protein-1. These results suggest that modulation of eNOS may be a novel therapeutic approach to treating lupus nephritis.
PMCID: PMC3669382  PMID: 23741359
10.  Inhibition of Sphingosine Kinase-2 in a Murine Model of Lupus Nephritis 
PLoS ONE  2013;8(1):e53521.
Sphingosine-1-phosphate (S1P), a potent bioactive lipid, is emerging as a central mediator in inflammation and immune responses. We have previously implicated S1P and its synthetic enzyme sphingosine kinase (SK) in inflammatory and autoimmune disorders, including inflammatory bowel disease and rheumatoid arthritis. Generation of S1P requires phosphorylation of sphingosine by SK, of which there are two isoforms. Numerous studies have implicated SK1 in immune cell trafficking, inflammation and autoimmune disorders. In this study, we set out to determine the role of SK and S1P in lupus nephritis (LN). To this end, we examined S1P and dihydro-S1P (dh-S1P) levels in serum and kidney tissues from a mouse model of LN. Interestingly dh-S1P was significantly elevated in serum and kidney tissue from LN mice, which is more readily phosphorylated by SK2. Therefore, we employed the use of the specific SK2 inhibitor, ABC294640 in our murine model of LN. Treatment with ABC294640 did not improve vascular or interstitial pathology associated with LN. However, mice treated with the SK2 inhibitor did demonstrate decreases in glomerular pathology and accumulation of B and T cells in the spleen these were not statistically different from lpr mice treated with vehicle. LN mice treated with ABC294640 did not have improved urine thromboxane levels or urine proteinuria measurements. Both S1P and dh-S1P levels in circulation were significantly reduced with ABC294640 treatment; however, dh-S1P was actually elevated in kidneys from LN mice treated with ABC294640. Together these data demonstrate a role for SKs in LN; however, they suggest that inhibition of SK1 or perhaps both SK isoforms would better prevent elevations in S1P and dh-S1P and potentially better protect against LN.
PMCID: PMC3536755  PMID: 23301082
11.  Mechanisms of the proteinuria induced by Rho GTPases 
Kidney International  2012;81(11):1075-1085.
Podocytes are highly differentiated cells that play an important role in maintaining glomerular filtration barrier integrity; a function regulated by small GTPase proteins of the Rho family. To investigate the role of Rho A in podocyte biology, we created transgenic mice expressing doxycycline-inducible constitutively active (V14Rho) or dominant-negative Rho A (N19Rho) in podocytes. Specific induction of either Rho A construct in podocytes caused albuminuria and foot process effacement along with disruption of the actin cytoskeleton as evidenced by decreased expression of the actin associated protein synaptopodin. The mechanisms of these adverse effects, however, appeared to be different. Active V14Rho enhanced actin polymerization, caused a reduction in nephrin mRNA and protein levels, promoted podocyte apoptosis, and decreased endogenous Rho A levels. In contrast, the dominant-negative N19Rho caused a loss of podocyte stress fibers, did not alter the expression of either nephrin or Rho A, and did not cause podocyte apoptosis. Thus, our findings suggest that Rho A plays an important role in maintaining the integrity of the glomerular filtration barrier under basal conditions, but enhancement of Rho A activity above basal levels promotes podocyte injury.
PMCID: PMC3352980  PMID: 22278020
12.  Diabetic Kidney Disease in FVB/NJ Akita Mice: Temporal Pattern of Kidney Injury and Urinary Nephrin Excretion 
PLoS ONE  2012;7(4):e33942.
Akita mice are a genetic model of type 1 diabetes. In the present studies, we investigated the phenotype of Akita mice on the FVB/NJ background and examined urinary nephrin excretion as a marker of kidney injury. Male Akita mice were compared with non-diabetic controls for functional and structural characteristics of renal and cardiac disease. Podocyte number and apoptosis as well as urinary nephrin excretion were determined in both groups. Male FVB/NJ Akita mice developed sustained hyperglycemia and albuminuria by 4 and 8 weeks of age, respectively. These abnormalities were accompanied by a significant increase in systolic blood pressure in 10-week old Akita mice, which was associated with functional, structural and molecular characteristics of cardiac hypertrophy. By 20 weeks of age, Akita mice developed a 10-fold increase in albuminuria, renal and glomerular hypertrophy and a decrease in the number of podocytes. Mild-to-moderate glomerular mesangial expansion was observed in Akita mice at 30 weeks of age. In 4-week old Akita mice, the onset of hyperglycemia was accompanied by increased podocyte apoptosis and enhanced excretion of nephrin in urine before the development of albuminuria. Urinary nephrin excretion was also significantly increased in albuminuric Akita mice at 16 and 20 weeks of age and correlated with the albumin excretion rate. These data suggest that: 1. FVB/NJ Akita mice have phenotypic characteristics that may be useful for studying the mechanisms of kidney and cardiac injury in diabetes, and 2. Enhanced urinary nephrin excretion is associated with kidney injury in FVB/NJ Akita mice and is detectable early in the disease process.
PMCID: PMC3319540  PMID: 22496773
13.  The benefit of targeted and selective inhibition of the alternative complement pathway for modulating autoimmunity and renal disease in MRL/lpr mice1 
Arthritis and rheumatism  2011;63(4):1076-1085.
Complement has both protective and pathogenic functions in lupus due to a balance between its role in the clearance of immune complexes (IC’s) and apoptotic cells versus inflammation. The classical pathway contributes to IC and apoptotic cell clearance, whereas the alternative pathway is a key mediator of renal inflammation. We investigated the effect of a new targeted inhibitor of the alternative pathway, CR2-fH, on lupus-like renal disease in MRL/lpr mice.
Mice were treated with either saline, CR2-fH, CR2-Crry (inhibits all complement pathways) or sCR2 (C3d-binding targeting vehicle). Sera were analyzed every 2 weeks for autoantibodies, circulating ICs and C3. Urinary albumin was also determined, and kidneys collected for histological evaluation at 23 weeks.
CR2-fH and CR2-Crry treatment improved survival and significantly reduced proteinuria, glomerular C3 deposition and circulating IC’s. CR2-fH, but not CR2-Crry, also significantly reduced glomerulonephritis, serum anti-dsDNA antibodiesa, and glomerular IgG and C1q deposition. Interestingly, sCR2 also significantly reduced levels of anti-dsDNA antibodies, circulating IC’s and glomerular deposition of IgG, C1q and C3, although there was no significant reduction in glomerulonephritis, proteinuria or mortality.
Targeted and selective inhibition of the alternative complement pathway is an effective treatment for murine lupus, and is more effective than blockade of all pathways. The data demonstrate benefits to leaving the classical/lectin pathways intact, and indicate distinct roles for the classical and alternative pathways of complement in progression of the disease. The sCR2 targeting vehicle contributes to therapeutic activity, possibly via modulation of autoimmunity.
PMCID: PMC3079315  PMID: 21452327
Complement; lupus; factor H; kidney; autoimmunity
15.  Biased Use of the IGHV4 Family and Evidence for Antigen Selection in Chlamydophila psittaci-Negative Ocular Adnexal Extranodal Marginal Zone Lymphomas 
PLoS ONE  2011;6(12):e29114.
Extranodal marginal zone lymphomas (EMZL) are the most common lymphomas in the ocular adnexa. The etiology and potential role for antigenic stimulation in these lymphomas are still controversial. We have examined IGHV gene usage and mutations in 67 Chlamydophila psittaci-negative ocular adnexal EMZL. Clonal IGHV gene sequences were identified in 43 tumors originating from the orbit (19), conjunctivae (18) and lacrimal gland (6). Forty four potentially functional clonal IGHV gene sequences were detected with overrepresentation of the IGHV4 family and IGHV4-34 gene. All but 3 sequences were mutated with the average percent homology to the germ line of 93.5±6.1. Multinomial model and Focused binomial test demonstrated evidence for positive and/or negative antigen selection in 59% of the potentially functional IGHV genes. Intraclonal variation was detected in 8 of 11 tumor specimens. Overall our findings demonstrate that C. psittaci-negative ocular adnexal EMZL exhibit biased usage of IGHV families and genes with evidence for intraclonal heterogeneity and antigen selection in multiple tumors, implicating B-cell receptor-mediated antigen stimulation in the pathogenesis of these lymphomas.
PMCID: PMC3246466  PMID: 22216179
16.  CD2AP in mouse and human podocytes controls a proteolytic program that regulates cytoskeletal structure and cellular survival 
The Journal of Clinical Investigation  2011;121(10):3965-3980.
Kidney podocytes are highly differentiated epithelial cells that form interdigitating foot processes with bridging slit diaphragms (SDs) that regulate renal ultrafiltration. Podocyte injury results in proteinuric kidney disease, and genetic deletion of SD-associated CD2-associated protein (CD2AP) leads to progressive renal failure in mice and humans. Here, we have shown that CD2AP regulates the TGF-β1–dependent translocation of dendrin from the SD to the nucleus. Nuclear dendrin acted as a transcription factor to promote expression of cytosolic cathepsin L (CatL). CatL proteolyzed the regulatory GTPase dynamin and the actin-associated adapter synaptopodin, leading to a reorganization of the podocyte microfilament system and consequent proteinuria. CD2AP itself was proteolyzed by CatL, promoting sustained expression of the protease during podocyte injury, and in turn increasing the apoptotic susceptibility of podocytes to TGF-β1. Our study identifies CD2AP as the gatekeeper of the podocyte TGF-β response through its regulation of CatL expression and defines a molecular mechanism underlying proteinuric kidney disease.
PMCID: PMC3195478  PMID: 21911934
18.  Recurrence of Type 1 Diabetes After Simultaneous Pancreas-Kidney Transplantation, Despite Immunosuppression, Is Associated With Autoantibodies and Pathogenic Autoreactive CD4 T-Cells 
Diabetes  2010;59(4):947-957.
To investigate if recurrent autoimmunity explained hyperglycemia and C-peptide loss in three immunosuppressed simultaneous pancreas-kidney (SPK) transplant recipients.
We monitored autoantibodies and autoreactive T-cells (using tetramers) and performed biopsy. The function of autoreactive T-cells was studied with in vitro and in vivo assays.
Autoantibodies were present pretransplant and persisted on follow-up in one patient. They appeared years after transplantation but before the development of hyperglycemia in the remaining patients. Pancreas transplant biopsies were taken within ∼1 year from hyperglycemia recurrence and revealed β-cell loss and insulitis. We studied autoreactive T-cells from the time of biopsy and repeatedly demonstrated their presence on further follow-up, together with autoantibodies. Treatment with T-cell–directed therapies (thymoglobulin and daclizumab, all patients), alone or with the addition of B-cell–directed therapy (rituximab, two patients), nonspecifically depleted T-cells and was associated with C-peptide secretion for >1 year. Autoreactive T-cells with the same autoantigen specificity and conserved T-cell receptor later reappeared with further C-peptide loss over the next 2 years. Purified autoreactive CD4 T-cells from two patients were cotransplanted with HLA-mismatched human islets into immunodeficient mice. Grafts showed β-cell loss in mice receiving autoreactive T-cells but not control T-cells.
We demonstrate the cardinal features of recurrent autoimmunity in three such patients, including the reappearance of CD4 T-cells capable of mediating β-cell destruction. Markers of autoimmunity can help diagnose this underappreciated cause of graft loss. Immune monitoring during therapy showed that autoimmunity was not resolved by the immunosuppressive agents used.
PMCID: PMC2844842  PMID: 20086230
19.  Podocyte-Specific Overexpression of Wild Type or Mutant Trpc6 in Mice Is Sufficient to Cause Glomerular Disease 
PLoS ONE  2010;5(9):e12859.
Mutations in the TRPC6 calcium channel (Transient receptor potential channel 6) gene have been associated with familiar forms of Focal and Segmental Glomerulosclerosis (FSGS) affecting children and adults. In addition, acquired glomerular diseases are associated with increased expression levels of TRPC6. However, the exact role of TRPC6 in the pathogenesis of FSGS remains to be elucidated. In this work we describe the generation and phenotypic characterization of three different transgenic mouse lines with podocyte-specific overexpression of the wild type or any of two mutant forms of Trpc6 (P111Q and E896K) previously related to FSGS. Consistent with the human phenotype a non-nephrotic range of albuminuria was detectable in almost all transgenic lines. The histological analysis demonstrated that the transgenic mice developed a kidney disease similar to human FSGS. Differences of 2–3 folds in the presence of glomerular lesions were found between the non transgenic and transgenic mice expressing Trpc6 in its wild type or mutant forms specifically in podocytes. Electron microscopy of glomerulus from transgenic mice showed extensive podocyte foot process effacement. We conclude that overexpression of Trpc6 (wild type or mutated) in podocytes is sufficient to cause a kidney disease consistent with FSGS. Our results contribute to reinforce the central role of podocytes in the etiology of FSGS. These mice constitute an important new model in which to study future therapies and outcomes of this complex disease.
PMCID: PMC2942896  PMID: 20877463
20.  25-Gauge transconjunctival sutureless vitrectomy for the diagnosis of intraocular lymphoma 
Diagnostic pars plana vitrectomy is a useful technique in the diagnosis of intraocular lymphoma (IOL); however, the role of transconjunctival sutureless vitrectomy (TSV) has not been fully explored for this indication. The purpose of this study was to review our experience with 25-gauge TSV for the diagnosis of IOL.
Patients who underwent 25-gauge TSV for the diagnosis of IOL (primary, secondary or recurrent) from two tertiary referral centres were reviewed. Demographic data and underlying medical conditions were reviewed. Preoperative and postoperative visual acuities (VA) and ophthalmic examination data were assessed. Cytopathology, flow cytometry, cytokine and gene rearrangement studies were assessed.
Twelve patients underwent 25-gauge diagnostic TSV with a median follow-up time of 37 weeks. B-cell or T-cell IOL was diagnosed based on cytology in 3/12 patients (25%, 95% CI 8.9 to 53.2%) and in eight patients (67%, 95% CI 39.1 to 86.1%) using adjunctive diagnostic testing. VA stabilised or improved in 11 eyes (92%). Mean VA improved from 20/95 to 20/66 (p=0.055, paired t test).
25-Gauge TSV is safe and effective for obtaining vitreous specimens for the evaluation of IOL. The availability of expert ophthalmic pathological consultation, flow cytometry, cytokine evaluation and gene rearrangement studies were essential to the diagnosis.
PMCID: PMC2928256  PMID: 20447965
21.  Glomerular type 1 angiotensin receptors augment kidney injury and inflammation in murine autoimmune nephritis 
Studies in humans and animal models indicate a key contribution of angiotensin II to the pathogenesis of glomerular diseases. To examine the role of type 1 angiotensin (AT1) receptors in glomerular inflammation associated with autoimmune disease, we generated MRL-Faslpr/lpr (lpr) mice lacking the major murine type 1 angiotensin receptor (AT1A); lpr mice develop a generalized autoimmune disease with glomerulonephritis that resembles SLE. Surprisingly, AT1A deficiency was not protective against disease but instead substantially accelerated mortality, proteinuria, and kidney pathology. Increased disease severity was not a direct effect of immune cells, since transplantation of AT1A-deficient bone marrow did not affect survival. Moreover, autoimmune injury in extrarenal tissues, including skin, heart, and joints, was unaffected by AT1A deficiency. In murine systems, there is a second type 1 angiotensin receptor isoform, AT1B, and its expression is especially prominent in the renal glomerulus within podocytes. Further, expression of renin was enhanced in kidneys of AT1A-deficient lpr mice, and they showed evidence of exaggerated AT1B receptor activation, including substantially increased podocyte injury and expression of inflammatory mediators. Administration of losartan, which blocks all type 1 angiotensin receptors, reduced markers of kidney disease, including proteinuria, glomerular pathology, and cytokine mRNA expression. Since AT1A-deficient lpr mice had low blood pressure, these findings suggest that activation of type 1 angiotensin receptors in the glomerulus is sufficient to accelerate renal injury and inflammation in the absence of hypertension.
PMCID: PMC2662542  PMID: 19287096
22.  Nephrotic syndrome and kidney failure due to immunocomplex-mediated renal damage in a patient with Waldenström's Macroglobulinemia: a case report 
Cases Journal  2008;1:333.
Unlike the quite frequent renal involvement seen in cases of Multiple Myeloma, the kidney is hardly ever compromised in patients with Waldenström's Macroglobulinemia. Nephrotic range proteinuria is a very unusual manifestation of renal injury in these patients and when present it is due to amyloid light-chain deposition most of the times.
Case presentation
A 60-year-old male patient presented to the hospital with nephrotic syndrome, renal insufficiency, hypertension and lymphadenopathy. The investigations led to the diagnosis of Waldenström's Macroglobulinemia with associated nephrotic syndrome and chronic kidney disease due to an unusual form of hypocomplementemic glomerulopathy with histopathological features similar to those seen in mesangiocapillary glomerulonephritis type III, but lacking proliferative changes.
We present an unusual case of immunologically-mediated renal damage in a patient with Waldenström's Macroglobulinemia, leading to non-amyloid nephrotic syndrome and chronic renal insufficiency.
PMCID: PMC2600791  PMID: 19019245
23.  Cytofluorographic and molecular identification of a CD8-positive, TCR-α/β-negative intraocular T cell lymphoma: a case report and review of the literature 
Cytofluorographic and molecular techniques are effective adjuncts in diagnosing intraocular lymphoma. Primary intraocular lymphoma is an uncommon entity predominantly of B cell origin and rarely with a T cell phenotype. The aim of the present paper is to report a case of a CD8-positive, TCR-α/β-negative intraocular T cell lymphoma and review the literature.
Case presentation
T cell neoplasia was detected based on flow cytometric demonstration of an abnormal T cell population and polymerase chain reactions for immunoglobulin and T-cell receptor rearrangements demonstrating evidence of monoclonality. Flow cytometry revealed a T cell population aberrantly expressing T-cell lineage markers. This T cell population expressed CD2, bright CD3, CD8, bright CD7, CD38, CD69, and variable CD25. T-cell receptor γ gene rearrangement studies demonstrated evidence of T-cell gene rearrangement confirming that the T cells were monoclonal.
We herein report the rare case of a TCR α/β-negative CD8+ intraocular T-cell lymphoma suggestive of gamma/delta origin diagnosed by flow cytometry and polymerase chain reaction.
PMCID: PMC2137928  PMID: 17963509
24.  Angiotensin II regulates cellular immune responses through a calcineurin-dependent pathway 
Journal of Clinical Investigation  1999;104(12):1693-1701.
The renin-angiotensin system (RAS) is a key regulator of vascular tone and blood pressure. In addition, angiotensin II also has a number of cellular effects that may contribute to disease pathogenesis. Using Agtr1a–/– mice, which lack AT1A receptors for angiotensin II, we have identified a novel function of the RAS to modulate the immune system. We find that angiotensin II, acting through type 1 (AT1) receptors on immune cells, triggers the proliferation of splenic lymphocytes. These actions contribute to the vigor of cellular alloimmune responses. Within lymphoid organs, sufficient components of the RAS are present to activate AT1 receptors during an immune response, promoting cell growth. These actions require activation of calcineurin phosphatase. In an in vivo model of cardiac transplantation, the absence of AT1 signaling accentuates the immunosuppressive effects of the calcineurin inhibitor cyclosporine. We conclude that inhibition of AT1 receptor signaling should be useful as an anti-inflammatory and immunosuppressive therapy. Furthermore, the actions of the RAS to promote lymphocyte activation may contribute to inflammation that characterizes a number of diseases of the heart and the vascular system.
J. Clin. Invest. 104:1693–1701 (1999).
PMCID: PMC409880  PMID: 10606623
25.  Multicolor Cytoenzymatic Evaluation of Dipeptidyl Peptidase IV (CD26) Function in Normal and Neoplastic Human T-Lymphocyte Populations 
Dipeptidyl peptidase IV (DPP IV), also identified as the glycoprotein CD26, is a transmembrane 110- to 120-kDa serine aminopeptidase involved in immune responses by influencing T-cell costimulation and by cleaving cytokines. Additionally, CD26 is a nonintegrin receptor that contains a binding site for extracellular matrix and other molecules. In order to further define the expression and functional activity of this membrane exopeptidase in human T cells, we developed a nondisruptive, four-color cytofluorogenic assay that utilizes three separate antibodies to cell-surface molecules (e.g., CD4/CD8/CD26 and CD19/CD56/CD26) along with a rhodamine 110-conjugated dipeptide substrate that allows the measurement of DPP IV activity in phenotypically defined cells. We found normal human thymi to have notable differences in time-dependent DPP IV activity among the thymocyte subsets defined by their CD4/CD8 phenotype, with CD4−/CD8− thymocytes containing less DPP IV activity than cells expressing CD4 and/or CD8 (i.e., maturing). CD26 positivity was moderately intense in thymocytes and tended to identify cells with higher DPP IV activity. The four-color technique was also used to examine mature peripheral blood lymphocytes, along with an assortment of leukemias and transformed T-cell lines. These experiments revealed that while DPP IV was consistently evident in normal T cells, neoplastic T cells could vary in their expression patterns. Furthermore, the presence (or intensity) of surface CD26 in some abnormal T cells and certain normal peripheral blood mononuclear cells was separable from the level of DPP IV measured intracellularly. Our results established that multicolor cytofluorographic analysis can be a practical means to measure DPP IV activity in various human cell populations. Furthermore, we found that DPP IV activity could vary in T cells according to their differentiation status and that under certain circumstances surface CD26 expression can be disassociated from the level of measured enzyme (i.e., DPP IV) activity.
PMCID: PMC104525  PMID: 9605992

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