Search

While natriuretic peptides (NPs) are primarily known for their renal and cardiovascular actions, NPs stimulate lipolysis in adipocytes and induce a thermogenic program in white adipose tissue (WAT) that resembles brown fat. The biologic effects of NPs are negatively regulated by the NP clearance receptor (NPRC), which binds and degrades NPs. Knockout (KO) of NPRC protects against diet induced obesity and improves insulin sensitivity in obese mice. To determine if pharmacologic blockade of NPRC enhanced the beneficial metabolic actions of NPs in type 2 diabetes, we blocked NP clearance in a mouse model of type 2 diabetes using the specific NPRC ligand ANP(4-23). We found that treatment with ANP(4-23) caused a significant decrease in body weight by increasing energy expenditure and reducing fat mass without a change in lean body mass. The decrease in fat mass was associated with a significant improvement in insulin sensitivity and reduced serum insulin levels. These beneficial effects were accompanied by a decrease in infiltrating macrophages in adipose tissue, and reduced expression of inflammatory markers in both serum and WAT. These data suggest that inhibiting NP clearance may be an effective pharmacologic approach to promote weight loss and enhance insulin sensitivity in type 2 diabetes. Optimizing the therapeutic approach may lead to useful therapies for obesity and type 2 diabetes.

Background Podocyte dysfunction is the main pathologic mechanism driving the development of FSGS and other morphologic types of steroid-resistant nephrotic syndrome (SRNS). Despite significant progress, the genetic causes of most cases of SRNS have yet to be identified. Methods Whole-genome sequencing was performed on 320 individuals from 201 families with familial and sporadic NS/FSGS with no pathogenic mutations in any known NS/FSGS genes. Results Two variants in the gene encoding regulator of calcineurin type 1 (RCAN1) segregate with disease in two families with autosomal dominant FSGS/SRNS. In vitro, loss of RCAN1 reduced human podocyte viability due to increased calcineurin activity. Cells expressing mutant RCAN1 displayed increased calcineurin activity and NFAT activation that resulted in increased susceptibility to apoptosis compared with wild-type RCAN1. Treatment with GSK-3 inhibitors ameliorated this elevated calcineurin activity, suggesting the mutation alters the balance of RCAN1 regulation by GSK-3β, resulting in dysregulated calcineurin activity and apoptosis. Conclusions These data suggest mutations in RCAN1 can cause autosomal dominant FSGS. Despite the widespread use of calcineurin inhibitors in the treatment of NS, genetic mutations in a direct regulator of calcineurin have not been implicated in the etiology of NS/FSGS before this report. The findings highlight the therapeutic potential of targeting RCAN1 regulatory molecules, such as GSK-3β, in the treatment of FSGS.

Background We previously reported that mutations in the anillin ( ANLN ) gene cause familial forms of FSGS. ANLN is an F-actin binding protein that modulates podocyte cell motility and interacts with the phosphoinositide 3-kinase (PI3K) pathway through the slit diaphragm adaptor protein CD2-associated protein (CD2AP). However, it is unclear how the ANLN mutations cause the FSGS phenotype. We hypothesized that the R431C mutation exerts its pathogenic effects by uncoupling ANLN from CD2AP. Methods We conducted in vivo complementation assays in zebrafish to determine the effect of the previously identified missense ANLN variants, ANLN R431C and ANLN G618C during development. We also performed in vitro functional assays using human podocyte cell lines stably expressing wild-type ANLN ( ANLN WT ) or ANLN R431C . Results Experiments in anln -deficient zebrafish embryos showed a loss-of-function effect for each ANLN variant. In human podocyte lines, expression of ANLN R431C increased cell migration, proliferation, and apoptosis. Biochemical characterization of ANLN R431C -expressing podocytes revealed hyperactivation of the PI3K/AKT/mTOR/p70S6K/Rac1 signaling axis and activation of mTOR-driven endoplasmic reticulum stress in ANLN R431C -expressing podocytes. Inhibition of mTOR, GSK-3 β , Rac1, or calcineurin ameliorated the effects of ANLN R431C . Additionally, inhibition of the calcineurin/NFAT pathway reduced the expression of endogenous ANLN and mTOR. Conclusions The ANLN R431C mutation causes multiple derangements in podocyte function through hyperactivation of PI3K/AKT/mTOR/p70S6K/Rac1 signaling. Our findings suggest that the benefits of calcineurin inhibition in FSGS may be due, in part, to the suppression of ANLN and mTOR. Moreover, these studies illustrate that rational therapeutic targets for familial FSGS can be identified through biochemical characterization of dysregulated podocyte phenotypes.

Selective modulation of Rho GTPase activity in podocytes recapitulates characteristic features of human nephrosis. Using a mouse model, Robins et al. found that high levels of Rac1 activation in podocytes caused podocyte detachment and glomerulosclerosis. Podocyte Rac1 activity was enhanced in biopsy specimens from patients with nephrosis, and serum from this patient population activated Rac1 in cultured podocytes. These data provide a causal link between podocyte Rac1 activation and human nephrotic diseases.

Gain-of-function mutations in TRPC6 cause familial focal segmental glomerulosclerosis, and TRPC6 is upregulated in glomerular diseases including diabetic kidney disease. We studied the effect of systemic TRPC6 knockout in the Akita model of type 1 diabetes. Knockout of TRPC6 inhibited albuminuria in Akita mice at 12 and 16 weeks of age, but this difference disappeared by 20 weeks. Knockout of TRPC6 also reduced tubular injury in Akita mice; however, mesangial expansion was significantly increased. Hyperglycemia and blood pressure were similar between TRPC6 knockout and wild-type Akita mice, but knockout mice were more insulin resistant. In cultured podocytes, knockout of TRPC6 inhibited expression of the calcium/calcineurin responsive gene insulin receptor substrate 2 and decreased insulin responsiveness. Insulin resistance is reported to promote diabetic kidney disease independent of blood glucose levels. While the mechanisms are not fully understood, insulin activates both Akt2 and ERK, which inhibits apoptosis signal regulated kinase 1 (ASK1)-p38–induced apoptosis. In cultured podocytes, hyperglycemia stimulated p38 signaling and induced apoptosis, which was reduced by insulin and ASK1 inhibition and enhanced by Akt or ERK inhibition. Glomerular p38 signaling was increased in TRPC6 knockout Akita mice and was associated with enhanced expression of the p38 gene target cyclooxygenase 2. These data suggest that knockout of TRPC6 in Akita mice promotes insulin resistance and exacerbates glomerular disease independent of hyperglycemia.

Expression of a constitutively active Rho A (V14Rho) in podocytes in vivo induces albuminuria and foot process (FP) effacement. These effects may be mediated by the Rho A effector Rho kinase (ROK); but inhibition of ROK with Y27632 failed to attenuate albuminuria or FP effacement in V14Rho mice. ROK activates LIM kinases (LIMKs), which phosphorylate and inhibit the actin depolymerizing factor cofilin 1 (CFL1). Sustained phosphorylation of CFL1 is implicated in human nephrotic diseases, but Y27632 did not inhibit phosphorylation of CFL1 in vivo, despite effective ROK inhibition. CFL1 is also phosphorylated by testis-specific kinase 1 (TESK1) on the same serine residue. TESK1 was expressed in podocytes, and, similar to the in vivo situation, Y27632 had little effect on phospho-CFL1 (pCFL1) levels in cultured podocytes. In contrast, Y27632 reduced pCFL1 levels in TESK1 knockout (KO) cells. ROK inhibition enhanced podocyte motility but, the motility promoting effect of Y27632 was absent in TESK1 KO podocytes. Thus, TESK1 regulates podocyte cytoskeletal dynamics in glomerular podocytes and may play an important role in regulating glomerular filtration barrier integrity in glomerular disease processes.

Familial forms of focal segmental glomerulosclerosis (FSGS) have been linked to gain-of-function mutations in the gene encoding the transient receptor potential channel C6 (TRPC6). GPCRs coupled to Gq signaling activate TRPC6, suggesting that Gq-dependent TRPC6 activation underlies glomerular diseases. Here, we developed a murine model in which a constitutively active Gq α subunit (Gq(Q209L), referred to herein as GqQ>L) is specifically expressed in podocytes and examined the effects of this mutation in response to puromycin aminonucleoside (PAN) nephrosis. We found that compared with control animals, animals expressing GqQ>L exhibited robust albuminuria, structural features of FSGS, and reduced numbers of glomerular podocytes. Gq activation stimulated calcineurin (CN) activity, resulting in CN-dependent upregulation of TRPC6 in murine kidneys. Deletion of TRPC6 in GqQ>L-expressing mice prevented FSGS development and inhibited both tubular damage and podocyte loss induced by PAN nephrosis. Similarly, administration of the CN inhibitor FK506 reduced proteinuria and tubular injury but had more modest effects on glomerular pathology and podocyte numbers in animals with constitutive Gq activation. Moreover, these Gq-dependent effects on podocyte injury were generalizable to diabetic kidney disease, as expression of GqQ>L promoted albuminuria, mesangial expansion, and increased glomerular basement membrane width in diabetic mice. Together, these results suggest that targeting Gq/TRPC6 signaling may have therapeutic benefits for the treatment of glomerular diseases.

BACKGROUND: We previously reported that mutations in the anillin (ANLN) gene cause familial forms of FSGS. ANLN is an F-actin binding protein that modulates podocyte cell motility and interacts with the phosphoinositide 3-kinase (PI3K) pathway through the slit diaphragm adaptor protein CD2-associated protein (CD2AP). However, it is unclear how the ANLN mutations cause the FSGS phenotype. We hypothesized that the R431C mutation exerts its pathogenic effects by uncoupling ANLN from CD2AP. METHODS: We conducted in vivo complementation assays in zebrafish to determine the effect of the previously identified missense ANLN variants, ANLN(R431C) and ANLN(G618C) during development. We also performed in vitro functional assays using human podocyte cell lines stably expressing wild-type ANLN (ANLN(WT)) or ANLN(R431C). RESULTS: Experiments in anln-deficient zebrafish embryos showed a loss-of-function effect for each ANLN variant. In human podocyte lines, expression of ANLN(R431C) increased cell migration, proliferation, and apoptosis. Biochemical characterization of ANLN(R431C)-expressing podocytes revealed hyperactivation of the PI3K/AKT/mTOR/p70S6K/Rac1 signaling axis and activation of mTOR-driven endoplasmic reticulum stress in ANLN(R431C)-expressing podocytes. Inhibition of mTOR, GSK-3β, Rac1, or calcineurin ameliorated the effects of ANLN(R431C). Additionally, inhibition of the calcineurin/NFAT pathway reduced the expression of endogenous ANLN and mTOR. CONCLUSIONS: The ANLN(R431C) mutation causes multiple derangements in podocyte function through hyperactivation of PI3K/AKT/mTOR/p70S6K/Rac1 signaling. Our findings suggest that the benefits of calcineurin inhibition in FSGS may be due, in part, to the suppression of ANLN and mTOR. Moreover, these studies illustrate that rational therapeutic targets for familial FSGS can be identified through biochemical characterization of dysregulated podocyte phenotypes.

Enhanced expression of cyclooxygenase 2 (COX2) in podocytes contributes to glomerular injury in diabetic kidney disease, but some basal level of podocyte COX2 expression might be required to promote podocyte attachment and/or survival. To investigate the role of podocyte COX2 expression in diabetic kidney disease, we deleted COX2 specifically in podocytes in a mouse model of Type 1 diabetes mellitus (Akita mice). Podocyte-specific knockout (KO) of COX2 did not affect renal morphology or albuminuria in nondiabetic mice. Albuminuria was significantly increased in wild-type (WT) and KO Akita mice compared with nondiabetic controls, and the increase in albuminuria was significantly greater in KO Akita mice compared with WT Akita mice at both 16 and 20 wk of age. At the 20-wk time point, mesangial expansion was also increased in WT and KO Akita mice compared with nondiabetic animals, and these histologic abnormalities were not improved by KO of COX2. Tubular injury was seen only in diabetic mice, but there were no significant differences between groups. Thus, KO of COX2 enhanced albuminuria and did not improve the histopathologic features of diabetic kidney disease. These data suggest that 1) KO of COX2 in podocytes does not ameliorate diabetic kidney disease in Akita mice, and 2) some basal level of podocyte COX2 expression in podocytes is necessary to attenuate the adverse effects of diabetes on glomerular filtration barrier function.

The emerging role of the transient receptor potential cation channel isotype 6 (TRPC6) as a central contributor to various pathological processes affecting podocytes has generated interest in the development of therapeutics to modulate its function. Recent insights into the regulation of TRPC6 have revealed PKG as a potent negative modulator of TRPC6 conductance and associated signaling via its phosphorylation at two highly conserved amino acid residues: Thr69/Thr70 (Thr69 in mice and Thr70 in humans) and Ser321/Ser322 (Ser321 in mice and Ser322 in humans). Here, we tested the role of PKG in modulating TRPC6-dependent responses in primary and conditionally immortalized mouse podocytes. TRPC6 was phosphorylated at Thr69 in nonstimulated podocytes, but this declined upon ANG II stimulation or overexpression of constitutively active calcineurin phosphatase. ANG II induced podocyte motility in an in vitro wound assay, and this was reduced 30–60% in cells overexpressing a phosphomimetic mutant TRPC6 (TRPC6T70E/S322E) or activated PKG ( P < 0.05). Pretreatment of podocytes with the PKG agonists S-nitroso- N-acetyl-dl-penicillamine (nitric oxide donor), 8-bromo-cGMP, Bay 41–2772 (soluble guanylate cyclase activator), or phosphodiesterase 5 (PDE5) inhibitor 4-{[3′,4′-(methylenedioxy)benzyl]amino}[7]-6-methoxyquinazoline attenuated ANG II-induced Thr69 dephosphorylation and also inhibited TRPC6-dependent podocyte motility by 30–60%. These data reveal that PKG activation strategies, including PDE5 inhibition, ameliorate ANG II-induced podocyte dysmotility by targeting TRPC6 in podocytes, highlighting the potential therapeutic utility of these approaches to treat hyperactive TRPC6-dependent glomerular disease.

To determine if augmenting podocyte injury promotes the development of advanced diabetic nephropathy (DN), we created mice that expressed the enzyme cytosine deaminase (CD) specifically in podocytes of diabetic Akita mice (Akita-CD mice). In these mice, treatment with the prodrug 5-flucytosine (5-FC) causes podocyte injury as a result of conversion to the toxic metabolite 5-fluorouracil (5-FU). We found that treatment of 4-5 week old Akita mice with 5-FC for 5 days caused robust albuminuria at 16 and 20 weeks of age compared to 5-FC treated Akita controls, which do not express CD (Akita CTLs). By 20 weeks of age, there was a significant increase in mesangial expansion in Akita-CD mice compared to Akita CTLs, which was associated with a variable increase in glomerular basement membrane (GBM) width and interstitial fibrosis. At 20 weeks of age, podocyte number was similarly reduced in both groups of Akita mice, and was inversely correlated with the albuminuria and mesangial expansion. Thus, enhancing podocyte injury early in the disease process promotes the development of prominent mesangial expansion, interstitial fibrosis, increased GBM thickness and robust albuminuria. These data suggest that podocytes play a key role in the development of advanced features of diabetic kidney disease.

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 (V14 Rho) or dominant-negative Rho A (N19 Rho) 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 V14 Rho 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 N19 Rho 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.

The molecular mechanisms responsible for aberrant calcium signaling in parathyroid disease are poorly understood. The loss of appropriate calcium-responsive modulation of PTH secretion observed in parathyroid disease is commonly attributed to decreased expression of the calcium-sensing receptor (CaSR), a G protein-coupled receptor. However, CaSR expression is highly variable in parathyroid adenomas, and the lack of correlation between CaSR abundance and calcium-responsive PTH kinetics indicates that mechanisms independent of CaSR expression may contribute to aberrant calcium sensing in parathyroid disease. To gain a better understanding of parathyroid tumors and the molecular determinants that drive parathyroid adenoma development, we performed gene expression profiling on a panel of 64 normal and neoplastic parathyroid tissues. The microarray data revealed high-level expression of genes known to be involved in parathyroid biology (PTH, VDR, CGA, CaSR, and GCM2). Moreover, our screen identified regulator of G protein signaling 5 (RGS5) as a candidate inhibitor of CaSR signaling. We confirmed RGS5 to be highly expressed in parathyroid adenomas relative to matched-pair normal glands. Transient expression of RGS5 in cells stably expressing CaSR resulted in dose-dependent abrogation of calcium-stimulated inositol trisphosphate production and ERK1/2 phosphorylation. Furthermore, we found that RGS5-nullizygous mice display reduced plasma PTH levels, an outcome consistent with attenuated opposition to CaSR activity. Collectively, these data suggest that RGS5 can act as a physiological regulator of calcium sensing by CaSR in the parathyroid gland. The abnormally elevated expression of RGS5 observed in parathyroid adenomas could thus represent a novel mechanism of CaSR desensitization in patients with primary hyperparathyroidism.

Intrarenal angiotensin II is increased in kidney diseases independently of plasma angiotensin II and is thought to promote progressive deterioration of renal architecture. Here we investigated the mechanism of enhanced renal angiotensin II generation in kidney glomerular diseases. For this, kidney- or liver-specific angiotensinogen gene (Agt) knockout was superimposed on the mouse model of inducible podocyte injury (NEP25). Seven days after induction of podocyte injury, renal angiotensin II was increased ninefold in NEP25 mice with intact Agt, accompanied by increases in urinary albumin and angiotensinogen excretion, renal angiotensinogen protein, and its mRNA. Kidney Agt knockout attenuated renal Agt mRNA but not renal angiotensin II, renal, or urinary angiotensinogen protein. In contrast, liver Agt knockout markedly reduced renal angiotensin II to 18.7% of that of control NEP25 mice, renal and urinary angiotensinogen protein, but not renal Agt mRNA. Renal angiotensin II had no relationship with renal Agt mRNA, or with renal renin mRNA, which was elevated in liver Agt knockouts. Kidney and liver dual Agt knockout mice showed phenotypes comparable to those of liver Agt knockout mice. Thus, increased renal angiotensin II generation upon severe podocyte injury is attributed to increased filtered angiotensinogen of liver origin resulting from loss of macromolecular barrier function of the glomerular capillary wall that occurs upon severe podocyte injury.

Activation of Wnt signaling pathways causes release and stabilization of the transcription regulator beta-catenin from a destruction complex composed of axin and the adenomatous polyposis coli (APC) protein (canonical signaling pathway). Assembly of this complex is facilitated by a protein-protein interaction between APC and a regulator of G protein signaling (RGS) domain in axin. Because G protein-coupled receptor kinase 2 (GRK2) has a RGS domain that is closely related to the RGS domain in axin, we determined whether GRK2 regulated canonical signaling. We found that GRK2 inhibited Wnt1-induced activation of a reporter construct as well as reduced Wnt3a-dependent stabilization and nuclear translocation of beta-catenin. GRK2 enzymatic activity was required for this negative regulatory effect, and depletion of endogenous GRK2 using small interfering RNA enhanced canonical signaling. GRK2-dependent inhibition of canonical signaling is relevant to osteoblast (OB) biology because overexpression of GRK2 attenuated Wnt/beta-catenin signaling in calvarial OBs. Coimmunoprecipitation studies found that: 1) GRK2 bound APC; 2) The GRK2-APC interaction was promoted by GRK2 enzymatic activity; and 3) Deletion of the RGS domain in GRK2 prevented both the GRK2-APC interaction and GRK2-dependent inhibition of canonical signaling. These data suggest that: 1) GRK2 negatively regulates Wnt signaling; 2) GRK2-dependent inhibition of canonical signaling requires a protein-protein interaction between the RGS domain in GRK2 and APC; and 3) Enzymatic activity promotes the GRK2-APC interaction and is required for the negative regulatory effect on canonical signaling. We speculate that inhibiting GRK2 activity in bone-forming OBs might be a useful therapeutic strategy for increasing bone mass.

Background and Aims: Rho kinase (ROCK) inhibition reduces systemic blood pressure (BP) and decreases renal damage in animal models of kidney disease. The aim of this study was to determine if ROCK inhibition might have beneficial effects in glomerular disease processes that are independent of systemic BP. Methods: We investigated the effects of the ROCK inhibitor Y27632 and hydralazine in murine puromycin aminonucleoside (PAN) nephrosis. Results: Treatment with either Y27632 or hydralazine similarly reduced systolic BP compared to vehicle-treated controls. Seven days after treatment with PAN, albuminuria, proteinuria and effacement of podocyte foot processes were significantly reduced in Y27632- and hydralazine-treated mice compared to vehicle-treated animals. Treatment with PAN significantly reduced expression of the podocyte proteins nephrin and Neph1, and the loss of glomerular nephrin was attenuated by treatment with Y27632 but not by treatment with hydralazine. In cultured podocytes, PAN potently activated both Rho and ROCK, and PAN-induced ROCK activation was prevented by Y27632. Conclusions: The ROCK inhibitor Y27632 attenuated glomerular nephrin loss in murine PAN nephrosis independent of its effects on systemic BP.

Diabetic nephropathy (DN) is a serious complication of both type 1 and type 2 diabetes mellitus. The disease is now the most common cause of end-stage kidney disease (ESKD) in developed countries, and both the incidence and prevalence of diabetes mellitus is increasing worldwide. Current treatments are directed at controlling hyperglycemia and hypertension, as well as blockade of the renin angiotensin system with angiotensin-converting enzyme inhibitors (ACEIs), and angiotensin receptor blockers. Despite these therapies, DN progresses to ESKD in many patients. As a result, much interest is focused on developing new therapies. It has been over two decades since ACEIs were shown to have beneficial effects in DN independent of their blood pressure-lowering actions. Since that time, our understanding of disease mechanisms in DN has evolved. In this review, we summarize major cell signaling pathways implicated in the pathogenesis of diabetic kidney disease, as well as emerging treatment strategies. The goal is to identify promising targets that might be translated into therapies for the treatment of patients with diabetic kidney disease.

Background/Aims: Activation of angiotensin II (ANG2) receptors stimulates extracellular signal-regulated kinases (ERKs) that, in some cell systems, are mediated by transactivating the epidermal growth factor (EGF) receptor (EGFR) through mechanisms involving matrix metalloprotease (MMP)-stimulated processing of heparin-binding EGF (HB-EGF) from its precursor. Methods: The signaling pathways linked to ANG2-dependent ERK activation were determined in an immortalized mouse podocyte cell line by monitoring ANG2-stimulated phosphorylation of ERK1/2. Results: ANG2 induced transient ERK phosphorylation that was maximal at 5 min and then rapidly dissipated. ANG2-dependent ERK activation was inhibited by: (1) the type-1 ANG2-selective antagonist losartan; (2) the type-2 ANG2-selective antagonist PD123319; (3) an inhibitor of MMP2/9; (4) the EGFR kinase inhibitor AG1478, and (5) the HB-EGF antagonists CRM197 and heparin. ANG2-dependent ERK activation was mediated by both protein kinase C (PKC)- and calcium-dependent mechanisms and was associated with tyrosine phosphorylation of EGFR. To determine if ANG2-dependent HB-EGF release could act in a paracrine fashion on adjacent cells, HEK293 cells were stably transfected with green fluorescent protein-tagged ERK2 (GFP-ERK2). In stably transfected HEK293 cells, EGF stimulated phosphorylation of endogenous ERK1/2 as well as GFP-ERK2. In contrast, ANG2 had no effect on ERK phosphorylation in stably transfected HEK293 cells. When podocytes were co-cultured with stably transfected HEK293 cells, however, treatment with ANG2 rapidly stimulated GFP-ERK2 phosphorylation. Both the MMP2/9 inhibitor and AG1478 attenuated ANG2-dependent phosphorylation of GFP-ERK2 in the co-culture system. Conclusions: These data indicate that ERK activation is induced by ANG2 in podocytes by mechanisms involving ANG2-dependent release of HB-EGF which, in turn, may act in an autocrine and paracrine fashion to stimulate ERK activity.

Focal and segmental glomerulosclerosis (FSGS) is a leading cause of glomerulonephritis and chronic kidney disease (CKD) in children and young adults ([1][1]). As much as one fifth of the ESRD population carries this diagnosis, and the proportion of ESRD attributed to FSGS has increased more than 10

Lipopolysaccharide (LPS) causes apoptotic deletion of CD4+CD8+thymocytes, a phenomenon that has been linked to immune dysfunction and poor survival during sepsis. Given the abundance of thromboxane-prostanoid (TP) receptors in CD4+CD8+thymocytes and in vitro evidence that thromboxane A2(TXA2) causes apoptosis of these cells, we tested whether enhanced generation of TXA2plays a role in LPS-induced thymocyte apoptosis. Mice injected with 50 μg of LPS intraperitoneally displayed a marked increase in generation of TXA2and prostaglandin E2in the thymus as well as apoptotic deletion of CD4+CD8+thymocytes. Administration of indomethacin or rofecoxib inhibited prostanoid synthesis but did not affect thymocyte death. In contrast, thymocyte apoptosis in response to LPS was significantly attenuated in TP-deficient mice. These studies indicate that TXA2mediates a portion of apoptotic thymocyte death caused by LPS. The absence of an effect of global inhibition of prostanoid synthesis suggests a complex role for prostanoids in this model.

In the past wise rabbis had stated: “The human body was given ten organs of which it is the task of the kidney to provide the human body with thought” (Talmud Brochot). This view of the kidney, although immensely flattering to nephrologists, is no longer universally accepted today. A remaining

To investigate the role of G protein-coupled receptor kinases (GRKs) in regulating bone formation in vivo, we overexpressed the potent G protein-coupled receptor (GPCR) regulator GRK2 in osteoblasts, using the osteocalcin gene-2 promoter to target expression to osteoblastic cells. Using the parathyroid hormone (PTH) receptor as a model system, we found that overexpression of GRK2 in osteoblasts attenuated PTH-induced cAMP generation by mouse calvaria ex vivo. This decrease in GPCR responsiveness was associated with a reduction in bone mineral density (BMD) in transgenic (TG) mice compared with non-TG littermate controls. The decrease in BMD was most prominent in trabecular-rich lumbar spine and was not observed in cortical bone of the femoral shaft. Quantitative computed tomography indicated that the loss of trabecular bone was due to a decrease in trabecular thickness, with little change in trabecular number. Histomorphometric analyses confirmed the decrease in trabecular bone volume and demonstrated reduced bone remodeling, as evidenced by a decrease in osteoblast numbers and osteoblast-mediated bone formation. Osteoclastic activity also appeared to be reduced because urinary excretion of the osteoclastic activity marker deoxypyridinoline was decreased in TG mice compared with control animals. Consistent with reduced coupling of osteoblast-mediated bone formation to osteoclastic bone resorption, mRNA levels of both osteoprotegrin and receptor activator of NF-κB ligand were altered in calvaria of TG mice in a pattern that would promote a low rate of bone remodeling. Taken together, these data suggest that enhancing GRK2 activity and consequently reducing GPCR activity in osteoblasts produces a low bone-turnover state that reduces bone mass.

The effects of GPCR systems in bone are regulated by a family of enzymes termed GRKs. We found that (1) GRK inhibition in osteoblasts has age-dependent effects on bone mass, and (2) the anabolic actions of GRK inhibition are revealed by treatment with PTH(1–34). Introduction: The effects of G-protein-coupled receptor (GPCR) systems in bone are modulated by a family of enzymes termed GPCR kinases (GRKs). These enzymes directly phosphorylate GPCR substrate and desensitize receptor signaling. We previously found that expression of a GRK inhibitor in osteoblasts using transgenic (TG) technologies enhanced bone remodeling, and in turn, increased BMD in 6-week-old TG mice compared with non-TG littermate controls, presumably because of enhanced GPCR function. The aim of this study was to determine the age-dependent effects of the transgene. Materials and Methods: BMD was monitored in TG mice and in controls at 6-week, 3-month, and 6-month time-points. To determine if the transgene enhanced responsiveness of bone to parathyroid hormone (PTH), we measured cyclic adenosine monophosphate (cAMP) generation by mouse calvaria ex vivo as well as the effects of treatment with PTH(1–34) on BMD, bone histomorphometry, and expression of the PTH-responsive gene RANKL in both TG mice and non-TG controls. Results: Consistent with our previous findings, we found that BMD was increased in TG mice compared with controls at 6 weeks of age. The increase in BMD was most prominent in trabecular-rich lumbar spine and was not observed in cortical bone of the femoral shaft. In contrast to younger animals, however, BMD in older TG mice was not statistically different compared with non-TG mice at 3 months of age and was similar to non-TG animals at 6 months of age. The GRK inhibitor seemed to promote GPCR activation in older mice, however, because (1) PTH-induced cAMP generation by mouse calvaria ex vivo was enhanced in TG mice compared with controls, (2) GRK inhibition increased responsiveness of lumbar spine to the osteoinductive actions of PTH(1–34), and (3) the enhanced anabolic effect of PTH(1–34) was associated with increased expression of the PTH-responsive gene RANKL in calvaria of the TG animals. Bone histomorphometry confirmed that PTH(1–34) increased trabecular bone volume in TG mice and found that this increase in bone mass was caused by enhanced bone formation, predominantly as a result of an increase in the mineral apposition rate (MAR). Conclusions: These data suggest that the anabolic effects of GRK inhibition are age dependent. The osteoinductive actions of the GRK inhibitor are, however, unmasked by treatment with PTH(1–34).

Metabolism of arachidonic acid by the cyclo-oxygenase (COX) pathway generates a family of prostanoid mediators. Nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibiting COX, thereby reducing prostanoid synthesis. The efficacy of these agents in reducing inflammation suggests a dominant proinflammatory role for the COX pathway. However, the actions of COX metabolites are complex, and certain prostanoids, such as PGE2, in some circumstances actually inhibit immune and inflammatory responses. In these studies, we examine the hypothesis that anti-inflammatory actions of NSAIDs may be due, in part, to inhibition of thromboxane A2 synthesis. To study the immunoregulatory actions of thromboxane A2, we used mice with a targeted disruption of the gene encoding the thromboxane-prostanoid (TP) receptor. Both mitogen-induced responses and cellular responses to alloantigen were substantially reduced in TP−/− spleen cells. Similar attenuation was observed with pharmacological inhibition of TP signaling in wild-type splenocytes, suggesting that reduced responsiveness was not due to subtle developmental abnormalities in the TP-deficient mice. The absence of TP receptors reduced immune-mediated tissue injury following cardiac transplant rejection, an in vivo model of intense inflammation. Taken together, these findings show that thromboxane augments cellular immune responses and inflammatory tissue injury. Specific inhibition of the TP receptor may provide a more precise approach to limit inflammation without some of the untoward effects associated with NSAIDs.

To test the effect of increased AT 1 receptor expression on blood pressure, we used gene targeting to generate mouse lines with a tandem duplication of the AT 1A receptor gene locus ( Agtr1a ) along with >10 kb of 5′ flanking DNA. By successive breeding, we generated mice with 3 and 4 copies of the Agtr1a gene locus on an inbred 129/Sv background. AT 1A mRNA expression and AT 1 -specific binding of 125 I-angiotensin II were increased in proportion to Agtr1a gene copy number. These animals survived in expected numbers, and their body, heart, and kidney weights were similar to wild-type, 2-copy control mice. Pressor responses to angiotensin II were blunted in the 4-copy mice compared with control mice. In male mice, there was no correlation between resting blood pressure and Agtr1a gene copy number or AT 1A mRNA levels. However, in female mice, there was a highly significant positive correlation between blood pressure and AT 1A receptor expression, paralleled by significant increases in aldosterone synthase expression with increase in gene copy number. Furthermore, in female but not male mice, there was a positive correlation between kallikrein and AT 1A receptor mRNA levels and an inverse correlation between renin mRNA and Agtr1a copy number. Thus, in female but not male mice, genetic variants that increase expression of AT 1 receptors affect blood pressure and gene expression programs. The impact of enhanced AT 1 receptor expression on blood pressure may be blunted by systemic compensatory responses and altered signal-effector coupling in the vasculature.

We investigated the role of Galphaq, filamin, Rho, the RhoGEF Lbc, and the C terminus of calcium-sensing receptor (CasR) in CasR signaling. We found that Ca(2+), Mg(2+), or the calcimimetic R isomer of N-(3-[2-chlorophenyl]propyl)-(R)-alpha-methyl-3-methoxybenzylamine (NPS-R568) stimulated serum response element (SRE) activity human embryonic kidney 293 cells transfected with CasR and an SRE-luciferase reporter construct. Coexpression of either the dominant negative Galphaq(305-359) minigene, regulators of G protein signaling (RGS)2 or RGS4, inhibited CasR-stimulated SRE activity, consistent with CasR activation of Galphaq. The cytoskeletal associated Rho protein is involved CasR activation of SRE, as evidenced by CasR-mediated increase in membrane-associated Rho A and by the ability of Clostridium botulinum C3 (C3) exoenzyme to inhibit both CasR and GalphaqQL-stimulated SRE activity. Overexpression of the RhoGEF Lbc, lacking either the Dbl-homology or Pleckstrin homology domain, as well as the filamin peptide (1530-1875) inhibited CasR-mediated activation of SRE. A carboxyl-terminal CasR minigene, CasR(906-980), encoding a filamin binding region, also blocked CasR- and GalphaqQL-stimulated SRE activity. Potential interactions between CasR, RhoGEF Lbc, Rho A, Galphaq, and filamin were demonstrated by reciprocal coimmunoprecipitation studies. Our results suggest that the C terminus of CasR may interact with filamin to create a cytoskeletal scaffold necessary for the spatial organization of Galphaq, RhoGEF Lbc, and Rho signaling pathways upstream of SRE activation.

Diseases affecting the glomerulus are the most common cause of end-stage kidney disease in developed countries (1). These disorders are characterized by significant proteinuria, and the level of proteinuria is an independent risk factor for disease progression (2). Podocytes are thought to play a key role in the pathogenesis of glomerular diseases (3, 4). The importance of podocytes in glomerular diseases is highlighted by genetic studies, which have identified mutant podocyte proteins that cause familial forms of nephrosis (5). Because podocytes are terminally differentiated cells with little capacity for replication, their ability to compensate for podocyte loss is limited (3). A component of current therapy is, therefore, focused on reducing podocyte injury by decreasing systemic blood pressure (BP) and inhibition of the renin–angiotensin system (2, 6).

FSGS is a clinical disorder characterized by focal scarring of the glomerular capillary tuft, podocyte injury, and nephrotic syndrome. Although idiopathic forms of FSGS predominate, recent insights into the molecular and genetic causes of FSGS have enhanced our understanding of disease pathogenesis. Here, we report a novel missense mutation of the transcriptional regulator Wilms' Tumor 1 (WT1) as the cause of nonsyndromic, autosomal dominant FSGS in two Northern European kindreds from the United States. We performed sequential genome-wide linkage analysis and whole-exome sequencing to evaluate participants from family DUK6524. Subsequently, whole-exome sequencing and direct sequencing were performed on proband DNA from family DUK6975. We identified multiple suggestive loci on chromosomes 6, 11, and 13 in family DUK6524 and identified a segregating missense mutation (R458Q) in WT1 isoform D as the cause of FSGS in this family. The identical mutation was found in family DUK6975. The R458Q mutation was not found in 1600 control chromosomes and was predicted as damaging by in silico simulation. We depleted wt1a in zebrafish embryos and observed glomerular injury and filtration defects, both of which were rescued with wild-type but not mutant human WT1D mRNA. Finally, we explored the subcellular mechanism of the mutation in vitro. WT1(R458Q) overexpression significantly downregulated nephrin and synaptopodin expression, promoted apoptosis in HEK293 cells and impaired focal contact formation in podocytes. Taken together, these data suggest that the WT1(R458Q) mutation alters the regulation of podocyte homeostasis and causes nonsyndromic FSGS.

FSGS is characterized by segmental scarring of the glomerulus and is a leading cause of kidney failure. Identification of genes causing FSGS has improved our understanding of disease mechanisms and points to defects in the glomerular epithelial cell, the podocyte, as a major factor in disease pathogenesis. Using a combination of genome-wide linkage studies and whole-exome sequencing in a kindred with familial FSGS, we identified a missense mutation R431C in anillin (ANLN), an F-actin binding cell cycle gene, as a cause of FSGS. We screened 250 additional families with FSGS and found another variant, G618C, that segregates with disease in a second family with FSGS. We demonstrate upregulation of anillin in podocytes in kidney biopsy specimens from individuals with FSGS and kidney samples from a murine model of HIV-1-associated nephropathy. Overexpression of R431C mutant ANLN in immortalized human podocytes results in enhanced podocyte motility. The mutant anillin displays reduced binding to the slit diaphragm-associated scaffold protein CD2AP. Knockdown of the ANLN gene in zebrafish morphants caused a loss of glomerular filtration barrier integrity, podocyte foot process effacement, and an edematous phenotype. Collectively, these findings suggest that anillin is important in maintaining the integrity of the podocyte actin cytoskeleton.

To investigate the domains of the parathyroid hormone (PTH) receptor required for regulation by G protein-coupled receptor kinases (GRKs), we created mutant PTH receptors lacking potential GRK-phosphorylation sites. Mutant #1 was truncated at amino acid 544 and, therefore, lacked nine hydroxyl group-containing amino acids at the C-terminus. In mutant #2, we replaced threonines 392 and 399 in the third intracellular loop with glycines. Co-transfection of HEK293 cells with the wild-type receptor and either GRK2, GRK3, or GRK5 inhibited PTH-induced cyclic (cAMP) generation; co-transfection of GRK4 or GRK6 had no effect on PTH receptor responsiveness. GRK2-mediated inhibition of PTH receptor signaling was associated with enhanced phosphorylation receptor proteins. Co-expression of GRK2 similarly reduced PTH-induced cAMP generation by the wild-type receptor and mutant #1, and caused phosphorylation of receptor proteins to a similar extent. Co-expression of GRK2 had little effect on PTH-induced cAMP generation by mutant #2 but enhanced agonist-induced phosphorylation of mutant #2 compared with that of either the wild-type receptor or mutant #1. Enhanced phosphorylation of mutant #2 was associated with a reduction in agonist-induced internalization of mutant #2 compared with the wild-type receptor. Thus, phosphorylation of mutant #2 failed to cause receptor desensitization and inhibited receptor internalization. These data are consistent with the notion that: (a) GRKs contribute to regulating PTH receptor responsiveness, and (b) domains in the third intracellular loop are not required for agonist-induced phosphorylation of PTH receptors, but are critical for both agonist-induced internalization of PTH receptors and GRK2-mediated regulation of PTH receptor signaling.

We isolated osteoblastic cell lines from wild-type (CasR(+/+)) and receptor null (CasR(-/-)) mice to investigate whether CasR is present in osteoblasts and accounts for their responses to extracellular cations. Osteoblasts from both CasR(+/+) and CasR(-/-) mice displayed an initial period of cell replication followed by a culture duration-dependent increase in alkaline phosphatase activity, expression of osteocalcin, and mineralization of extracellular matrix. In addition, a panel of extracellular cations, including aluminum and the CasR agonists gadolinium and calcium, stimulated DNA synthesis, activated a transfected serum response element-luciferase reporter construct, and inhibited agonist-induced cAMP in CasR(-/-) osteoblasts. The functional responses to these cations were identical in CasR(+/+) and CasR(-/-) osteoblasts. Thus, the absence of CasR alters neither the maturational profile of isolated osteoblast cultures nor their in vitro responses to extracellular cations. In addition, CasR transcripts could not be detected by reverse transcription-polymerase chain reaction with mouse specific primers in either CasR(+/+) or CasR(-/-) osteoblasts, and immunoblot analysis with a CasR-specific antibody was negative for CasR protein expression in osteoblasts. The presence of a cation-sensing response in osteoblasts from CasR(-/-) mice indicates the existence of a novel osteoblastic extracellular cation-sensing mechanism.

Leukotrienes, the 5-lipoxygenase (5LO) products of arachidonic acid metabolism, have many proinflammatory actions that have been implicated in the pathogenesis of a variety of inflammatory diseases. To investigate the role of LTs in autoimmune disease, we generated an MRL-lpr/lpr mouse line with a targeted disruption of the 5lo gene. MRL-lpr/lpr mice spontaneously develop autoimmune disease that has many features resembling human systemic lupus erythematosus, including sex-related survival differences; female MRL-lpr/lpr mice experience significant early mortality compared with males. Unexpectedly, we found that mortality was accelerated in male 5LO-deficient MRL-lpr/lpr mice compared with male wild-type MRL-lpr/lpr animals. In contrast, the 5lo mutation had no effect on survival in females. Mortality was also accelerated in male MRL-lpr/lpr mice that were treated chronically with a pharmacological inhibitor of LT synthesis. Furthermore, LT-dependent inflammatory responses are enhanced in male MRL-lpr/lpr mice compared with females, and the 5lo mutation has greater impact on these responses in males. Because immune complex-mediated glomerulonephritis is the major cause of death in MRL-lpr/lpr mice and has been related to arachidonic acid metabolites, we also assessed kidney function and histopathology. In male MRL-lpr/lpr mice, renal plasma flow was significantly reduced in the 5lo−/− compared with the 5lo+/+ group, although there were no differences in the severity of renal histopathology, lymphoid hyperplasia, or arthritis between the groups. These findings suggest that the presence of a functional 5lo gene confers a survival advantage on male MRL-lpr/lpr mice and that, when 5LO function is inhibited, either genetically or pharmacologically, this advantage is abolished.

Aluminum is a weak agonist for the calcium-sensing receptor. Background Aluminum (Al 3+ ) has diverse biological effects mediated through activation of a putative extracellular cation-sensing receptor. A recently identified calcium-sensing receptor (CaSR), which has been identified in target tissues for Al 3+ , may transduce some of the biological effects of Al 3+ . Methods To test this possibility, we transfected human embryonic kidney 293 (HEK 293) cells with a cDNA encoding the rat CaSR and evaluated CaSR expression by Western blot analysis and function by measurement of intracellular calcium ([Ca 2+ ] i ) levels and inositol monophosphate (IP1) generation following stimulation with Al 3+ and a panel of CaSR agonists. Results The CaSR protein was detected by immunoblot analysis in cells transfected with the CaSR cDNA but not in nontransfected HEK 293 cells. In addition, [Ca 2+ ] i levels and IP1 generation were enhanced in a dose-dependent fashion by additions of the CaSR agonists calcium (Ca 2+ ), magnesium (Mg 2+ ), gadolinium (Gd 3+ ), and neomycin only in cells transfected with CaSR. To determine if Al 3+ activated CaSR, we stimulated cells transfected with rat CaSR with 10 μ m to 1 m m concentrations of Al 3+ . Concentrations of Al 3+ in the range of 10 μ m to 100 μ m had no effect on [Ca 2+ ] i levels or IP1 generation. In contrast, 1 m m Al 3+ induced small but significant increases in both parameters. Whereas Gd 3+ antagonized calcium-mediated activation of CaSR, pretreatment with Al 3+ failed to block subsequent activation of rat CaSR by Ca 2+ , suggesting a distinct mechanism of Al 3+ action. Conclusion Al 3+ is not a potent agonist for CaSR. Because Al 3+ affects a variety of target tissues at micromolar concentrations, it seems unlikely that CaSR mediates these cellular actions of Al 3+ .

Diabetic nephropathy is the most common cause of ESRD in the United States.[1][1] Although current strategies can slow disease progression,[2][2],[3][3] development of renal failure requiring renal replacement therapy is a distressingly common outcome in patients with diabetes.[1][1] As a result

To investigate the role of C-terminal hydroxyamino acids in desensitization of the receptor for thromboxane A2 (TxA2), we created a mutant TxA2 receptor (TP receptor) in which serines at positions 321, 322, and 328 were replaced with either alanine or glycine. Mutant and wild type receptors were expressed in a mesangial cell line, and clones expressing similar numbers of receptors were studied. Affinity and specificity of TxA2 binding to the mutant receptor were identical to wild type receptors. In contrast, TxA2-induced inositol trisphosphate generation by the mutant receptor was enhanced compared with the wild type. Prior treatment with the TxA2agonist U46619 reduced subsequent U46619-induced increases in inositol trisphosphate generation by both receptors; however, the extent of desensitization was significantly reduced in the receptor mutant. Protein kinase C (PKC) inhibitors attenuated TxA2-induced desensitization of wild type receptors, but had little effect on TxA2-induced desensitization of mutant receptors. Pretreatment with the phorbol ester phorbol 12,13-dybutyrate (PDBu) (100 nm) decreased subsequent responsiveness of wild type but not mutant TP receptors. U46619-induced desensitization of wild type receptors was associated with enhanced phosphorylation of receptor proteins. This agonist-specific phosphorylation of the TP receptor was largely prevented by inhibitors of PKC. Treatment with 100 nm PDBu increased phosphorylation of both wild type and mutant TP receptors, but the extent of phosphorylation of the receptor mutant was reduced compared with the wild type. Increasing the concentration of PDBu from 100 nm to 1 μmPDBu reduced responsiveness of both mutant and wild type receptors without enhancing phosphorylation of either of the receptor proteins. These data suggest that 1) phosphorylation of C-terminal serines contributes to agonist-specific desensitization of the TP receptor, 2) PKC-induced desensitization of TP receptors is caused, in part, by phosphorylation of C-terminal serines, and 3) desensitization of TP receptors by PKC is complex and involves mechanisms that may not require direct phosphorylation of receptor proteins.

We examined the effects of thromboxane A2(TxA2) on the activities of the plasminogen-plasmin system in glomerular mesangial cells. When mesangial cells are exposed to the TxA2agonist U-46619, a substantial increase in production of plasminogen activator inhibitor-1 (PAI-1) protein is observed that is significantly greater than that induced by 10% serum alone. This increase in PAI-1 protein production is accompanied by an increase in steady-state levels of PAI-1 mRNA. This stimulation is specifically mediated by TxA2(thromboxane prostanoid, TP) receptors, since U-46619 also stimulates PAI-1 expression in cells that are transfected with TP receptors, and this stimulation of PAI-1 production is completely blocked by the TxA2receptor antagonist, SQ-29,548. Despite the increase in PAI-1 production, there was net stimulation of plasmin activity in the medium of mesangial cells that had been exposed to U-46619. Furthermore, U-46619 also caused an increase in tissue plasminogen activator (tPA) mRNA levels. Thus TxA2stimulates the production of PAI-1 and plasminogen activators by mesangial cells through a receptor-dependent mechanism. In inflammatory renal diseases, the balance of these effects may modulate glomerular thrombosis and renal fibrosis.

Desensitization of G-protein coupled receptors limits the physiologic effects of an agonist. Short-term desensitization mechanisms are critically dependent on receptor phosphorylation by protein kinases. The effectiveness of these regulatory mechanisms might be limited by substrate (receptor) availability. To investigate the role of receptor number in the desensitization of G-protein coupled receptors, we transfected a mouse mesangial cell line with a genomic clone encoding the mouse thromboxane A2 (TxA2) receptor and obtained cell lines that expressed low (approximately 250-500 fmol/mg protein) or high (2500-4000 fmol/mg protein) levels of TxA2 receptors. Activation of TxA2 receptors stimulated phosphoinositide (PI) hydrolysis and increased intracellular calcium ([Ca2+]i) levels. Prior exposure to the TxA2 agonist (15S)-hydroxy-11alpha,9alpha-(epoxymethano)prosta-5Z,+ ++13E-dienoic acid (U46619) reduced subsequent (15S)-hydroxy- 11alpha,9alpha-(epoxymethano)prosta-5Z,13E-dieno ic acid-induced increases in inositol trisphosphates and intracellular calcium levels by approximately 50% in clones expressing low numbers of TxA2 receptors, but had little effect on TxA2 receptor responsiveness in clones expressing high receptor numbers. Failure of TxA2 receptors to desensitize caused sustained increases in intracellular calcium levels and phosphoinositide hydrolysis. Thus, homologous desensitization of TxA2 receptors is attenuated in cells expressing high levels of receptors for TxA2. These data suggest that receptor number plays a key role in the short-term regulation of G-protein coupled receptors.

MRL-lpr/lpr mice spontaneously develop an autoimmune disease similar to human systemic lupus erythematosus (SLE). Disease manifestations include anti-DNA autoantibody production, arthritis, vasculitis, and an immune-complex glomerulonephritis. The development of autoimmune disease is associated with massive, generalized lymphadenopathy caused by accumulation of an abnormal T-cell population in peripheral lymphoid organs. In MRL-lpr/lpr mice, treatment with E-series prostaglandins ameliorates renal disease and reduces peripheral lymphadenopathy. Little is known about mechanisms of action of E-series prostaglandins in murine lupus or the effects of these agents on other clinically important manifestations of SLE, such as arthritis and vasculitis. To further investigate the effects of an E-series prostaglandin in murine SLE, we administered the prostaglandin, E(1) (PGE(1)) analog misoprostol (1 mg kg(minus sign1) day(minus sign1)) to MRL-lpr/lpr mice for 8 weeks by twice daily subcutaneous injection. At 20 weeks of age, treatment with misoprostol reduced the severity of renal disease and arthritis but did not affect the extent or severity of vasculitis. The beneficial effects of misoprostol on arthritis and renal disease were associated with a significant decrease in splenic and lymph node weight in mice given the PGE(1) analog. This decrease in lymphoproliferation resulted primarily from a generalized reduction in the number of T cells in peripheral lymphoid organs. Thus, T-cell depletion was associated with beneficial effects on arthritis and nephritis in MRL-lpr/lpr mice, supporting a role for T lymphocytes in these disease processes. The ability of E-series prostaglandins to favorably modify autoimmune disease in this murine model suggests that misoprostol may be a useful adjunct to current therapies for the treatment of patients with SLE.

MRL-lpr/lpr mice spontaneously develop an autoimmune disease with nephritis similar to human systemic lupus erythematosus. In these animals, treatment with E-series prostaglandins ameliorates renal disease and prolongs survival, perhaps by modulating production of cytokines or eicosanoids. To further define the mechanisms of action of E-series prostaglandins in established murine lupus nephritis, we administered the prostaglandin E1 (PGE1) analog misoprostol to 20-week-old MRL-lpr/lpr mice by twice-daily subcutaneous injection. After 2 days of treatment, misoprostol reduced renal cortical interleukin-1 beta (IL-1 beta) mRNA levels compared to vehicle-treated controls (0.19 +/- 0.06 (misoprostol) vs 0.50 +/- 0.04 (vehicle) densitometry units; P0.005). A similar reduction in cortical IL-1 beta mRNA levels was found in left kidneys harvested from MRL-lpr/lpr mice following 2 days of treatment with misoprostol compared to right kidneys harvested from the same animal prior to the first dose of PGE1 analog (0.12 +/- .05 (left) vs 0.39 +/- 0.18 (right) densitometry units; P0.05). This reduction in cortical IL-1 beta mRNA levels was not associated with alterations in renal production of thromboxane B2, PGE2, or leukotriene B4 or with significant changes in the severity of renal inflammatory cell infiltrates. Time-course studies indicated that IL-1 beta mRNA levels were decreased within 24 hr of initiating misoprostol therapy. This reduction in IL-1 beta mRNA levels was transient because levels were not reduced after 1 week of treatment with the PGE1 analog.(ABSTRACT TRUNCATED AT 250 WORDS)

G protein-coupled receptor kinase interacting protein 2 (GIT2) is a signaling scaffold protein involved in the regulation of cytoskeletal structure, membrane trafficking, and G protein-coupled receptor internalization. Since dynamic cytoskeletal reorganization plays key roles both in osteoblast differentiation and in the maintenance of osteoclast polarity during bone resorption, we hypothesized that skeletal physiology would be altered in GIT2(-/-) mice. We found that adult GIT2(-/-) mice have decreased bone mineral density and bone volume in both the trabecular and cortical compartments. This osteopenia was associated with decreased numbers of mature osteoblasts, diminished osteoblastic activity, and increased marrow adiposity, suggesting a defect in osteoblast maturation. In vitro, mesenchymal stem cells derived from GIT2(-/-) mice exhibited impaired differentiation into osteoblasts and increased adipocyte differentiation, consistent with a role for GIT2 in mesenchymal stem cell fate determination. Despite elevated osteoclast inducing cytokines and osteoclast numbers, GIT2(-/-) mice also exhibit impaired bone resorption, consistent with a further role for GIT2 in regulating osteoclast function. Collectively, these findings underscore the importance of the cytoskeleton in both osteoblast and osteoclast function and demonstrate that GIT2 plays essential roles in skeletal metabolism, affecting both bone formation and bone resorption in vivo.

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.

Aim: The goal of this study was to examine the capacity for glomerular repair after a podocyte-depleting injury. Methods: We created transgenic (TG) mice expressing the yeast enzyme cytosine deaminase specifically in glomerular podocytes. In these TG animals, the prodrug 5-flucytosine (5-FC) is converted to 5-fluorouracil and promotes cell death. Results: Treatment with increasing dosages of 5-FC caused graded increases in proteinuria 1–2 weeks after treatment, which returned to control levels by the 10-week time point. Light microscopic examination revealed minimal pathology at the 2-week time point, but electron microscopy revealed found foot process effacement as well as focal areas of glomerular basement membrane duplication, and immunohistochemical studies detected podocyte apoptosis and a decrease in the number of Wilms’ tumor protein 1 (WT1)-positive cells. By the 10-week time point, however, the number of WT1-positive cells was similar to controls and a few mice had developed focal areas of glomerulosclerosis. Consistent with the effects of 5-FC on podocyte number, expression of the podocyte mRNAs for nephrin, podocin, synaptopodin and podocalyxin were altered in a similar temporal fashion. Conclusion: The glomerulus has a significant capacity for repair after a podocyte-depleting injury.

To determine the role of Gq signaling and calcineurin (CN) activation in promoting apoptosis of glomerular podocytes, constitutively active Gq [Gq(+)] or CN [CN(+)] proteins were introduced into cultured podocytes using protein transduction by tagging the proteins with the transactivator of transcription peptide. To investigate the role of CN in promoting podocyte apoptosis in vivo, a genetic model of type 1 diabetes mellitus (Akita mice) was treated with the CN inhibitor FK506. In cultured podocytes, Gq(+) stimulated nuclear translocation of nuclear factor of activated T cells (NFAT) family members, activated an NFAT reporter construct, and enhanced podocyte apoptosis in a CN-dependent fashion. CN(+) similarly promoted podocyte apoptosis, and apoptosis induced by either angiotensin II or endothelin-1 was blocked by FK506. Induction of apoptosis required NFAT-induced gene transcription because apoptosis induced by either Gq(+) or CN(+) was blocked by an inhibitor that prevented CN-dependent NFAT activation without affecting CN phosphatase activity. Podocyte apoptosis was mediated, in part, by the NFAT-responsive gene cyclooxygenase 2 (COX2) and prostaglandin E(2) generation because apoptosis induced by Gq(+) was attenuated by either COX2 inhibition or blockade of the Gq-coupled E-series prostaglandins receptor. The findings appeared relevant to podocyte apoptosis in diabetic nephropathy because apoptosis was significantly reduced in Akita mice by treatment with FK506. These data suggest that Gq stimulates CN and promotes podocyte apoptosis both in vitro and in vivo. Apoptosis requires NFAT-dependent gene transcription and is mediated, in part, by CN-dependent COX2 induction, prostaglandin E(2) generation, and autocrine activation of the Gq-coupled E-series prostaglandins receptor.

MRL-lpr/lpr mice develop an inflammatory arthritis in association with other manifestations of autoimmunity. Although a variety of immune cell disturbances have been described in these mice, the relationship of these abnormalities to the pathogenesis of arthritis has not yet been determined; the role of T cells is especially unclear since synovial hypertrophy and joint erosions have been noted in some studies in the absence of a significant T cell infiltrate. Therefore, to determine if T cells are required for arthritis in MRL-lpr/lpr mice, we evaluated the effects of prolonged treatment with a monoclonal anti-CD4 antibody. Knee joints from treated mice had markedly reduced arthritis compared to saline-treated control animals as measured by the degree of synovial hypertrophy and inflammation. Nephritis in these mice was concomitantly reduced. In contrast, rheumatoid factor levels were not affected by CD4+ cell depletion, despite significant effects on anti-DNA. These results indicate that in MRL-lpr/lpr mice anti-CD4 therapy can inhibit arthritis, suggesting an important role of T cells in the pathogenesis of this lesion.