Significant Increase In Proteinuria Research Articles

The Expression of Toll-like Receptors (TLR7 and TLR9) in Class III and Class IV of Recently Diagnosed Lupus Nephritis with 12-Month Follow-Up.

Renal involvement is an important cause of morbidity and mortality in systemic lupus erythematosus (SLE). The present study included patients with recently diagnosed Class III and Class IV lupus nephritis (LN) treated by Rheumatology who, upon the detection of alterations in their kidney function, were referred to Nephrology for the joint management of both medical specialties. The purpose of this study was to compare the plasma expression of Toll-Like Receptor 7 (TLR7) and TLR9 in healthy control (HC) subjects and newly diagnosed Class III and Class IV LN patients with 12-month follow-ups. The plasma expression of TLR7 and TLR9 proteins was determined by the ELISA method. A significant increase in the expression of TLR7 protein was found in Class III LN in the basal determination compared to the expression in the HC (p = 0.002) and at 12 months of follow-up (p = 0.03) vs. HC. The expression of TLR9 showed a behavior opposite to that of TLR7. TLR9 showed decreased protein expression in LN Class III patients' baseline and final measurements. The result was similar in the basal and final determinations of LN Class IV compared to the expression in HC. A significant decrease in SLEDAI -2K was observed at 12 months of follow-up in patients in Class III (p = 0.01) and Class IV (p = 0.0001) of LN. Complement C3 levels improved significantly at 12-month follow-up in Class IV patients (p = 0.0001). Complement C4 levels decreased significantly at 12-month follow-up in LN Class III compared to baseline (p = 0.01). Anti-DNA antibodies decreased significantly at 12 months of follow-up in Class IV LN (p = 0.01). A significant increase in proteinuria was found at 12 months of follow-up in Class III LN, compared to the baseline determination (p = 0.02). In LN Class IV, proteinuria decreased at 12 months of follow-up compared to baseline (p = 0.0001). Albuminuria decreased at 12 months of follow-up in LN Class IV (p = 0.006). Class IV LN, albuminuria also decreased at 12 months of follow-up (p = 0.009). Hematuria persisted in all patients and the glomerular filtration rate did not change. Three Class IV patients died before 12 months of follow-up from various causes. In conclusion, although the rheumatologic data appeared to improve, the renal function data remained inconsistent. Decreased expression of TLR9 and increased expression of TLR7 could be useful in the early diagnosis of Class III and Class IV LN is correct.

Impact of Khat Chewing on Serum Uric Acid and Albuminuria Levels in Yemeni Type II Diabetes Mellitus Patients

Background: Diabetes mellitus is the major cause of end-stage renal disease and is a common endocrine illness defined by chronic hyperglycemia. In addition to diabetes, substance addiction is considered to be a cause of renal issues. The World Health Organization has classed khat (Catha edulis) as an illicit substance. Khat interferes with regular physiological activities, which may have negative health impacts on organs and systems. 
 Objectives: To determine the effect of khat and uric acid on nephropathy in type II diabetes mellitus.
 Materials and Methods: This is an analytical, cross-sectional study that was conducted on 215 males aged 35 to 55 years who had previously been diagnosed with type II diabetes mellitus and were visiting AL- Thawra General Hospital in Ibb City. The diabetic person was corresponded in age and BMI by the control participant. The subjects were divided into two groups. There were 105 people with type II diabetes mellitus (59% chewing Khat and 46% not chewing Khat), 110 people were healthy and did not have type II diabetes (44% of them chewed Khat and 66% did not chew Khat). 
 Results: A significant increase in albuminuria and proteinuria within the normal range in the diabetes mellitus Khat Chewer group compared to the diabetes mellitus Non-Khat Chewer group (p˂0.001). However, no significant differences were seen in the healthy control group.
 Conclusion: Khat chewing has a strong effect on those with type II diabetes and increases the progression of kidney nephropathy. There was an association between khat chewing and higher uric acid levels in both diabetic and non-diabetic patients.

Validation of Townes As Mice As a Model of Chronic Kidney Disease and Venous Thrombosis Associated with Sickle Cell Trait

Individuals with sickle cell trait (SCT) are heterozygous carriers of the sickle beta-globin gene. Systematic analyses of large cohort studies over the past decade have established that SCT is a risk factor for a limited number of complications, most notably chronic kidney disease (CKD) and venous thrombosis (VT). SCT red blood cells (RBCs) can undergo sickling, although the degree and duration of hypoxia required to produce sickling are much greater than in sickle cell disease due to the lower intra-erythrocyte sickle hemoglobin beta (HbS) concentration of 25-45%. Hypoxia-mediated HbS polymerization can be enhanced by cellular dehydration, hyperosmolarity and/or acidosis that may be encountered in the renal inner medulla. Furthermore, prolonged exposure of SCT RBCs to profound hypoxia, such as in the nidus of a venous clot, will also result in sickling. The objective of this study was to determine if Townes AS mice manifest kidney dysfunction analogous to their SCT human counterparts. Furthermore, we evaluated whether VT was more pronounced in a well-described model of inferior vena cava (IVC) stasis. We used humanized male sickle cell trait (AS) and age-matched genetic control (AA) mice (n=7-16). Analysis of urinary renal biomarkers (albuminuria, proteinuria, kidney injury marker 1 (KIM-1), osmolality) and kidney function (transcutaneous measurement of glomerular filtration rate (GFR)) were conducted at 4-week intervals from 8 to 40 weeks of age. VT was induced by complete occlusion of the IVC in AA and AS mice. Sickling of RBCs was determined by histological and electron microscopy analysis of the IVC clot. Senicapoc, an oral Gardos channel inhibitor, was administered twice daily (20mg/kg, bid) for 2 weeks to prevent AS RBC dehydration and subsequent sickling. Human HbS accounts for 30.1% of total Hb expression in AS mice. Consistent with human SCT, peripheral blood counts did not differ from AA controls. Basal plasma thrombin-antithrombin complex levels were also not different between AA and AS mice. We performed a longitudinal study of renal structure and function in AA and AS mice over 40 weeks. At 8 weeks of age, AS mice exhibited no differences in any parameter compared to AA controls. However, AS mice subsequently developed albuminuria and a moderate but statistically significant increase in GFR (Figure 1). At 36 and 40 weeks of age, we observed progressive loss of kidney function demonstrated by statistically significant decreased GFR accompanied by a significant increase in albuminuria in AS mice (Figure 1). Over time, proteinuria gradually increased in AS mice, and had doubled by 40 weeks of age compared to AA controls (4.6 ±1.1 vs. 2.3 ±0.4 mg/24h, p<0.05). Increased levels of urinary KIM-1, a marker of tubular damage, were observed in AS mice throughout the duration of the study, with the highest levels at 16 weeks (228.3 ± 32.6 pg/24h vs. 53.8 ± 8.5 pg/24h, respectively, p˂0.05). Urine osmolality was not different at 8 weeks of age, but progressively declined in AS mice with statistically significant changes vs. controls starting at 24 weeks until the end of study (1946 ±63 vs. 2672 ±135 mOsm/kg at 40 weeks, p=0.002). Histologic analysis confirmed glomerular injury (congestion, glomerulosclerosis) and tubular injury (medullary congestion, interstitial fibrosis) in 40 week old AS mice. Following 24 hours of IVC stasis, thrombus weight was significantly increased (p<0.01) in AS mice (mean±SEM; 29.0 mg +/- 1.7; n=8) compared to AA controls (20.0 mg ± 1.7; n=7). Histologic and electron microscopic evaluation demonstrated that severe hypoxia (pO 2 ≈ 10 mm Hg) present in the nidus of venous thrombi was associated with extensive sickling of RBCs in AS mice. To determine if the enhancement of VT observed in AS mice was mediated by RBC sickling, mice were pretreated with Senicapoc or vehicle. Compared to vehicle-treated AS mice (27.3 mg ± 2.2 n=9) pre-treatment with Senicapoc significantly reduced (p<0.05) clot weight (18.1mg ± 1.1; n=7) to that observed in untreated AA controls. In aggregate, our data demonstrate that Townes AS murine nephropathy closely mimics the renal abnormalities (impaired urinary concentration and albuminuria with later onset loss of GFR) described in humans with SCT. Furthermore, we have established a mouse model of the venous thrombotic complications associated with SCT and have demonstrated that hypoxia-induced sickling of RBCs may contribute to enhanced VT observed in AS mice.

Plasma Lipid and Lipoprotein Concentrations in Pregnancy Induced Hypertension

Background and aim: Variation in serum lipid profile is associated with hypertension. Abnormal lipid profile directly affects the endothelial dysfunction and significantly associated with atherosclerotic cardiovascular diseases. Hypertension is the pregnancy toxaemia associated key feature which is considered to be caused by vasospastic phenomena in placenta, kidney, brain, and uterus. The current study aimed to assess plasma lipid and lipoprotein concentrations in pregnant women with hypertension (PIH). Methods: This study was conducted on 66 (36 PIH cases, 30 control cases) age matched women in the Department of Obstetrics and Gynecology, Moulvi Ameer Shah Memorial Hospital, Peshawar from October 2021 to June 2022. The criteria for preeclampsia were proteinuria, and proteinuria after pregnancy, hypertension, edema, and reversal of hypertension. Subjects' fasting venous blood samples were collected. Plasma was isolated from heparine anticoagulated blood. In a preservative bottle (500 mg/1000 mL urine) of Na2B4O7, urine was collected after 24 hours. Urine and plasma were kept at -80°C until the assay. Total cholesterol levels, HDL-C, and triglyceride were enzymatically measured. The amount of spectrophotometric and malondialdehyde was measured for plasma lipid peroxidation. SPSS version 26 was used for data analysis. Results: There was a significant increase in proteinuria, mean diastolic, and systolic pressure of preeclampsia patients than the control group. Total triglyceride, malondialdehyde (MDA), low density lipoprotein cholesterol (LDL-C), and apolipoprotein B (apo-B) in a study group were significantly higher as compared to control. Whereas the study group had lower high density lipoprotein cholesterol (HDL-C) than that of control group. The total cholesterol concentration, plasma protein, gestational age, apo-A, and hemoglobin had no statistical significance in both groups. Conclusion: The present study suggested that abnormal lipid metabolism, high triglycerides, specifically LDL-C and lipid peroxides, and low HDL-C concentrations, may lead to the vascular dysfunction and preeclampsia is associated with oxidative stress. There are only minor differences in lipoprotein concentrations and serum lipid between PIH women and women with uncomplicated pregnancy. Keywords: Plasma lipid, Lipoprotein concentration, Pregnancy-induced hypertension

Symposium 2-1 - Role of exosomes in placenta-maternal body axis for organ specific damage of preeclampsia

Exosomes are extra-cellular vesicles that are released from many cells, which are taken up by distant organs and alter those cell phenotype. Preeclampsia (PE) is characterized by hypertension and diverse organ damage. However, the underlying causes of specific maternal organ damage remain largely unexplained. We focused on exosomes released by placenta and aimed to investigate whether they have a role in maternal organ damage in PE. We obtained exosomes from placentas of pregnant women with and without PE who gave birth at the University of Tokyo Hospital. Isolated exosomes were administered to pregnant mice from E11.5 to examine their effects on the mother and fetuses. A significant increase in proteinuria was observed in pregnant mice treated with exosomes derived from placenta of PE patients (PE group) compared to those of normal pregnancies (normal group). Whereas, blood pressure and the fetus size and numbers were not affected. This phenomenon was due to the selective uptake of PE exosomes in kidney, which was mediated by surface molecules on the exosomes. Gene expression analysis of kidney in PE group showed pathway changes associated with potential damage in kidney function. Moreover, pathological analysis revealed glomerular endotheliosis, which is a characteristic of PE. In other organs, pathological changes that could be related to the pathogenesis of PE were observed in the brain and liver in the PE group. Our results indicate the existence of an exosome-mediated organ-specific connection between placenta and maternal organs, which may explain organ-selective damages in PE patients.

MO178: Renal Function Impairment in Diabetic Patients after Intravitreal Injection of Anti-Vascular endothelial Growth Factor

Abstract BACKGROUND AND AIMS Intravitreal administration of vascular endothelial growth factor (VEGF) inhibitors is the treatment of a wide variety of retinal diseases. Kidney damage caused by systemic administration is widely known. However, there is not enough information in the literature on the renal effect of these drugs after intravitreal injection. The aim of the study is to analyse the effect of intravitreal anti-VEGF on kidney function in diabetic patients. METHOD A prospective cohort study of diabetic patients with and without chronic kidney disease (CKD) who received intravitreal anti-VEGF from January 2018 to December 2019. Clinical and analytical parameters were analysed. The follow-up time was 24 months. RESULTS We included 45 patients (55.6% male) with a mean age of 74.4 ± 11.5 (50–91) years. Approximately 64.4% of patients had CKD [estimated glomerular filtration rate (eGFR) <60 mL/min]. The types of anti-VEGF used were: bevacizumab 57.8% and ranibizumab 42.2%. The average number of doses administered was 7.6 ± 4.8 (1–22). The initial eGFR was 48.7 ± 25.3 mL/min and the Alb/Cr ratio 145 (49.45) mg/g. A significant decrease in the eGFR was observed at 6, 12 and 24 months (Table 1). There was a significant increase in proteinuria at 12 and 24 months. This drop in eGFR was independent of the presence of CKD, the anti-VEGF type or the number of doses. After the admistration of the first dose, five patients (17.2%) in the CKD group required renal replacement therapy (mean time 22 ± 12 months). CONCLUSION A significant decrease in eGFR and an increase in proteinuria are observed after administration of anti-VEGF in diabetic patients. For this reason, a close monitoring of renal function is needed to establish an early diagnosis and management of possible complications.

(Pro)renin receptor antagonist PRO20 attenuates nephrectomy-induced nephropathy in rats via inhibition of intrarenal RAS and Wnt/β-catenin signaling.

Introduction(Pro)renin receptor has emerged as a new member of the renin‐angiotensin system implicated in the pathogenesis of chronic kidney disease (CKD). Herein we report characterization of the therapeutic potential of (pro)renin receptor (PRR) antagonist PRO20 in 5/6 nephrectomy (5/6Nx) rats.MethodsMale Wistar rats underwent 5/6Nx followed by treatment with vehicle or received daily injections of a PRR inhibitor PRO20 (700 μg/kg) via the 3 s.c. Sham group served as a control.ResultsAs compared with the sham control, the 5/6Nx rats exhibited significant increases in proteinuria, glomerulosclerosis, tubular injury, and interstitial inflammation in the remnant kidneys. Treatment with PRO20 significantly attenuated these abnormalities, as evidenced by reduced expression of fibronectin, α‐SMA, collagen 1, TGF‐β1, IL‐6, IL‐8, IL‐1β, MCP‐1 and increased expression of E‐cadherin. Increased urinary/renal levels of renin activity, angiotensinogen (AGT), and Angiotensin II (Ang II) by 5/6Nx, which were all ameliorated by PRO20. Renal PRR, the secreted proteolytic fragment of PRR (sPRR) in renal and urinary, were all elevated in 5/6Nx rats. Moreover, our results revealed that renal Wnt3A and β‐catenin expression were upregulated during 5/6Nx, which were all attenuated by PRO20.ConclusionsOverall we conclude that in vivo antagonism of PRR with PRO20 will improve 5/6Nx‐induced CKD mainly through inhibition of intrarenal RAS and Wnt/β‐catenin signaling pathway.

Differentiating Acute Rejection From Preeclampsia After Kidney Transplantation.

To evaluate the clinical and laboratory characteristics in pregnancy that differentiate preeclampsia from acute renal allograft rejection and to investigate the maternal, neonatal, and graft sequelae of these diagnoses. We conducted a retrospective case-controlled registry study of data abstracted from Transplant Pregnancy Registry International deliveries between 1968 and 2019. All adult kidney transplant recipients with singleton pregnancies of at least 20 weeks of gestation were included. Acute rejection was biopsy proven and preeclampsia was diagnosed based on contemporary criteria. Variables were compared using χ2, Fisher exact, and Wilcoxon rank sum tests as appropriate. Multivariable linear regression was used to analyze preterm birth. Kaplan-Meier curves with log-rank test and Cox proportional hazards model were used to compare graft loss over time. There were 26 pregnant women with biopsy-confirmed acute rejection who were matched by the year they conceived to 78 pregnant women with preeclampsia. Recipients with acute rejection had elevated peripartum serum creatinine levels (73% vs 14%, P<.001), with median intrapartum creatinine of 3.90 compared with 1.15 mg/dL (P<.001). Conversely, only patients with preeclampsia had a significant increase in proteinuria from baseline. Although there were no significant differences in maternal outcomes, graft loss within 2 years postpartum (42% vs 10%) and long-term graft loss (73% vs 35%) were significantly increased in recipients who experienced acute rejection (P<.001 for both). The frequency of delivery before 32 weeks of gestation was 53% with acute rejection and 20% with preeclampsia. After controlling for hypertension and immunosuppressant use, acute rejection was associated with higher frequency of delivery at less than 32 weeks of gestation (adjusted odds ratio 4.04, 95% CI 1.10-15.2). In pregnancy, acute rejection is associated with higher creatinine levels, and preeclampsia is associated with increased proteinuria. Acute rejection in pregnancy carries a risk of prematurity and graft loss beyond that of preeclampsia for kidney transplant recipients. The Transplant Pregnancy Registry International is supported in part by an educational grant from Veloxis Pharmaceuticals.

Long-Term but Not Short-Term Maternal Fasting Reduces Nephron Number and Alters the Glomerular Filtration Barrier in Rat Offspring.

The present study examined the effects of maternal Ramadan-type fasting during selected days in the first, second, or third trimester, or during the entire pregnancy, on the kidney structure of male rat offspring. Pregnant rats were provided with food ad libitum during pregnancy (control group, C), or they were exposed to 16 h of fasting/day for three consecutive days in the middle of the first (FT1), second (FT2), or third trimester (FT3), or during whole pregnancy (FWP). Our results showed that dams in the FWP group demonstrated lower food intake and body weight during gestation. Litter size was unaltered by fasting in all groups; however, litter weight was significantly reduced only in the FWP group. Nephron number was decreased in the FWP group, but it remained unchanged in the other fasting groups. The ultrastructure of the glomerular filtration barrier indicated that the kidneys of offspring of the FWP group demonstrated wider diameters of fenestrations and filtration slits and smaller diameters of basement membranes. This was reflected by a significant increase in proteinuria in FWP only. These results suggest that, unlike with short-term fasting, which seems to be safe, maternal long-term fasting induces structural changes that were non-reversible, and that may contribute to impaired renal function, leading to chronic diseases in later life.

Proteinuria and Bilirubinuria as Potential Risk Indicators of Acute Kidney Injury during Running in Outpatient Settings.

Background and objectives: The purpose of this study was to explore which urinary markers could indicate acute kidney injury (AKI) during prolonged trail running in outpatient settings. Materials and Methods: Twenty-nine experienced trail runners (age 39.1 ± 8.8 years, weight 71.9 ± 11 kg, height 171.9 ± 8.3 cm) completed a 35 km event (cumulative positive ascend of 1815 m, altitude = 906 to 1178 m.a.s.l.) under a temperature of 25.52 ± 1.98 °C and humidity of 79.25 ± 7.45%). Two participant groups (AKI = 17 and No-AKI = 12) were made according to AKI diagnosis criteria based on pre- and post-race values of serum creatinine (sCr) (an increase of 1.5 times from baseline). Blood and urinalysis were performed immediately pre- and post-race. Results: Pre- vs. post-race differences in sCr and sBUN were found in both AKI and No-AKI groups (p < 0.01). Differences in post-race values were found between groups (p = 0.03). A total of 52% of AKI runners presented significant increases in proteinuria (χ2 = 0.94, p = 0.01) and 47% in bilirubinuria (χ2 = 0.94, p = 0.04). Conversely, No-AKI participants presented no significant increases in urine markers. Conclusions: These study’s findings may suggest the potential use of urinalysis as an accessible alternative in the outpatient setting to early identify transitional AKI until a clinical confirmation is performed.

Cardio-renal protective effect of the xanthine oxidase inhibitor febuxostat in the 5/6 nephrectomy model with hyperuricemia

Although hyperuricemia has been shown to be associated with the progression of cardiovascular disorder and chronic kidney disease (CKD), there is conflicting evidence as to whether xanthine oxidase (XO) inhibitors confer organ protection besides lowering serum urate levels. In this study, we addressed the cardio-renal effects of XO inhibition in rodent CKD model with hyperuricemia. Sprague-Dawley rats underwent 5/6 nephrectomy and received a uricase inhibitor oxonic acid for 8 weeks (RK + HUA rats). In some rats, a XO inhibitor febuxostat was administered orally. Compared with control group, RK + HUA group showed a significant increase in albuminuria and renal injury. Febuxostat reduced serum uric acid as well as urinary albumin levels. Histological and immunohistochemical analysis of the kidney revealed that febuxostat alleviated glomerular, tubulointerstitial, and arteriolar injury in RK + HUA rats. Moreover, in the heart, RK + HUA showed individual myofiber hypertrophy and cardiac fibrosis, which was significantly attenuated by febuxostat. We found that renal injury and the indices of cardiac changes were well correlated, confirming the cardio-renal interaction in this model. Finally, NF-E2-related factor 2 (Nrf2) and the downstream target heme oxygenase-1 (HO-1) protein levels were increased both in the heart and in the kidney in RK + HUA rats, and these changes were alleviated by febuxostat, suggesting that tissue oxidative stress burden was attenuated by the treatment. These data demonstrate that febuxostat protects against cardiac and renal injury in RK + HUA rats, and underscore the pathological importance of XO in the cardio-renal interaction.

P0046ZHX2 DOWNREGULATION DUE INSERTIONS AND DELETIONS IN THE HAS2-ZHX2 INTERGENIC REGION PREDISPOSE TO PODOCYTE DISEASE RELAPSE

Abstract Background and Aims ZHX2 transcriptional factor is normally found in podocyte membrane forming complexes with ZHX1 and APA or ZHX3 and Ephrin B1. Altered ZHX2 expression disrupts these interactions and is related with the worsening of different primary glomerular diseases such as focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD). A small percentage of Hodgkin lymphoma (HL) patients develop nephrotic syndrome (NS). Studies of Reed-Sternberg cell line L-1236 reveals a chromosomal rearrangement that leads to ZHX2 downregulation suggesting a possible role of ZHX2 in the development of NS in these patients. Human podocyte disease relapse is common after a common cold probably mediated by cytokines released by immune cells and the rhinovirus. Our aim is to study how the presence of insertions and deletions (InDels) between HAS2 and ZHX2 in patients with MCD, FSGS and HL could alter ZHX2 expression and the implication in podocyte disease relapse after a common cold and in HL. Method Genomic DNA between HAS2 and ZHX2 (Chr8:122624000-124001000 from UCSC hg19 GRCh37) was sequenced from 28 NS patients and 27 controls using Agilent Custom capture and high throughput Illumina sequencing. The CLC Genomics software was used to identify InDels (3-20 bp) present exclusively in patients. One of the identified InDels was replicated in human podocytes using CRISPR Cas9 and homology directed repair technology to study changes in ZHX2. A common cold cytokine cocktail containing IL-2, IL-4R, IL-6, IL-10, INF-γ, TNF-α and ICAM-1 was injected into control (BALB/c, n=5) and ZHX2 deficient mice (BALB/cJ, n=5) (Dose X); podocyte specific ZHX2 deficient (ZHX2 flox/flox cre+/+, n=3) and floxed control mice (ZHX2 flox/flox, n=3) (dose X/15). Buffalo Mna (B. Mna) rats were also injected with the cytokine cocktail (X/50) to study relapse in FSGS (n=7). Cell supernatant from HL Reed Sternberg cells (L-1236) (200µg of total protein) was injected into BALB/c (n=5) and BALB/cJ mice (n=5) and kidney function was assessed. Results Multiple InDels were found exclusively in the patient population, two of them, shared by two or more patients. The insertion at 122,533,694 was presented in patients with MCD, FSGS and HL with NS and also in L-1236 cells. This insertion was replicated in human podocytes using CRISPR/Cas9 (CRISPR B). Another insertion noted both in patients and controls was generated for comparison (CRISPR A). A reduced ZHX2 expression was detected in CRISPR B but not in CRISPR A single cell clones. The shared insertion at 122,787,088 was presented within the ZHX2 gene intron 1. BALB/cJ mice has lower ZHX2 expression in liver and podocytes due to an insertion in intron 1. To study whether this insertion could be related with relapse of MCD and FSGS following a common cold, a cytokine cocktail was injected into BALB/cJ and BALB/c mice. BALB/cJ mice developed acute albuminuria after cytokine treatment (65.3±24.3 μg per 18h), but not control BALB/c mice (10.8±1.5 μg per 18h), when compared with baseline values (BALB/cJ 5.1±1.1 μg per 18h; BALB/c 6.5±1.1 μg per 18h) (p&amp;lt;0.05). BALB/cJ mice had also higher nuclear expression of ZHX1. The cytokine cocktail also induced albuminuria in ZHX2 flox/flox/cre+/+ mice but not in control ZHX2 flox/flox, suggesting that ZHX2 deficiency in podocytes is responsible of kidney injury after cytokine injection. To study common cold related relapse in FSGS, B. Mna rats were injected with a rat cytokine cocktail, showing a significant increase in proteinuria (61.5±4.2 mg per 18h) compared with baseline (47±4.1 mg per 18h). Cell culture supernatant from L-1236 was injected into BALB/cJ and BALB/c mice to study the effect of secreted soluble mediators in NS. L-1236 supernatant (200 µg) had a nephrogenic effect in ZHX2 deficient mice but not in controls. Conclusion InDels between HAS2 and ZHX2 genes presented in patients with MCD, FSGS and HL, alter ZHX2 expression and are related to relapse following a common cold and in HL.

Knockout of Per1 Exacerbates the Hypertensive Phenotype of the Dahl Salt Sensitive Rat

The dynamic between blood pressure, its circadian rhythm, and the circadian clock genes has been considered as a factor in the subsequent renal injury that accompanies hypertension. Various circadian clock proteins such as BMAL1 and PERIOD (PER) function in a marriage of positive and negative feedback loops that control transcriptional regulation. Of particular interest is PER1 which naturally acts in the negative loop of the cycle as a repressor, but recent findings show that it may have additional roles as a transcription activator. PER1 has a role in renal sodium handling as a target of aldosterone, and it has been linked to the expression of the α‐subunit of the epithelial sodium channel (α‐ENaC). Investigators have found that knockout of PER1 in 129/sv mice causes a decrease in mean arterial pressure (MAP); however, when the gene is knocked out in the C57BL/6J mouse on a 4% NaCl diet (high salt, HS) with deoxycorticosterone pivalate (DOCP) treatment, there is a significant increase in MAP with animals displaying a “non‐dipping” pattern of MAP. The wildtype mice that were on HS with DOCP treatment had a 11.4 ± 1.9% decrease in MAP from night to day whereas the knockout mice with the same conditions had only a 5.7 ± 1.4% decrease. To test the contribution of PER1 in the development of salt‐induced hypertension, we created the Per1 knockout rat on the Dahl Salt‐Sensitive rat background (SS; knock out SSPer1−/−). Eight to 9‐week old SS and SSPer1−/− male rats were implanted with DSI telemetry units to measure blood pressure on a 4% NaCl diet (high salt, HS) for 3 weeks. MAP in SSPer1−/− rats was significantly higher compared to wild type SS rats (191 ± 7 vs. 168 ± 4 mmHg, P = 0.031; respectively) after 3 weeks of HS. We also revealed a significant increase in albuminuria in SSPer1−/− rats on HS compared to SS rats on HS (0.10 ± 0.01 vs. 0.05 ± 0.01 Alb/Cre Clearance ratio, P = 0.027; respectively). Knockout of Per1 in SS rats on HS also resulted in a significant increase in cholesterol (200.0 ± 11.8 vs. 135.0 ± 9.7 mg/Dl, P = 0.003; respectively) and alkaline phosphatase (37.5 ± 3.6 vs. 77.6 ± 3.3 U/L, P = &lt;0.001; respectively). Furthermore, SSPer1−/− rats after 3 weeks of HS diet had significant decreases in creatinine clearance compared to SS rats (65 ± 6 vs. 127 ± 24 mL/hour, P = 0.036; respectively). Here, we show that lack of PER1 causes a dramatization of the salt‐sensitive phenotype of the SS rat with a decline in renal function. These data propose that the role PER1 plays in sodium homeostasis in the kidney is pivotal for proper blood pressure regulation.Support or Funding InformationNational Institutes of Health Grants R35 HL135749 (AS) and R01 DK109570 (MLG) and by the American Heart Association 18PRE34030127 (DS)

Changes in proteinuria and diagnosing preeclampsia in CKD pregnancy.

The diagnosis of preeclampsia superimposed on underlying kidney disease is complicated by physiological changes in renal function, blood pressure and proteinuria during pregnancy. Previous studies have demonstrated a significant increase in proteinuria during pregnancy in women with diabetic nephropathy in the absence of superimposed preeclampsia. There is a paucity of studies addressing changes in proteinuria during pregnancy in other conditions causing chronic kidney disease. A retrospective audit of changes in urine protein:creatinine ratios in 68 pregnancies to women with preconception proteinuria was performed, to determine whether changes in proteinuria may be useful in diagnosing preeclampsia. A 100% or greater increase in proteinuria from baseline increase was regarded as a significant change. In 51 pregnancies (75%) the peak urine protein:creatinine ratio did not rise significantly from values at the start of pregnancy. Where a greater than doubling of urine protein:creatinine ratio was seen, 12 of 17 pregnancies (70%) were judged clinically to have superimposed preeclampsia A significant increase in proteinuria also occurred in 3 women with lupus nephritis, and 2 with diabetic nephropathy, in the absence of preeclampsia. Larger studies of women with chronic kidney disease would be important to determine whether changes in proteinuria during pregnancy may be of diagnostic value for superimposed preeclampsia.

NOX2-derived reactive oxygen species in immune cells exacerbates salt-sensitive hypertension

Previous studies utilizing the SSp67phox−/− rat have demonstrated the importance of systemic NADPH oxidase NOX2-derived reactive oxygen species (ROS) production in the pathogenesis of Dahl Salt-Sensitive (SS) hypertension and renal damage. It is established that the immune system contributes to the development of SS hypertension and our laboratory has observed an enrichment of NOX2 subunits in infiltrating T cells. However, the contribution of immune cell-derived ROS in SS hypertension remains unknown. To assess the role of ROS in immune cells, SSp67phox−/− rats underwent total body irradiation and received bone marrow transfer from either SS (+SS) or SSp67phox−/− (+SSp67phox−/−) donor rats. Demonstrated in a respiratory burst assay, response to phorbol 12-myristate 13-acetate stimulus (135 μM) was 10.2-fold greater in peritoneal macrophages isolated from +SS rats compared to nonresponsive + SSp67phox−/− cells, validating that + SS rats were capable of producing NOX2-derived ROS in cells of hematopoietic origin. After 3 weeks of high salt challenge, there was an exacerbated increase in mean arterial pressure in +SS rats compared to + SSp67phox−/− control rats (176.1 ± 4.7 vs 147.9 ± 8.4 mmHg, respectively), which was accompanied by a significant increase in albuminuria (168.3 ± 23.7 vs 107.0 ± 20.4 mg/day) and renal medullary protein cast formation (33.2 ± 4.7 vs 8.1 ± 3.5%). Interestingly, upon analysis of renal immune cells, there was trending increase of CD11b/c + monocytes and macrophages in the kidney of +SS rats (4.7 ± 0.4 vs 3.5 ± 0.5 × 106 cells/kidney, +SS vs + SSp67phox−/−, p = 0.06). These data altogether demonstrate that immune cell production of NOX2-derived ROS is sufficient to exacerbate Dahl SS hypertension, renal damage, and renal inflammation.

Gebe sıçanlara bevacizumab uygulayarak preeklampsi çalışmalarında kullanılabilecek deneysel hayvan modeli oluşturulabilir.

INTRODUCTION: To develop a rat model of preeclampsia by administering bevacizumab, an angiogenesis inhibitor. METHODS: Sixteen pregnant rats were randomly allocated to intraperitoneal injection of 10 mg/kg bevacizumab or 0.1 cc intraperitoneal serum physiologic on the 4th and 8th days of gestation. Blood pressure, body weight, and proteinuria were measured on both day 0 (D0) and day 20 (D20). Blood samples were collected on D20 for analysis, including for determining vascular endothelial growth factor (VEGF) and soluble Fms-like tyrosine kinase 1 (sFlt-1) levels. On the same day, the mice were euthanized, the placentas and pups were weighted, and the angiogenesis markers and microvessel density were evaluated using immunohistochemical methods. RESULTS: Lower serum VEGF (p = 0.038) and higher SFlt-1 (p = 0.015) levels were observed in bevacizumab-treated pregnant rats. Bevacizumab-treated pregnant rats had significantly higher systolic (p = 0.050) and diastolic (p = 0.046) blood pressures compared to the controls. Additionally, the bevacizumab group showed a significant increase in proteinuria on D20 compared to that on D0 (p = 0.026). Although higher serum AST, ALT, BUN, and creatinine levels and renal glomerular endotheliosis scores as well as lower placental VEGF and microvessel density were noted in bevacizumab-treated rats, these differences were not statistically significant (p > 0.05 for each). DISCUSSION AND CONCLUSION: The promising results of this trial show that bevacizumab treatment in pregnant rats might provide a model to study human preeclampsia

Abstract 123: Effect of Immune Cell-Derived Reactive Oxygen Species on Dahl SS Hypertension

Previous studies utilizing the SS p67 phox–/– rat have demonstrated the importance of systemic NADPH oxidase NOX2-derived reactive oxygen species (ROS) production in the pathogenesis of Dahl Salt-Sensitive (SS) hypertension and renal damage. Our laboratory has established a key role for renal immune cell infiltration in the development of SS hypertension and observed an enrichment of NOX2 subunits in infiltrating T cells. The contribution of ROS specifically derived from immune cells in SS hypertension remains unknown. To assess the role of ROS in immune cells, SS p67 phox–/– rats underwent total body irradiation and received bone marrow transfer from either SS p67 phox–/– (p67/p67) or SS (p67/SS) donor rats. p67/SS rats would be capable of producing NOX2-derived ROS only in the transferred SS cells of hematopoietic origin while the p67/p67 control group would be unable to produce NOX2-derived ROS globally. Mean arterial pressure (MAP) was not different between the p67/SS and p67/p67 rats when fed a 0.4% NaCl diet (112.6±1.4 vs 110.7±2.4 mmHg, n=4-5). After 3 weeks of high salt, however, there was a significantly greater increase in MAP in the p67/SS rats (176.1±4.7 mmHg) compared to the p67/p67 control rats (147.9±8.4 mmHg), which was accompanied by a significant increase in albuminuria (230.3±13.9 vs 153.1±14.9 mg/day, p67/SS vs p67/p67, n=5-6). Interestingly, upon analysis of the immune cells in the kidney, no differences were detected in the number of CD45+ total leukocytes, CD11b/c+ monocytes/macrophages, CD3+ T cells, and CD45R+ B cells between the p67/SS and p67/p67 (n=4-5), indicating that the recruitment of immune cells into the kidney during hypertension and renal damage is independent of NOX2-derived ROS production from the renal parenchyma. The respiratory burst response to phorbol 12-myristate 13-acetate stimulus (135 μM) in peritoneal macrophages isolated from p67/SS was 4.0-fold greater compared to nonresponsive p67/p67 cells, and was completely inhibited by superoxide dismutase, demonstrating that the reconstituted bone marrow cells in the p67/SS are capable of producing superoxide. In summary, the production of NOX2-derived ROS from immune cells alone is sufficient for the development of Dahl SS hypertension and renal damage.

Angiotensin II responsiveness after preeclampsia: translational data from an experimental rat model and early-onset human preeclampsia.

Formerly preeclamptic women have an increased risk for cardiovascular and renal disease later in life. It is unknown which mechanisms contribute to this increased risk and whether this is induced by preeclampsia or by prepregnancy factors. We hypothesized that the increased risk for cardiovascular disease is partly due to an increased angiotensin II (ang II) responsiveness postpartum and that preeclampsia itself is involved in inducing this increased ang II responsiveness. In never-pregnant, formerly healthy pregnant rats and rats with former experimental preeclampsia [experimental preeclampsia model induced by low-dose endotoxin infusion on day 14 of pregnancy; endotoxin-infused pregnant rats (EP-rats)], ang II responsiveness was studied by measuring changes in blood pressure (BP) and proteinuria after chronic ang II infusion with osmotic minipumps (200 ng/kg per min). In addition, we measured BP and responses to ang II (0.3, 1.0 and 3.0 ng/kg per min) in 18 formerly early-onset preeclamptic, without comorbidities, and 18 formerly healthy pregnant women (controls). In rats, a significantly higher systolic BP at termination was observed in formerly EP-rats vs. never-pregnant rats after ang II infusion (159.5 ± 29.5 vs. 136.7 ± 16.8; P = 0.049). In response to ang II, there was a significant increase in proteinuria in formerly EP-rats vs. healthy pregnant and never-pregnant rats (P < 0.01 for both). In humans, 1.0 ng/kg per min ang II showed a trend towards an increased mean arterial BP response in formerly preeclamptic women vs. controls (P = 0.057). Our data show an increased ang II responsiveness following (experimental) preeclampsia and support a role for preeclampsia itself in altered ang II responsiveness postpartum.

Evaluation of inducible nitric oxide synthase inhibition on kidney function and structure in high-fat diet-induced kidney disease.

What is the central question of this study? The metabolic pathways regulating the effects of obesity on the kidney remain unknown. We sought to determine whether inducible nitric oxide synthase (iNOS) is involved in the underlying mechanisms of high-fat diet-induced kidney disease using a specific iNOS inhibitor, N6-(1-iminoethyl)-l-lysine hydrochloride (L-NIL). What is the main finding and its importance? We did not demonstrate an upregulation of iNOS renal expression after high caloric intake, suggesting that iNOS might not be a crucial player in the development of obesity-induced kidney disease. Although L-NIL treatment clearly ameliorated systemic metabolic parameters, the effect on loss of renal function, impairment of tubular integrity, oxidative stress and inflammation appeared to be more moderate. Central obesity is related to caloric excess, promoting deleterious cellular responses in targeted organs. Nitric oxide (NO) has been determined as a key player in the pathogenesis of metabolic diseases. Here, we investigated the implication of inducible NO synthase (iNOS) in the development of obesity-induced kidney disease. C57Bl/6male mice were randomized to a low-fat diet (LFD) or a high-fat diet (HFD) and treated with N6-(1-iminoethyl)-l-lysine hydrochloride (L-NIL), a specific iNOS inhibitor, for 16weeks. Mice fed an HFD exhibited a significant increase in body weight, fasting blood glucose and plasma concentrations of non-esterified fatty acids, triglyceride and insulin. Inhibition of iNOS prevented these changes in mice fed an HFD. Interestingly, the significant increase in albuminuria and mesangial matrix expansion were not ameliorated with L-NIL, whereas a significant decrease in proteinuria, N-acetyl-β-d-glucosaminidase excretion and renal triglyceride content were found, suggesting that iNOS inhibition is more suitable for tubular function than glomerular function. The urinary concentration of hydrogen peroxide, a stable product of reactive oxygen species production, that was found to be increased in mice fed an HFD, was significantly reduced with L-NIL. Finally, despite a moderate effect of L-NIL on inflammatory processes in the kidney, we demonstrated a positive impact of this treatment on adipocyte hypertrophy and on adipose tissue inflammation. These results suggest that inhibition of iNOS leads to a moderate beneficial effect on kidney function in mice fed an HFD. Further studies are needed for better understanding of the role of iNOS in obesity-induced kidney disease.

Abstract P268: Overexpression of miR-210 Induces Preeclampsia-like Symptoms in Pregnant Mice by Attenuating the STAT6/IL-4 Pathway

Preeclampsia (PE) is a pregnancy-related hypertensive disorder that affects 5-8% of all pregnancies worldwide. Placental microRNA (miRNA) expression is known to be dysregulated during PE which includes miR-210. However, the role of miR-210 in the development of PE is still unknown. We have previously demonstrated STAT6, a transcription factor, to be a direct target of miR-210. In addition, STAT6 modulates levels of IL-4, an anti-inflammatory cytokine which is known to be beneficial during pregnancy. Given that the STAT6/IL-4 pathway is important for normal pregnancy, we hypothesized that an overexpression of miR-210 in mice will contribute to PE-like symptoms by inhibiting the STAT6/IL-4 pathway. Systolic blood pressures (SBP) were measured using the tail-cuff method on both WT (C57Bl/6J) and miR-210 TG mice. The miR-210 TG mice exhibited increased SBP compared to the WT mice (WT: 95±2 mmHg, miR-210 TG: 115±2.4 mmHg, p&lt;0.05). The miR-210 TG mice also displayed endothelial dysfunction measured by aortic vascular reactivity using wire myography (WT: 93% miR-210 TG: 68% relaxation, p&lt;0.05 vs. WT) and a significant increase in proteinuria (4 fold, p&lt;0.05 vs. WT). Furthermore, the miR-210 TG mice exhibit placental necrosis and a significant increase in the number of malformed pups. Placental STAT6 levels decreased in miR-210 TG compared to WT mice (2.6 fold, p&lt;0.05 vs. WT) as determined by immunoblotting. In addition, qRT-PCR analysis of the miR-210 TG placentas exhibited a (4.8 fold, p&lt;0.05 vs. WT) decrease in IL-4 expression. Immunoblotting and immunohistochemistry (IHC) studies showed a decrease in IL-4 levels (2.4 fold, p&lt;0.05 vs. WT) and immunoreactivity in the miR-210 TG placentas as well as. IHC studies in human placentas also showed a decrease in both IL-4 and STAT6. Based on our results, miR-210 overexpression induces PE-like symptoms including hypertension, endothelial dysfunction, proteinuria, and malformed pups in pregnant mice. In addition, miR-210 overexpression attenuated the STAT6/IL-4 anti-inflammatory pathway. Our data suggests that either targeting miR-210 or the STAT6/1L-4 pathway could be a potential therapeutic in mitigating the symptoms of PE.