Detection of Podocin in Human Urine Sediment Samples by Charge Derivatization and LC-MS-MRM Method.

Detection of podocytes in urine might serve as a useful diagnostic tool in both primary and secondary glomerular diseases. The utility of podocyturia has been confirmed for both pre-eclampsia and glomerulonephritis. Here, we present a new and sensitive method for qualitative LC-MS-multiple-reaction-monitoring (MRM) analysis of podocin, serving as a podocyturia biomarker in urine sediments. The following podocin tryptic peptides with the 169LQTLEIPFHEIVTK182, 213AVQFLVQTTMK223, 240SIAQDAK246, and 292MIAAEAEK299 sequences were applied as a model. The selective chemical derivatization of the ε amino group of C-terminal lysine residue in tryptic peptides, by 2,4,6-triphenylpyrylium salt (TPP) as a fixed charge tag, was employed to increase the ionization efficiency, in routine ESI-MS analysis. Additionally, the generation of a reporter ion, in the form of a protonated 2,4,6-triphenylpyridinium cation, makes the derivatized peptide analysis in the MRM mode unambiguous. Identification of derivatized and non-derivatized peptides were performed, and the obtained results suggest that the peptide with the 292MIAAEAEK299 sequence may serve as a marker of podocyturia.

Tetramethylpyrazine reduces the consequences of nitric oxide inhibition in pregnant rats.

Pre-eclampsia (PE) is closely associated with perinatal morbidity and mortality and we want to investigate tetramethylpyrazine (TMP)'s effects on PE. Pregnant Sprague-Dawley rats were randomly divided into five groups: normal pregnant (PC), PE, PE+TMP 20 mg/kg, PE+TMP 40 mg/kg, and PE+TMP 60 mg/kg group. The PE rat model was established via L-NAME treatment. Systolic blood pressures (SBP) and urinary protein concentration were detected via the tail-cuff method and CBB kit, respectively. mRNA levels of key genes were analyzed via quantitative PCR and protein levels of key genes were measured by ELISA or western blot. TMP decreased SBP and urinary protein concentration of PE rats. TMP inhibited L-NAME-induced decrease in pups alive ratio, pups weight, and the ratio of pups/placenta weight and reversed L-NAME induced changes in placental histology, whereas it had little effect on placental weight. Urinary nephrin and podocin expressions were enhanced and serum placental growth factor level was decreased in PE rats, whereas TMP inhibited the above phenomena. TMP suppressed L-NAME-induced sFlt-1 upregulation in serums and kidneys of PE rats, whereas it downregulated IL-6 and MCP-1 expression in PE rats' serums, placentas and kidneys. TMP also suppressed the increase in placental sFlt-1 and vascular endothelial growth factor level caused by L-NAME. In addition, TMP inhibited CHOP and GRP78 expressions and decreased the ratio of p-elF2α/elF2α in PE rats. TMP attenuated the consequences of NO inhibition in pregnant rats.

Immunoglobulin Binding Protein 1 as a Potential Urine Biomarker in Patients with Lupus Nephritis.

We evaluated the role of immunoglobulin binding protein 1 (IGBP1), a phosphoprotein associated with the B cell receptor (BCR) complex, as a urine biomarker in lupus nephritis (LN). The IGBP1 concentrations in plasma and urine of patients with LN, systemic lupus erythematosus (SLE) without nephritis and healthy controls were estimated by ELISA. IGBP1 expression in the kidneys of LN patients and transplantation donors was detected by immunohistochemistry. Microarray-based global gene expression profile of HK-2 cells with IGBP1 knock-down and fluorescence-activated cell sorting (FACS) for intracellular IGBP1 expression in human peripheral blood mononuclear cells (PBMCs) was performed. Urine IGBP1 levels were elevated significantly in LN patients, and it correlated with the clinical activity indices (complement 3 (C3) level, anti-dsDNA antibodies titer, SLE Disease Activity Index-2000 (SLEDAI-2K) and histological activity index. IGBP1 expression was increased in LN patients as compared to the donors and was detected mainly in the tubules by histopathology. In microarray analysis, several genes related to SLE pathogenesis (PPME1, ROCK2, VTCN1, IL-17R, NEU1, HLA-DM, and PTX3) responded to siRNA-mediated IGBP1 silencing. In FACS, IGBP1 was expressed mainly in the CD14+ cells. The overall expression of IGBP1 in PBMCs was higher in LN patients as compared with that in SLE patients without nephritis. Conclusively, urinary IGBP1 may be a novel biomarker reflecting the clinical and histological activities in LN.

Evaluation of Tryptic Podocin Peptide in Urine Sediment Using LC-MS-MRM Method as a Potential Biomarker of Glomerular Injury in Dogs with Clinical Signs of Renal and Cardiac Disorders.

The early asymptomatic stage of glomerular injury is a diagnostic challenge in the course of renal and extra-renal disease, e.g., heart insufficiency. It was found that podocin, a podocyte-specific protein present in the urine, may serve as a biomarker in the diagnosis of glomerular disease in humans and animals including glomerulonephritis, glomerulosclerosis, amyloidosis, or nephropathy. Therefore, there is a need of development of the sensitive and straightforward method of urinary podocin identification. In this work, we report our extended research under the glomerular injury investigation in dogs by application of clinical examination and LC-MS-MRM method in the identification of canine podocin in urine samples. The LC-MS-MRM method is based on the identification of podocin tryptic peptide with the 218H-AAEILAATPAAVQLR-OH232 sequence. The model peptide was characterized by the highest ionization efficiency of all the proposed model podocin tryptic peptides in a canine urine sediment according to the LC-MS/MS analysis. The obtained results revealed the presence of the model peptide in 40.9% of dogs with MMVD (active glomerular injury secondary to heart disease = cardiorenal syndrome-CRS) and 33.3% dogs with chronic kidney disease. The potential applicability of the developed methodology in the analysis of podocin in canine urine sediments was confirmed.

Effects of childbirth on podocyturia in women with normotensive, uncomplicated pregnancies.

Changes in hemodynamics and blood pressure occur shortly before and after childbirth regardless of the mode of delivery. This study aimed to test the hypothesis that parturition induces a temporal increase in podocyturia monitored by podocyte-specific protein podocin mRNA expression levels (Pod-mRNA). A total of 105 urine specimens, consisting of 43 and 62 from 18 and 20 otherwise healthy women with vaginal delivery (VD) and elective cesarean delivery (ECS), respectively, were studied. Determination of urine protein and creatinine (Cr) concentrations and quantitative analyses of Pod-mRNA, nephrin mRNA (Nep-mRNA), synaptopodin mRNA (Syn-mRNA), and aquaporin 2 mRNA expression were performed using RT-PCR in pelleted urine samples. Levels of mRNA expression were corrected by urine Cr concentration. Podocyturia increased significantly, concomitant with a significantly decreased Nep:Pod-mRNA ratio (NPR) in the urine, collected immediately before or after childbirth regardless of the delivery mode compared with urine collected before commencement of labor or on postpartum day 3 or later. Podocyturia was significantly negatively correlated with NPR [correlation coefficient (r) = -0.614/-0.750 for VD/ECS women, respectively], as well as the Syn:Pod-mRNA ratio. Systolic blood pressure exceeded 140 mmHg during labor in 50% of VD women, and mean arterial pressure was significantly positively correlated with podocyturia during labor in VD women (r = 0.733). Thus parturition induces a transient increase in urine podocytes with reduced Nep- and Syn-mRNA expressions. Glomerular podocytes with reduced Nep- and Syn-mRNA levels were suggested to be likely to detach from the glomerular basement membrane around childbirth.

Accelerated podocyte detachment and progressive podocyte loss from glomeruli with age in Alport Syndrome.

Podocyte depletion is a common mechanism driving progression in glomerular diseases. Alport Syndrome glomerulopathy, caused by defective α3α4α5 (IV) collagen heterotrimer production by podocytes, is associated with an increased rate of podocyte detachment detectable in urine and reduced glomerular podocyte number suggesting that defective podocyte adherence to the glomerular basement membrane might play a role in driving progression. Here a genetically phenotyped Alport Syndrome cohort of 95 individuals [urine study] and 41 archived biopsies [biopsy study] were used to test this hypothesis. Podocyte detachment rate (measured by podocin mRNA in urine pellets expressed either per creatinine or 24-hour excretion) was significantly increased 11-fold above control, and prior to a detectably increased proteinuria or microalbuminuria. In parallel, Alport Syndrome glomeruli lose an average 26 podocytes per year versus control glomeruli that lose 2.3 podocytes per year, an 11-fold difference corresponding to the increased urine podocyte detachment rate. Podocyte number per glomerulus in Alport Syndrome biopsies is projected to be normal at birth (558/glomerulus) but accelerated podocyte loss was projected to cause end-stage kidney disease by about 22 years. Biopsy data from two independent cohorts showed a similar estimated glomerular podocyte loss rate comparable to the measured 11-fold increase in podocyte detachment rate. Reduction in podocyte number and density in biopsies correlated with proteinuria, glomerulosclerosis, and reduced renal function. Thus, the podocyte detachment rate appears to be increased from birth in Alport Syndrome, drives the progression process, and could potentially help predict time to end-stage kidney disease and response to treatment.

The effects of sildenafil citrate on urinary podocin and nephrin mRNA expression in an L-NAME model of pre-eclampsia.

We investigated the effects of sildenafil citrate (SC) on podocyturia in N ω-nitro-L-arginine methyl ester hydrochloride (L-NAME) model of pre-eclampsia (PE). One hundred and twenty Sprague-Dawley rats (SDR) were divided into five groups like pregnant control (PC), early-onset PE (EOPE), late-onset PE(LOPE), early and late-onset PE with SC-treated groups [EOPE (SC); LOPE (SC)]. PE was induced in SDR by oral administration of L-NAME in drinking water for 4-8 days for EOPE and 8-14 day for LOPE. The blood pressure, urine volume and total urine protein were increased in EOPE and LOPE groups when compared to PC, and all the above parameters decreased in EOPE (SC) and LOPE (SC) groups when compared to EOPE and LOPE groups, respectively. The EOPE and LOPE groups showed an increase in urinary nephrin mRNA and podocin mRNA levels compared to PC group. Increases in serum and renal soluble fms-like tyrosine kinase-1 (sFlt-1) expression levels and decreases in renal vascular endothelial growth factor (VEGF) expression and serum placenta growth factor (PlGF) levels were observed in EOPE and LOPE groups when compared to PC group. In addition, decreases in serum and renal sFlt-1 expression levels and increases in renal VEGF expression and serum PlGF levels were observed in EOPE (SC) and LOPE (SC) groups when compared to EOPE and LOPE groups, respectively. The light microscopy showed that the renal tissue of L-NAME-treated rats had extensive glomerular damage, tubular damage and infiltration by mononuclear cells when compared to PC group. Therefore, SC ameliorated podocyturia through its effects on the antiangiogenic/angiogenic status in this animal model.

Post-partum podocyturia following pre-eclamptic pregnancy.

AIM: Urine podocin mRNA expression and urine podocin : nephrin mRNA expression ratio (PNR) increase with increasing proteinuria during pregnancy complicated with pre-eclampsia (PE). This suggests that urine podocytes with reduced nephrin mRNA expression are abundant in pathological podocyturia. The aim of this study was therefore to determine post-partum changes in podocyturia and PNR in relation to proteinuria after pre-eclampsia (PE). METHODS: A total of 137 peripartum urine specimens, consisting of 72 and 65 from 24 and 30 women with PE and normotensive control pregnancies (NCP), respectively, were studied. Determination of urine protein and creatinine concentration and quantitative analysis of podocyte-specific podocin and nephrin mRNA expression were carried out using reverse transcription-polymerase chain reaction in pelleted urine samples. Podocyturia was monitored via urine podocin mRNA expression. Podocyturia and proteinuria were normalized by urine creatinine concentration. RESULTS: Podocyturia and urine PNR decreased with decreasing proteinuria as well as with increasing time after delivery in the urine from PE women. In physiological proteinuria (i.e. protein : creatinine ratio [P/Cr] 0.005-0.1 mg/mg), however, both podocyturia and PNR were significantly greater in the urine from PE women compared with NPC women, although P/Cr was similar between the groups (median, 0.037 mg/mg for PE vs 0.029 mg/mg for NCP). CONCLUSIONS: Podocyturia decreases with decreasing proteinuria in PE women after childbirth. In PE women, however, pathological podocyturia consisting of podocytes with decreased nephrin mRNA expression persisted even after proteinuria decreased to a level similar to that in NCP women.

Urinary-cell mRNA profile and acute cellular rejection in kidney allografts.

BACKGROUND: The standard test for the diagnosis of acute rejection in kidney transplants is the renal biopsy. Noninvasive tests would be preferable. METHODS: We prospectively collected 4300 urine specimens from 485 kidney-graft recipients from day 3 through month 12 after transplantation. Messenger RNA (mRNA) levels were measured in urinary cells and correlated with allograft-rejection status with the use of logistic regression. RESULTS: A three-gene signature of 18S ribosomal (rRNA)-normalized measures of CD3ε mRNA and interferon-inducible protein 10 (IP-10) mRNA, and 18S rRNA discriminated between biopsy specimens showing acute cellular rejection and those not showing rejection (area under the curve [AUC], 0.85; 95% confidence interval [CI], 0.78 to 0.91; P<0.001 by receiver-operating-characteristic curve analysis). The cross-validation estimate of the AUC was 0.83 by bootstrap resampling, and the Hosmer-Lemeshow test indicated good fit (P=0.77). In an external-validation data set, the AUC was 0.74 (95% CI, 0.61 to 0.86; P<0.001) and did not differ significantly from the AUC in our primary data set (P=0.13). The signature distinguished acute cellular rejection from acute antibody-mediated rejection and borderline rejection (AUC, 0.78; 95% CI, 0.68 to 0.89; P<0.001). It also distinguished patients who received anti-interleukin-2 receptor antibodies from those who received T-cell-depleting antibodies (P<0.001) and was diagnostic of acute cellular rejection in both groups. Urinary tract infection did not affect the signature (P=0.69). The average trajectory of the signature in repeated urine samples remained below the diagnostic threshold for acute cellular rejection in the group of patients with no rejection, but in the group with rejection, there was a sharp rise during the weeks before the biopsy showing rejection (P<0.001). CONCLUSIONS: A molecular signature of CD3ε mRNA, IP-10 mRNA, and 18S rRNA levels in urinary cells appears to be diagnostic and prognostic of acute cellular rejection in kidney allografts. (Funded by the National Institutes of Health and others.).

IMAC fractionation in combination with LC-MS reveals H2B and NIF-1 peptides as potential bladder cancer biomarkers.

Improvement in bladder cancer (BC) management requires more effective diagnosis and prognosis of disease recurrence and progression. Urinary biomarkers attract special interest because of the noninvasive means of urine collection. Proteomic analysis of urine entails the adoption of a fractionation methodology to reduce sample complexity. In this study, we applied immobilized metal affinity chromatography in combination with high-resolution LC-MS/MS for the discovery of native urinary peptides potentially associated with BC aggressiveness. This approach was employed toward urine samples from patients with invasive BC, noninvasive BC, and benign urogenital diseases. A total of 1845 peptides were identified, corresponding to a total of 638 precursor proteins. Specific enrichment for proteins involved in nucleosome assembly and for zinc-finger transcription factors was observed. The differential expression of two candidate biomarkers, histone H2B and NIF-1 (zinc finger 335) in BC, was verified in independent sets of urine samples by ELISA and by immunohistochemical analysis of BC tissue. The results collectively support changes in the expression of both of these proteins with tumor progression, suggesting their potential role as markers for discriminating BC stages. In addition, the data indicate a possible involvement of NIF-1 in BC progression, likely as a suppressor and through interactions with Sox9 and HoxA1.

Urine podocin:nephrin mRNA ratio (PNR) as a podocyte stress biomarker.

BACKGROUND: Proteinuria and/or albuminuria are widely used for noninvasive assessment of kidney diseases. However, proteinuria is a nonspecific marker of diverse forms of kidney injury, physiologic processes and filtration of small proteins of monoclonal and other pathologic processes. The opportunity to develop new glomerular disease biomarkers follows the realization that the degree of podocyte depletion determines the degree of glomerulosclerosis, and if persistent, determines the progression to end-stage kidney disease (ESKD). Podocyte cell lineage-specific mRNAs can be recovered in urine pellets of model systems and in humans. In model systems, progressive glomerular disease is associated with decreased nephrin mRNA steady-state levels compared with podocin mRNA. Thus, the urine podocin:nephrin mRNA ratio (PNR) could serve as a useful progression biomarker. The use of podocyte-specific transcript ratios also circumvents many problems inherent to urine assays. METHODS: To test this hypothesis, the human diphtheria toxin receptor (hDTR) rat model of progression was used to evaluate potentially useful urine mRNA biomarkers. We compared histologic progression parameters (glomerulosclerosis score, interstitial fibrosis score and percent of podocyte depletion) with clinical biomarkers [serum creatinine, systolic blood pressure (BP), 24-h urine volume, 24-h urine protein excretion and the urine protein:creatinine ratio(PCR)] and with the novel urine mRNA biomarkers. RESULTS: The PNR correlated with histologic outcome as well or better than routine clinical biomarkers and other urine mRNA biomarkers in the model system with high specificity and sensitivity, and a low coefficient of assay variation. CONCLUSIONS: We concluded that the PNR, used in combination with proteinuria, will be worth testing for its clinical diagnostic and decision-making utility.

Protective effects of sinomenine against doxorubicin-induced nephrosis in rats.

Sinomenine (SN, 1) is a pure compound extracted from the Sinomenium acutum plant. We investigated the protective effects and mechanism of action of SN in a rat model of doxorubicin (DOX)-induced nephrosis. Nephrosis was induced by a single dose of 5 mg/kg DOX, and DOX-treated rats received a daily i.p. injection of 10 or 30 mg/kg SN, or saline (n = 6). Urine and serum biochemical parameters, serum TNF-α and IL-1ß levels, nephrin, podocin, α-actinin-4, and peroxisome proliferator-activated receptor-α (PPAR-α) protein expression, and renal ultrastructure were examined at day 28. Compound 1 significantly attenuated the effect of DOX on urine and serum biochemical parameters. Electron microscopy demonstrated that 1 suppressed DOX-induced increases in foot process width. Compared with those in control rats, nephrin, podocin, and PPAR-α protein expressions decreased in the glomeruli of DOX-treated rats, and this effect was significantly attenuated by 1. However, no appreciable alterations were observed in the expression level of α-actinin-4. DOX significantly increased serum TNF-α and IL-1ß compared with those in control rats, and 1 significantly reduced the serum levels of TNF-α and IL-1ß. SN ameliorates DOX-induced nephrotic syndrome in rats, resulting in a modulation of renal nephrin, podocin expression, and thereby protecting podocytes from injury.

Increasing urinary podocyte mRNA excretion and progressive podocyte loss in kidney contribute to the high risk of long-term renal disease caused by preterm birth.

Podocyte abnormalities are common mechanism driving the progression of glomerular diseases, which account for most chronic kidney diseases (CKDs). However, the role of podocyte in the mechanism of high-risk long-term CKD caused by prematurity has not been well clarified. In present study, urine samples of 86 preterm infants and 32 full-term infants were collected, and podocyte-specific podocin mRNA levels in urine pellet were applied to indicate urinary podocyte mRNA excretion. In addition, in a preterm animal rat model, preterm rats were identified by delivery 2 days early. From the age of 3 weeks-12 months, urine samples were collected to examine podocyte mRNA excretion by measuring podocyte-specific podocin mRNA levels. Kidney samples at the age of 3 weeks, 2 months, and 12 months were collected from 8, 5 and 6 preterm rats and 9, 6 and 8 full-term rats, respectively, to examine podocyte density and podocyte area by measuring the podocyte specific nuclear marker WT-1 and the podocyte specific marker synaptopodin. As results, a more than threefold increase of urinary podocyte-specific podocin mRNA excretion rate was found in preterm infants compared with full-term infants. In addition, there was negative correlation between gestational age at birth and urinary podocin mRNA excretion. In preterm rats, a reduction in the total number of differentiated podocytes in glomeruli and an increased podocyte podocin mRNA excretion rate in urine were detected at the end of kidney differentiation. Moreover, long-term follow-up data in preterm rats showed there was an increased the risk of renal disease indicated by persistent podocyte mRNA loss, proteinuria, and enlarged glomeruli. In conclusion, increasing podocyte mRNA excretion in urine and podocyte loss in kidney led by prematurity drive the progression of long-term abnormal kidney function and could potentially explain the high risk of long-term CKD in preterm infants.

Evaluation of podocin in urine in horses using qualitative and quantitative methods.

No sensitive method for diagnosing early kidney dysfunction in horses has been identified so far. Many studies carried out in humans and small animals show that podocin can be useful to diagnose various kidney diseases, mainly affecting the glomeruli. The aim of this study was to perform a qualitative and quantitative analysis of podocin in urine samples obtained from healthy horses, horses with clinical kidney dysfunction and horses at risk of acute kidney injury. The study objectives aimed to assess: (1) whether the selected podocin tryptic peptide for LC-MS-MRM allows for podocin detection in horse; and (2) whether the species-specific ELISA test makes this detection possible as well;, (3) whether the chosen methods are sensitive enough to detect kidney dysfunction and glomerular injury, (4) whether the results of the tests applying both methods correspond with one another, (5) whether the results correlate with the hematological and biochemical data. The signals that may indicate the presence of trypsin fragments of podocin were found in three healthy horses, all the horses diagnosed with kidney dysfunction and half of the animals at risk for acute kidney injury. The concentration of podocin, diagnosed with the ELISA test was as follows: from 0.19 to 1.2 ng/ml in healthy animals, from 0.19 to 20.0 ng/ml in AKI horses, from 0.29 to 5.71 ng/ml in horses at risk for acute kidney injury. The results of both methods corresponded significantly. Podocin may be a potential biomarker of clinical kidney disease in horses and may be used in the detection of glomerular injury. However, its use is limited by the possibility of physiological podocyturia. LC-MS-MRM seems to be a more sensitive method to evaluate the presence of podocin than the ELISA test, whilst selected tryptic peptides of podocin appear to apply to horses. The ELISA test showed greater effectiveness in excluding the disease than in confirming it.

Ameliorative Effects of Bredemolic Acid on Markers Associated with Renal Dysfunction in a Diet-Induced Prediabetic Rat Model.

Recently, studies have shown that renal dysfunction is associated not only with overt diabetes but also with the preceding stage known as prediabetes. Diet and pharmacological interventions are the therapeutic approaches to managing prediabetes, but the compliance in combining the two interventions is low. Hence, the efficacy of pharmacological intervention is reduced without diet modification. In our previous study, we established that bredemolic acid (BA) ameliorated glucose homeostasis via increased GLUT 4 expression in the skeletal muscle of prediabetic rats in the absence of diet intervention. However, the effects of bredemolic acid on renal function in prediabetic condition are unknown. Therefore, this study was aimed at investigating the ameliorative effects of bredemolic acid on renal dysfunction in a diet-induced prediabetic rat model. Thirty-six Sprague-Dawley male rats (150-180 g) were divided into two groups: the nonprediabetic (n = 6) and prediabetic (n = 30) groups which were fed normal diet (ND) and high-fat high-carbohydrate (HFHC) diet, respectively, for 20 weeks. After the 20th week, the prediabetic groups were subdivided into prediabetic control (PD) and 4 other prediabetic groups which were treated with either BA (80 mg/kg) or metformin (MET, 500 mg/kg) for further 12 weeks (21st to 32nd). Plasma, urine, and kidney samples were collected for biochemical analysis. The untreated prediabetic (PD) rats presented increased fluid intake and urine output; increased creatinine, urea, and uric acid plasma concentrations; albuminuria; proteinuria; sodium retention; potassium loss; increased aldosterone and kidney injury molecule (KIM-1) concentration; and increased urinary podocin mRNA expression. However, BA administration attenuated the renal markers and oxidative stress and decreased the urinary podocin mRNA expression. In conclusion, BA administration, regardless of diet modification, attenuates renal dysfunction in an experimentally induced prediabetic state.

Integrin beta1-mediated matrix assembly and signaling are critical for the normal development and function of the kidney glomerulus.

The human kidneys filter 180 l of blood every day via about 2.5 million glomeruli. The three layers of the glomerular filtration apparatus consist of fenestrated endothelium, specialized extracellular matrix known as the glomerular basement membrane (GBM) and the podocyte foot processes with their modified adherens junctions known as the slit diaphragm (SD). In this study we explored the contribution of podocyte beta1 integrin signaling for normal glomerular function. Mice with podocyte specific deletion of integrin beta1 (podocin-Cre beta1-fl/fl mice) are born normal but cannot complete postnatal renal development. They exhibit detectable proteinuria on day 1 and die within a week. The kidneys of podocin-Cre beta1-fl/fl mice exhibit normal glomerular endothelium but show severe GBM defects with multilaminations and splitting including podocyte foot process effacement. The integrin linked kinase (ILK) is a downstream mediator of integrin beta1 activity in epithelial cells. To further explore whether integrin beta1-mediated signaling facilitates proper glomerular filtration, we generated mice deficient of ILK in the podocytes (podocin-Cre ILK-fl/fl mice). These mice develop normally but exhibit postnatal proteinuria at birth and die within 15 weeks of age due to renal failure. Collectively, our studies demonstrate that podocyte beta1 integrin and ILK signaling is critical for postnatal development and function of the glomerular filtration apparatus.

Urinary GADD45gamma expression is associated with progression of lgA nephropathy.

BACKGROUND: Growth arrest and DNA damage-45gamma (GADD-45gamma) is induced in response to environmental stresses and functions in the regulation of cell cycles. Previous findings by our group suggested that GADD45gamma contributed to renal tubular cell damage through induction of inflammatory and fibrogenic molecules. We examined the effects of urinary GADD45gamma expression on the decline in renal function with IgA nephropathy in the present study. METHODS: Patients (n = 62) were followed for a total of 710.3 +/- 287.5 days. The rate of renal function decline was assessed by the slopes of inverse serum creatinine and estimated glomerular filtration rate (GFR) plotted against time. Renal survival was calculated by Kaplan-Meier analysis and the primary endpoint was an increase in serum creatinine levels by 50% or more. RESULTS: Kidney function declined more rapidly in the GADD45gamma-positive group compared to the GADD45gamma-negative group. Kidney survival estimates at the end of the study period were 82.9% in the GADD45gamma-positive group and 100% in the negative group (p = 0.03). This difference remained significant in the group with GFR values <90 ml/min/1.73 m2 when adjusted to stratification factors. CONCLUSION: The results suggest that urinary GADD45gamma expression is associated with progression of renal disease.

The urine podocin/creatinine ratio as a novel biomarker of cardiorenal syndrome in dogs due to degenerative mitral valve disease.

Dysfunction of heart leads inevitable to the dysfunction of kidney which is termed as the cardiorenal syndrome (CRS). Previous studies have confirmed existence of CRS in dogs with degenerative mitral valve disease (DMVD). The goal of the study was to assess the usefulness of commercial test to measure podocyturia in dogs and test the urine podocine/creatinine ratio (UPoC) as an early marker of kidney injury. Urine podocine/creatinine ratio was calculated because numbers of podocytes is dependent on the urine concentration. Fifty dogs was divided into three groups: fifteen healthy (control group), twenty nine with DMVD class C-chronic according to ACVIM (heart group) and six with chronic kidney disease (kidney group). Each dog underwent a clinical examination: electrocardiography, echocardiography, chest radiograph, abdominal ultrasound, blood haematological and biochemical analysis including symmetric dimethylarginine (SDMA) and cystatin C (Cyst C), routine urine analysis and analysis of podocytes using an ELISA test. UPoC was calculated. Mean value ± standard deviation for UPoC was respectively 9.7 ± 4.8 x 10-10 for control group, 49.0 ± 80.0 x 10-10 for heart group, 33.7 ± 18.0 x 10-10 for kidney group. The UPoC in the heart and kidney group was significantly higher than in the control group (P < 0.0001, sensivity 0.83, specyfity 0.20). Commercial ELISA tests may be used to assess podocyturia in dogs. An UPoC increase exceeding 12.93 x 10-10 indicates glomerular damage in DMVD dogs. Based on UPoC, 79.3% of dogs with C-chronic stage of DMVD developed CRS.

Tandem mass spectrometry analysis of urinary podocalyxin and podocin in the investigation of podocyturia in women with preeclampsia and Fabry disease patients.

BACKGROUND: Podocytes are highly differentiated visceral cells, and several related specific proteins, such as podocalyxin and podocin are potential tools for the evaluation of podocyturia. However, precise quantitation of podocyturia-related proteins is complex and often unreliable. METHOD: A reversed-phase ultra-performance liquid chromatography coupled to tandem mass spectrometry method was developed and validated to quantify podocalyxin and podocin levels in urine supernatant by using specific cleavable peptides and standards. Urine samples from women with normotensive or hypertensive pregnancies, gestational diabetes and preeclampsia, as well as treated and untreated Fabry patients, and gender-matched controls were investigated. RESULTS: The multiplex analysis shows that podocalyxin levels were higher than podocin levels in patients, the former being particularly higher in pregnant women. Women with preeclampsia had abnormal urine levels of both proteins with a higher sensitivity for podocalyxin. Slightly increased levels of podocin were also observed in Fabry males, while both proteins were increased in untreated Fabry females. Correlations were established between podocalyxin and podocin levels and clinical parameters associated with Fabry disease and preeclampsia. CONCLUSIONS: This methodology makes possible the precise, simultaneous and reliable analysis of podocalyxin and podocin levels, and offers a valuable tool for the evaluation of podocyturia.

Upregulation of NDRG1 predicts poor outcome and facilitates disease progression by influencing the EMT process in bladder cancer.

N-myc downstream regulated gene 1 (NDRG1) is an intracellular protein involved in cell differentiation and was recently reported to exert various effects in several cancers. However, its expression and role in bladder cancer remain unclear. Our study enrolled 100 bladder cancer patients to detect NDRG1 expression in tumour tissues by immunohistochemistry. Correlations between NDRG1 expression and clinical factors were analysed. An NDRG1 overexpression plasmid and NDRG1 siRNAs were transfected into bladder cancer cell lines. Cell biological behaviours were assessed by CCK-8, flow cytometry, wound healing and Transwell assays. Additionally, the influence of NDRG1 on epithelial-mesenchymal transition (EMT) was investigated by western blotting and real-time PCR. NDRG1 expression in urine from bladder cancer patients was examined by ELISA. NDRG1 protein levels were significantly increased in bladder cancer patients and correlated with tumour stage (p = 0.025), lymph node metastasis (p = 0.034) and overall survival (p = 0.016). Patients with high NDRG1 expression had poorer outcomes than those with low NDRG1 expression. NDRG1 overexpression was associated with increased cell proliferation, migration, and invasion and decreased apoptotic cell numbers; NDRG1 knockdown resulted in the inverse effects. Moreover, upregulated NDRG1 expression was associated with downregulated Cytokeratin 7 and Claudin-1 expression and upregulated N-cad, ß-catenin and slug expression. Downregulated NDRG1 expression was associated with the inverse effects. Urine protein levels could distinguish bladder cancer patients from healthy controls, with an area under the curve of 0.909. NDRG1 promoted EMT in bladder cancer and could be an effective diagnostic and prognostic biomarker in bladder cancer patients.