Relationship between polymorphisms and mutations at rs7125942 and rs3736228 of LRP5 gene and bone metabolism in postmenopausal women.

OBJECTIVE: To analyze the relationship between the polymorphism and mutation of rs7125942 and rs3736228 locus in the low-density lipoprotein receptor-related protein 5 (LRP5) genotype and bone mineral density (BMD) in postmenopausal women in Xinjiang, China, to provide a basis for prevention and treatment of the disease. METHODS: According to the results of dual-energy X-ray (DEXA) determination of BMD, the 136 subjects were divided into three groups: Group A: normal bone mass, Group B: osteopenia, Group C: osteoporosis. 1. Age, body, mass index (BMI), and menopause of all subjects were recorded. 2. Fasting blood glucose (FBG), glycosylated hemoglobin (HbA1c), calcium (Ca), phosphorus (P), alkaline phosphatase (ALP), and clinical biochemical data were determined. 3. LRP5 locus polymorphisms were determined by time-of-flight mass spectrometry. RESULTS: 1. Compared with group A, the age, ALP, Cr, and BUN levels in group B and group C were increased, but UA levels were lower (P < 0.05), and Serum P was higher in the group C (P < 0.05). 2. There was no statistically significant difference in the prevalence of diabetes between the three groups (P > 0.05). 3. The ROC curves for different BMD sites such as L1, L2, L3, L4, L total, and femoral neck were 0.929, 0.955, 0.901, 0.914, 0.885, and 0.873 (P < 0.01). 4. At rs7125942 locus, there was statistically significant difference in the distribution of wild-type (CC) and mutant (CG) with the normal bone mass (NBM) group and the abnormal bone mass (ABM) group (P < 0.05). 5. At rs7125942 locus, compared with wild-type (CC), mutant (CG) had lower LDL and FPG in NBM group (P < 0.05), and lower serum ALP in the ABM group (P < 0.05). At rs3736228 locus, the BMD (Femoral neck) of mutant (CT/TT) was lower than that of wild-type (CC) in the NBM group (P < 0.05). 6. Age and menopausal years were negatively correlated with BMD of the femoral neck and L1-4 (P < 0.05), and BMI and TG were positively (P < 0.05), and the results of multiple linear regression analysis showed that age, BMI, and TG were both independent factors affecting BMD (P < 0.05).

Visual outcome in patients of keratomycosis, at a tertiary care centre in Larkana, Pakistan.

OBJECTIVE: To study the post-treatment visual outcome of fungal keratitis. METHODS: This prospective study was carried out at Chandka Medical College and Hospital, Larkana, Pakistan, from March 2005 to March 2016. Patients with clinical features of fungal keratitis, with positive corneal scrapings for fungi, and those who followed up for a minimum period of three months after recovery from infection were included.Other causes of infectious keratitis were excluded. The clinical diagnosis of fungal keratitis was based on risk factor identification and characteristic non-specific and specific corneal features. Treatment included antifungal preparations, topical and if necessary systemic, in addition to symptomatic measures. SPSS 20 was used for data analysis. RESULTS: Of the 1,130 patients, 750(66.37%) were males and 380(33.63%) were females. The overall mean age was 39.44±12.46 years (range:16-74 years). After the completion of treatment, 590(52.21%) of the eyes just retained visual acuity of not more than counting fingers and 126(11.15%) patients lost their globe. Patients with remaining corneal opacity needed keratoplasty. CONCLUSIONS: Most of the eyes just retained visual acuity of counting fingers while some patients lost their globe.

Types of Traumatic Lens Dislocations at Larkana.

The objective of this study was to determine the pattern of traumatic lens dislocations presenting at our institute. This may help develop the preventive strategies. The number of cases of traumatic lens dislocations, presented at the Department of Ophthalmology, Chandka Medical College, Larkana, Pakistan, from January 2002 to June 2015, were 59 including 61.02% (n=36) males and 38.98% (n=23) females. Cause of trauma was wood or plant impalement in 35.6% (n=21) cases, cracker blast in 13.55% (n=8) cases, fall on ground in 11.86% (n=7) cases, penetrating injuries with needle, scissors or knife in 10.16% (n=6) cases, road traffic accidents in 10.16% (n=6) cases, sports injuries (cricket ball and gulle danda) in 8.47% (n=5) cases, firearm injuries in 5.1% (n=3) cases, and fist hitting in 5.1% (n=3) cases. Lens was dislocated posteriorly in 33.90% (n=20) cases, anteriorly in 25.42% (n=15) cases, inferiorly in 11.86% (n=7) cases, medially in 10.17% (n=6) cases, laterally in 10.17% (n=6) cases, superiorly in 6.78% (n=4) cases, and a single (1.69%) case of lenticele was seen.

Factors associated with pterygium based on history and clinical examination of patients in Pakistan.

PURPOSE: The purpose of this study was to determine the factors associated with Pterygium, utilizing history and examination. METHODS: In this prospective case series study, a total of 1227 patients with Pterygium presenting at the Department of Ophthalmology, Chandka Medical College Hospital Larkana, Pakistan, from January 1997 to January 2015 were included. A standard proforma containing proposed risk factors was filled in for every patient. Clinical examination was performed on slit-lamp biomicroscope to confirm presence of pterygium. RESULTS: Out of the total 1227 patients, 656 (53.46%) were males, and 571 (46.54%) were females. Mean age ± standard deviation was 53.12 years ± 15.85 years, and the age range was 20-79 years. 1063 (86.63%) patients belonged to areas with hot and dry weather, 421 (34.31%) patients had a positive family history for Pterygium, 740 (60.31%) patients had history of previous exposure to toxic chemicals, and 364 (29.67%) patients had dry eye. CONCLUSION: This study points towards the simultaneous role of multiple risk factors including sun exposure, hot climate, toxic material exposure, familial transmission, and dry eye in association with pterygium.

The Expression Level of mRNA, Protein, and DNA Methylation Status of FOSL2 of Uyghur in XinJiang in Type 2 Diabetes.

Objective. We investigated the expression levels of both FOSL2 mRNA and protein as well as evaluating DNA methylation in the blood of type 2 diabetes mellitus (T2DM) Uyghur patients from Xinjiang. This study also evaluated whether FOSL2 gene expression had demonstrated any associations with clinical and biochemical indicators of T2DM. Methods. One hundred Uyghur subjects where divided into two groups, T2DM and nonimpaired glucose tolerance (NGT) groups. DNA methylation of FOSL2 was also analyzed by MassARRAY Spectrometry and methylation data of individual units were generated by the EpiTyper v1.0.5 software. The expression levels of FOS-like antigen 2 (FOSL2) and the protein expression levels were analyzed. Results. Significant differences were observed in mRNA and protein levels when compared with the NGT group, while methylation rates of eight CpG units within the FOSL2 gene were higher in the T2DM group. Methylation of CpG sites was found to inversely correlate with expression of other markers. Conclusions. Results show that a correlation between mRNA, protein, and DNA methylation of FOSL2 gene exists among T2DM patients from Uyghur. FOSL2 protein and mRNA were downregulated and the DNA became hypermethylated, all of which may be involved in T2DM pathogenesis in this population.

Tubular cell phenotype in HIV-associated nephropathy: role of phospholipid lysophosphatidic acid.

Collapsing glomerulopathy and microcysts are characteristic histological features of HIV-associated nephropathy (HIVAN). We have previously reported the role of epithelial mesenchymal transition (EMT) in the development of glomerular and tubular cell phenotypes in HIVAN. Since persistent tubular cell activation of NFκB has been reported in HIVAN, we now hypothesize that HIV may be contributing to tubular cell phenotype via lysophosphatidic acid (LPA) mediated downstream signaling. Interestingly, LPA and its receptors have also been implicated in the tubular interstitial cell fibrosis (TIF) and cyst formation in autosomal dominant polycystic kidney disease (PKD). Primary human proximal tubular cells (HRPTCs) were transduced with either empty vector (EV/HRPTCs), HIV (HIV/HRPTCs) or treated with LPA (LPA/HRPTC). Immunoelectrophoresis of HIV/HRPTCs and LPA/HRPTCs displayed enhanced expression of pro-fibrotic markers: a) fibronectin (2.25 fold), b) connective tissue growth factor (CTGF; 4.8 fold), c) α-smooth muscle actin (α-SMA; 12 fold), and d) collagen I (5.7 fold). HIV enhanced tubular cell phosphorylation of ILK-1, FAK, PI3K, Akt, ERKs and P38 MAPK. HIV increased tubular cell transcriptional binding activity of NF-κB; whereas, a LPA biosynthesis inhibitor (AACOCF3), a DAG kinase inhibitor, a LPA receptor blocker (Ki16425), a NF-κB inhibitor (PDTC) and NFκB-siRNA not only displayed downregulation of a NFκB activity but also showed attenuated expression of profibrotic/EMT genes in HIV milieu. These findings suggest that LPA could be contributing to HIV-induced tubular cell phenotype via NFκB activation in HIVAN.

AT1R blockade in adverse milieus: role of SMRT and corepressor complexes.

ANG II type 1 receptor blockade (AT1R-BLK) is used extensively to slow down the progression of proteinuric kidney diseases. We hypothesized that AT1R-BLK provides podocyte protection through regulation of silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and vitamin D receptor (VDR) expression under adverse milieus such as high glucose and human immunodeficiency virus infection. Both AT1R-BLK and VDR agonists (VDAs) stimulated VDR complex formation that differed not only in their composition but also in their functionality. AT1R-BLK-induced VDR complexes contained predominantly unliganded VDR, SMRT, and phosphorylated histone deacetylase 3, whereas VDA-VDR complexes were constituted by liganded VDR and CREB-binding protein/p300. AT1R-BLK-induced complexes attenuated podocyte acetyl-histone 3 levels as well as cytochrome P-450 family 24A1 expression, thus indicating their deacetylating and repressive properties. On the other hand, VDA-VDR complexes not only increased podocyte acetyl-histone 3 levels but also enhanced cytochrome P-450 family 24A1 expression, thus suggesting their acetylating and gene activation properties. AT1R-BLK- induced podocyte SMRT inhibited expression of the proapoptotic gene BAX through downregulation of Wip1 and phosphorylation of checkpoint kinase 2 in high-glucose milieu. Since SMRT-depleted podocytes lacked AT1R-BLK-mediated protection against DNA damage, it appears that SMRT is necessary for DNA repairs during AT1R-BLK. We conclude that AT1R-BLK provides podocyte protection in adverse milieus predominantly through SMRT expression and partly through unliganded VDR expression in 1,25(OH)2D-deficient states; on the other hand, AT1R-BLK contributes to liganded VDR expression in 1,25(OH)2D-sufficient states.

Hyperglycemia enhances kidney cell injury in HIVAN through down-regulation of vitamin D receptors.

In the present study, we evaluated the effect of short term hyperglycemia on renal lesions in a mouse model (Tg26) of HIV-associated nephropathy (HIVAN). Control and Tg26 mice in groups (n=6) were administered either normal saline (FVBN or Tg) or streptozotocin (FVBN+STZ or Tg26+STZ). After two weeks, biomarkers were collected and kidneys were harvested. FVBN+ STZ and Tg26+STZ displayed elevated serum glucose levels when compared to FVBN and Tg26 respectively. Tg26+STZ displayed elevated (P<0.05) blood urea nitrogen (BUN) levels (P<0.05) and enhanced (P<0.01) proteinuria when compared to Tg26. Tg26+STZ displayed enhanced (P<0.001) number of sclerotic glomeruli and microcysts vs. Tg26. Renal tissues of Tg26 displayed down regulation of vitamin D receptor (VDR) expression and enhanced Ang II production when compared to FVBN mice. Hyperglycemia exacerbated down regulation of VDR and production of Ang II in FVBN and Tg mice. Hyperglycemia increased kidney cell reactive oxygen species (ROS) production and oxidative DNA damage in both FVBN and Tg26 mice. In in vitro studies, HIV down regulated podocyte VDR expression and also enhanced renin angiotensin system activation. In addition, both glucose and HIV stimulated kidney cell ROS generation and DNA damage and compromised DNA repair; however, tempol (superoxide dismutase mimetic), losartan (Ang II blocker) and EB1089 (VDR agonist) provided protection against DNA damaging effects of glucose and HIV. These findings indicated that glucose activated the RAS and inflicted oxidative stress-mediated DNA damage via down regulation of kidney cell VDR expression in HIV milieu both in vivo and in vitro.

Renin modulates HIV replication in T cells.

HIV is known to subvert cellular machinery to enhance its replication. Recently, HIV has been reported to enhance TC renin expression. We hypothesized that HIV induces and maintains high renin expression to promote its own replication in TCs. Renin enhanced HIV replication in TCs in a dose-dependent manner. (P)RR-deficient TCs, as well as those lacking renin, displayed attenuated NF-κB activity and HIV replication. TCs treated with renin and Hpr displayed activation of the (P)RR-PLZF protein signaling cascade. Renin, HIV, and Hpr activated the PI3K pathway. Both renin and Hpr cleaved Agt (a renin substrate) to Ang I and also cleaved Gag polyproteins (protease substrate) to p24. Furthermore, aliskiren, a renin inhibitor, reduced renin- and Hpr-induced cleavage of Agt and Gag polyproteins. These findings indicate that renin contributes to HIV replication in TCs via the (P)RR-PLZF signaling cascade and through cleavage of the Gag polyproteins.

Modulation of renin angiotensin system predominantly alters sclerotic phenotype of glomeruli in HIVAN.

HIV-associated nephropathy (HIVAN) is a common complication of HIV-1 infection in patients with African ancestry in general and with APOL1 gene risk variants in particular. Although collapsing glomerulopathy is considered a hallmark of HIVAN, significant numbers of glomeruli in patients with HIVAN also display other variants of focal segmental glomerulosclerosis (FSGS). We propose that collapsed glomeruli as well as glomeruli with other variants of FSGS are manifestations of HIVAN and their prevalence depends on associated host factors. We explored the role of the renin-angiotensin system (RAS) in the manifestation of any specific glomerular phenotype in HIVAN. To evaluate the role of the RAS we have used a genetically engineered mouse model of HIVAN (Tg26) with two and four copies of angiotensinogen (Agt) gene (Tg26/Agt2 and Tg26/Agt4). In Tg26/Agt2, 1 out of 6 glomeruli exhibited sclerosed phenotype, whereas 1 out of 25 glomeruli displayed collapsed phenotype; on the other hand, in Tg26/Agt4, 1 out of 3 glomeruli exhibited sclerotic phenotype and only 1 out of 7 glomeruli showed collapsed phenotype. To inhibit the effect of RAS, Tg26/Agt2 were administered captopril, aliskiren, aliskiren plus captopril or aliskiren plus telmisartan by miniosmotic pumps for 4 weeks. In all experimental groups there was a significant reduction in percentage of sclerosed glomeruli and only minimal reduction in collapsed glomeruli compared to normal saline receiving Tg26/Agt2. These findings suggest that the manifestation of the sclerosed phenotype in HIVAN is predominantly dependent on activation of the RAS.

Renin angiotensin system modulates mTOR pathway through AT2R in HIVAN.

Mammalian target of rapamycin (mTOR) has been reported to contribute to the development of HIV-associated nephropathy (HIVAN). We hypothesized that HIV may be activating renal tissue mTOR pathway through renin angiotensin system (RAS) via Angiotensin Receptor Type II receptor (AT2R). Renal tissues of Vpr transgenic and Tg26 (HIVAN) mice displayed enhanced phosphorylation of mTOR and p70S6K. Aliskiren, a renin inhibitor attenuated phosphorylation of both mTOR and p70S6K in renal tissues of HIVAN mice. Interestingly, Angiotensin Receptor Type I (AT1R) blockade did not modulate renal tissue phosphorylation of mTOR in HIVAN mice; on the other hand, AT2R blockade attenuated renal tissue phosphorylation of mTOR in HIVAN mice. In vitro studies, both renin and Ang II displayed enhanced mouse tubular cell (MTC) phosphorylation of p70S6K in a dose dependent manner. HIV/MTC also displayed enhanced phosphorylation of both mTOR and p70S6K; interestingly this effect of HIV was further enhanced by losartan (an AT1R blocker). On the other hand, AT2R blockade attenuated HIV-induced tubular cell phosphorylation of mTOR and p70S6K, whereas, AT2R agonist enhanced phosphorylation of mTOR and p70S6K. These findings indicate that HIV stimulates mTOR pathway in HIVAN through the activation of renin angiotensin system via AT2R.

Bone-derived mesenchymal stromal cells from HIV transgenic mice exhibit altered proliferation, differentiation capacity and paracrine functions along with impaired therapeutic potential in kidney injury.

Mesenchymal stem cells (MSCs) secrete paracrine factors that could be cytoprotective and serve roles in immunoregulation during tissue injury. Although MSCs express HIV receptors, and co-receptors, and are susceptible to HIV infection, whether HIV-1 may affect biological properties of MSCs needs more study. We evaluated cellular proliferation, differentiation and paracrine functions of MSCs isolated from compact bones of healthy control mice and Tg26 HIV-1 transgenic mice. The ability of MSCs to protect against cisplatin toxicity was studied in cultured renal tubular cells as well as in intact mice. We successfully isolated MSCs from healthy mice and Tg26 HIV-1 transgenic mice and found the latter expressed viral Nef, Vpu, NL4-3 and Vif genes. The proliferation and differentiation of Tg26 HIV-1 MSCs was inferior to MSCs from healthy mice. Moreover, transplantation of Tg26 HIV-1 MSCs less effectively improved outcomes compared with healthy MSCs in mice with acute kidney injury. Also, Tg26 HIV-1 MSCs secreted multiple cytokines, but at significantly lower levels than healthy MSCs, which resulted in failure of conditioned medium from these MSCs to protect cultured renal tubular cells from cisplatin toxicity. Therefore, HIV-1 had adverse biological effects on MSCs extending to their proliferation, differentiation, function, and therapeutic potential. These findings will help in advancing mechanistical insight in renal injury and repair in the setting of HIV-1 infection.

mTOR plays a critical role in p53-induced oxidative kidney cell injury in HIVAN.

Oxidative stress has been implicated to contribute to HIV-induced kidney cell injury; however, the role of p53, a modulator of oxidative stress, has not been evaluated in the development of HIV-associated nephropathy (HIVAN). We hypothesized that mammalian target of rapamycin (mTOR) may be critical for the induction of p53-mediated oxidative kidney cell injury in HIVAN. To test our hypothesis, we evaluated the effect of an mTOR inhibitor, rapamycin, on kidney cell p53 expression, downstream signaling, and kidney cell injury in both in vivo and in vitro studies. Inhibition of the mTOR pathway resulted in downregulation of renal tissue p53 expression, associated downstream signaling, and decreased number of sclerosed glomeruli, tubular microcysts, and apoptosed and 8-hydroxy deoxyguanosine (8-OHdG)-positive (+ve) cells in Tg26 mice. mTOR inhibition not only attenuated kidney cell expression of p66ShcA and phospho-p66ShcA but also reactivated the redox-sensitive stress response program in the form of enhanced expression of manganese superoxide dismutase (MnSOD) and catalase. In in vitro studies, the mTOR inhibitor also provided protection against HIV-induced podocyte apoptosis. Moreover, mTOR inhibition downregulated HIV-induced podocyte (HP/HIV) p53 expression. Since HP/HIV silenced for mTOR displayed a lack of expression of p53 as well as attenuated podocyte apoptosis, this suggests that mTOR is critical for kidney cell p53 activation and associated oxidative kidney cell injury in the HIV milieu.

Rapamycin-induced modulation of miRNA expression is associated with amelioration of HIV-associated nephropathy (HIVAN).

Recent studies suggested that miRNAs are involved in the development of the pathogenesis of HIV-associated nephropathy (HIVAN). Rapamycin, a widely used mTOR inhibitor, has been demonstrated to slow down the progression of HIVAN. However, the role of miRNA in the regulation of these processes has not been investigated so far. In the current study, we have used a microarray-based approach in combination with real-time PCR to profile the miRNA expression patterns in rapamycin-treated HIVAN mice (Tg26). Our results demonstrated that 19 miRNAs belonging to 13 different families expressed differentially in renal tissues of rapamycin-receiving Tg26 mice when compared to Tg26 mice-receiving saline only. The patterns of miRNAs expression in rapamycin-receiving Tg26 mice took a reverse turn. These miRNAs were classified into 8 functional categories. In in vitro studies, we examined the expression of specific miRNAs in HIV-1 transduced human podocytes (HIV/HPs). HIV/HPs displayed attenuation of expression of miR-99a, -100a, -199a and miR-200, whereas, rapamycin inhibited this effect of HIV. These findings suggest that rapamycin-mediated up-regulation of specific miRNAs could contribute to amelioration of renal lesions in HIVAN mice.

Morphine induces albuminuria by compromising podocyte integrity.

Morphine has been reported to accelerate the progression of chronic kidney disease. However, whether morphine affects slit diaphragm (SD), the major constituent of glomerular filtration barrier, is still unclear. In the present study, we examined the effect of morphine on glomerular filtration barrier in general and podocyte integrity in particular. Mice were administered either normal saline or morphine for 72 h, then urine samples were collected and kidneys were subsequently isolated for immunohistochemical studies and Western blot. For in vitro studies, human podocytes were treated with morphine and then probed for the molecular markers of slit diaphragm. Morphine-receiving mice displayed a significant increase in albuminuria and showed effacement of podocyte foot processes. In both in vivo and in vitro studies, the expression of synaptopodin, a molecular marker for podocyte integrity, and the slit diaphragm constituting molecules (SDCM), such as nephrin, podocin, and CD2-associated protein (CD2AP), were decreased in morphine-treated podocytes. In vitro studies indicated that morphine modulated podocyte expression of SDCM through opiate mu (MOR) and kappa (KOR) receptors. Since morphine also enhanced podocyte oxidative stress, the latter seems to contribute to decreased SDCM expression. In addition, AKT, p38, and JNK pathways were involved in morphine-induced down regulation of SDCM in human podocytes. These findings demonstrate that morphine has the potential to alter the glomerular filtration barrier by compromising the integrity of podocytes.

HIV compromises integrity of the podocyte actin cytoskeleton through downregulation of the vitamin D receptor.

Alterations in the podocyte actin cytoskeleton have been implicated in the development of proteinuric kidney diseases. In the present study, we evaluated the effect of HIV on the podocyte actin cytoskeleton and the mechanism involved. We hypothesized that HIV may be compromising the actin cytoskeleton via downregulation of the vitamin D receptor (VDR) of conditionally immortalized differentiated human podocytes (CIDHPs). HIV-transduced podocytes (HIV/CIDHPs) not only displayed downregulation of VDR but also showed activation of the renin-angiotensin system (RAS) in the form of enhanced expression of renin and increased production of ANG II. Moreover, CIDHPs lacking VDR displayed enhanced ANG II production, and treatment of HIV/CIDHPs with EB1089 (vitamin D3; VD) attenuated ANG II production. HIV/CIDHPs as well as ANG II-treated CIDHPs exhibited enhanced expression of cathepsin (CTS) L. Additionally, losartan (an ANG II type I receptor blocker) inhibited both HIV- and ANG II-induced podocyte cathepsin L expression. Furthermore, VD downregulated HIV-induced podocyte CTSL expression. Both losartan and free radical scavengers attenuated HIV- and ANG II-induced podocyte reactive oxygen species (ROS) generation. HIV also led to cytosolic CTSL accumulation through enhancement of podocyte lysosomal membrane permeabilization; on the other hand, VD, losartan, and superoxide dismutase (SOD) attenuated HIV-induced enhanced podocyte cytosolic CTSL accumulation. Morphological evaluation of HIV/CIDHPs revealed sparse actin filaments and attenuated expression of dynamin. Interestingly, podocytes lacking CTSL displayed enhanced dynamin expression, and HIV/CIDHPs expressing CTSL exhibited downregulation of dynamin. These findings indicate that HIV-induced downregulation of podocyte VDR and associated RAS activation and cytosolic CTSL accumulation compromised the actin cytoskeleton.

Deficit of p66ShcA restores redox-sensitive stress response program in cisplatin-induced acute kidney injury.

Overwhelming oxidative stress and compromised tubular cell antioxidant response have been incriminated for cisplatin (Cis)-induced acute kidney injury (AKI). We hypothesized that Cis-induced AKI was the outcome of the deactivated redox-sensitive stress response program (RSSRP). Wild type (WT) and heterozygous p66ShcA(p66(+/-)) mice in groups of six were administered either normal saline (WT) or Cis (12.5 mg/kg, intraperitoneal, Cis/WT). Renal biomarkers were collected and kidneys were harvested for renal histology. Cis/WT showed elevated blood urea nitrogen levels and enhanced tubular cell apoptosis, necrosis, and dilated tubules filled with casts when compared to Cis/p66(+/-). Cis/p66(+/-) developed only a clinically occult AKI (normal blood urea levels and only microscopic alterations). Immunoblots from the lysates of renal tissues of Cis/WT displayed enhanced expression of phospho-p66ShcA, and phospho-Foxo3A but attenuated expression of MnSOD and catalase; conversely, p66 deficit prevented these alterations in Cis milieu. In in vitro studies, Cis treated mouse proximal tubular cells (MPTCs) displayed enhanced phosphorylation of p66ShcA and no increase in tubular cell expression of MnSOD. In addition, renal tissues of Cis/WT and Cis-treated MPTCs displayed enhanced phosphorylation of p53 and Bax expression. However, MPTC partially silenced for p66ShcA displayed partial inhibition of Cis-induced tubular cell apoptosis as well as necrosis. These findings indicate that Cis-induced AKI is the outcome of the deactivated RSSRP (attenuated anti-oxidant response) and activation of pro-apoptotic (p53-induced Bax expression) pathway.

Transplantation of bone marrow-derived MSCs improves cisplatinum-induced renal injury through paracrine mechanisms.

Mesenchymal stem cells (MSCs) have been reported to preserve renal function in various models of acute kidney injury (AKI). Different routes were used to transplant MSCs but the role of cell transplantation routes in directing outcomes has been unknown. In the present study, we evaluated organ bio-distributions of transplanted MSCs, and correlated survival of transplanted cells with outcomes in mice with cisplatinum-induced AKI. We found that after intravenous administration, MSCs were largely localized in pulmonary capillaries and only a minute fraction of MSCs entered kidneys and the cells survived only transiently. Therefore, we also transplanted MSCs via intraperitoneal and renal subcapsular routes. Transplanted MSCs survived longer in peritoneal cavity and renal subcapsular space. Interestingly, when MSC transplantation was followed by cisplatinum-induced AKI, renal morphology and renal functions were better preserved, irrespective of the cell transplantation route. As transplanted MSCs did not migrate to kidneys from either peritoneal cavity or renal subcapsular space, this finding suggested that migration of cells was not required for the beneficial response. The possibility of indirect mechanisms was confirmed when administration of the conditioned medium from MSCs also protected renal tubular cells from cisplatinum-induced cytotoxicity. We identified presence of over forty regulatory cytokines in the conditioned medium obtained from MSCs. Since paracrine factors released by transplanted cells accounted for improvements, it appears that the route of cell transplantation is not critical for realizing benefits of cell therapy with MSCs in AKI. Studies of specific cytokines secreted by MSCs will help to obtain new therapeutic mechanisms for renal protection.

Ethanol and vitamin D receptor in T cell apoptosis.

Ethanol has been demonstrated to cause T cell apoptosis. In the present study, we evaluated the role of VDR and the renin angiotensin system (RAS) in oxidative stress-induced T cell apoptosis. Ethanol-treated human T cells displayed down regulation of vitamin D receptor (VDR) and the activation of the RAS in the form of enhanced T cell renin expression and angiotensin II (Ang II) production. The silencing of VDR with siRNA displayed the activation of the RAS, and activation of the VDR resulted in the down regulation of the RAS. It suggested that ethanol-induced T cell RAS activation was dependent on the VDR status. T cell ROS generation by ethanol was found to be dose dependent. Conversely, ethanol-induced ROS generation was inhibited if VDR was activated or Ang II was blocked by an angiotensin II type 1 (AT1) receptor blocker (Losartan). Furthermore, it was observed that ethanol not only induced double strand breaks in T cells but also attenuated DNA repair response, whereas, VDR activation inhibited ethanol-induced double strand breaks and also enhanced DNA repairs. Since free radical scavengers inhibited ethanol-induced DNA damage, it would indicate that ethanol-induced DNA damage was mediated through ROS generation. These findings indicated that ethanol-induced T cell apoptosis was mediated through ROS generation in response to ethanol-induced down regulation of VDR and associated activation of the RAS.

Rapamycin-induced modulation of HIV gene transcription attenuates progression of HIVAN.

HIV-associated nephropathy (HIVAN) is the manifestation of HIV gene expression by kidney cells in the presence of specific host factors. Recently, rapamycin (sirolimus) has been demonstrated to modulate the progression of HIVAN. We hypothesized that rapamycin would modulate the progression of HIVAN by attenuating HIV gene expression. To test our hypothesis, three weeks old Tg26 mice (n=6) were administered either vehicle or rapamycin (5 mg/kg, every other day, intraperitoneal) for eight weeks. At the end of the experimental period, the kidneys were harvested. In in vitro studies, human podocytes were transduced with either HIV-1 (NL4-3) or empty vector (EV), followed by treatment with either vehicle or rapamycin. Total RNA and proteins were extracted from renal tissues/cellular lysates and HIV gene transcription/translation was measured by real time PCR and Western blotting studies. Renal histological slides were graded for glomerular sclerosis and tubular dilatation with microcyst formation. Rapamycin attenuated both glomerular and tubular lesions in Tg26 mice. Rapamycin decreased transcription of HIV genes both in renal tissues as well as in HIV-1 transduced podocytes. Our data strongly indicate that HIV-1 long terminal repeat-mediated transcriptional activity was targeted by rapamycin. Rapamycin enhanced podocyte NF-κB and CREB activities but then it decreased AP-1 binding activity. Since expression of HIV genes by kidney cells has been demonstrated to be the key factor in the development HIVAN, it appears that rapamycin-induced altered transcription of HIV genes might have partly contributed to its disease modulating effects.