Developmental origins for kidney disease due to Shroom3 deficiency

CKD is a significant health concern with an underlying genetic component. Multiple genome-wide association studies (GWASs) strongly associated CKD with the shroomfamilymember 3 (SHROOM3) gene, which encodes an actin-associated protein important in epithelial morphogenesis. However, the role of SHROOM3 in kidney development and function is virtually unknown. Studies in zebrafish and rat showed that alterations in Shroom3 can result in glomerular dysfunction. Furthermore, human SHROOM3 variants can induce impaired kidney function in animal models. Here, we examined the temporal and spatial expression of Shroom3 in the mammalian kidney. We detected Shroom3 expression in the condensing mesenchyme, Bowman\u27s capsule, and developing and mature podocytes in mice. Shroom3 null (Shroom3Gt/Gt) mice showed marked glomerular abnormalities, including cystic and collapsing/degenerating glomeruli, and marked disruptions in podocyte arrangement and morphology. These podocyte-specific abnormalities are associated with altered Rho-kinase/myosin II signaling and loss of apically distributed actin. Additionally, Shroom3 heterozygous (Shroom3Gt/+) mice showed developmental irregularities that manifested as adult-onset glomerulosclerosis and proteinuria. Taken together, our results establish the significance of Shroom3 in mammalian kidney development and progression of kidney disease. Specifically, Shroom3 maintains normal podocyte architecture in mice via modulation of the actomyosin network, which is essential for podocyte function. Furthermore, our findings strongly support the GWASs that suggest a role for SHROOM3 in human kidney disease

Effectiveness of group cognitive therapy about opium addict complications on attitude of adolescents with drug dependent parents

Background and Aim: Statistics show that 30% to 40 % of &nbsp;opium addicted fathers&rsquo; children are prone to substance abuse in the future. The present study aimed at assessing the effectiveness of cognitive therapy approach &nbsp;to attitude changing of adolescents with substance dependent fathers. Materials and Methods: &nbsp;In this controlled. field-trail randomized study. .data collection tool was &ldquo;attitude to addiction questionnaire&rdquo;. The study population was all male students in the first grade of high school in Maneh - Samalghan city. . Six sessions of group cognitive therapy based on the effectiveness of drug side-effects in drug-addicted fathers&rsquo; adolescent children&rsquo;s attitude were held. The above-mentioned questionnaire was filled out before and after intervention. The obtained data &nbsp;was fed into SPSS software (V: 16) using. Independent t-test .and paired t-test were used for analysis and P<0.05 was taken as the significant level. Results: &nbsp;There were no significant differences between the two groups in pre-test regarding their attitude about drug abuse (P=.20%). Mean score variance from pre-test to post-test in the intervention group decreased, but in the control group, it showed a slight increase. This means that the intervention reduced the positive attitude towards drugs, but the changes were not statistically significant (p=0.57). Besides, among ten factors decisive in an individual&rsquo;s attitude about addiction, only group cognitive therapy &nbsp;was able&nbsp; to decrease mean points of an individual&rsquo;s attitude about drug abuse .. Significantly (P = 0.04). Conclusion: It was found that group cognitive therapy education about opium&nbsp; addict complicationsdidn`t have a significant effect on the attitude of the students with addicted fathers. Thus, a change of adolescents&rsquo; attitude requires more research

Effects of Vocational Consultation on Relapse Rate and Hope among Drug Dependents in Bojnurd, Iran

Introduction: Drug addiction is one of the most flagrant social damages that can easily enervate the socio- cultural foundation of a country as well as endanger human dynamism. One of the prevalent problems among most addicted people is their low hope and relapse of drug dependence. The aim of this study was to assess the effect of vocational consultation (for training on problem-solving skills) on hope and relapse rate of patients treated in methadone maintenance clinics. Methods: This experiment was conducted on 60 drug abusers treated in a methadone maintenance program in drug addiction centers in Bojnurd, Iran, in 2014. The patients were randomly and equally allocated into two study and control groups. All patients completed the Miller Hope Questionnaire before and after the intervention. Ten sessions of vocational consultation were held for the study group while the control group received no special treatment. Patients were followed up on for relapses for six months. Data were analyzed using SPSS (version 16) and the paired-samples t-test technique. Results: The results indicated that the mean and standard deviation of hope on the pre-test in the study group increased on the post-test (from M=175.5, SD=31.8, to M=198.5, SD=20.4), while in the control group the mean of hope decreased from the pre-test to past-test stage (M=184.7, SD=27.7, to M=183.3, SD=26.1), showing a significant relationship, t(56)= 5.657, p<0.05. The relapse rate was not significantly different in the two groups. Conclusion: The vocational consultation positively affects hope among drug dependents but did not affect their relapse rate during the six-month follow-up. Increasing the hope in these groups of patients may be effective in other aspects of treatment success in long-term follow-up

Minimal Kidney Disease Phenotype in Heterozygous Null Mice

Background: Shroom family member 3 (SHROOM3) encodes an actin-associated protein that regulates epithelial morphology during development. Several genome-wide association studies (GWAS) have identified genetic variances primarily in the 5’ region of SHROOM3, associated with chronic kidney disease (CKD) and poor transplant outcomes. These genetic variants are associated with alterations in Shroom3 expression. Objective: Characterize the phenotypic abnormalities associated with reduced Shroom3 expression in postnatal day 3-, 1-month and 3-month-old mice. Methods: The Shroom3 protein expression pattern was determined by immunofluorescence. We generated Shroom3 heterozygous null mice ( Shroom3 Gt/+ ) and performed comparative analyses with wild type littermates based on somatic and kidney growth, gross renal anatomy, renal histology, renal function at postnatal day 3, 1 month, and 3 months. Results: The Shroom3 protein expression localized to the apical regions of medullary and cortical tubular epithelium in postnatal wild type kidneys. Co-immunofluorescence studies confirmed protein expression localized to the apical side of the tubular epithelium in proximal convoluted tubules, distal convoluted tubules, and collecting ducts. While Shroom3 heterozygous null mice exhibited reduced Shroom3 protein expression, no differences in somatic and kidney growth were observed when compared to wild type mice. Although, rare cases of unilateral hypoplasia of the right kidney were observed at postnatal 1 month in Shroom3 heterozygotes. Yet renal histological analysis did not reveal any overt abnormalities in overall kidney structure or in glomerular and tubular organization in Shroom3 heterozygous null mice when compared to wild type mice. Analysis of the apical-basolateral orientation of the tubule epithelium demonstrated alterations in the proximal convoluted tubules and modest disorganization in the distal convoluted tubules at 3 months in Shroom3 heterozygotes. Additionally, these modest abnormalities were not accompanied by tubular injury or physiological defects in renal and cardiovascular function. Conclusion: Taken together, our results describe a mild kidney disease phenotype in adult Shroom3 heterozygous null mice, suggesting that Shroom3 expression and function may be required for proper structure and maintenance of the various tubular epithelial parenchyma of the kidney

Stromally Expressed β-Catenin Modulates Wnt9b Signaling in the Ureteric Epithelium

<div><p>The mammalian kidney undergoes cell interactions between the epithelium and mesenchyme to form the essential filtration unit of the kidney, termed the nephron. A third cell type, the kidney stroma, is a population of fibroblasts located in the kidney capsule, cortex and medulla and is ideally located to affect kidney formation. We found β-catenin, a transcriptional co-activator, is strongly expressed in distinctive intracellular patterns in the capsular, cortical, and medullary renal stroma. We investigated β-catenin function in the renal stroma using a conditional knockout strategy that genetically deleted β-catenin specifically in the renal stroma cell lineage (β-cat^s-/-). <i>β-cat^s-/-</i> mutant mice demonstrate marked kidney abnormalities, and surprisingly we show β-catenin in the renal stroma is essential for regulating the condensing mesenchyme cell population. We show that the population of induced mesenchyme cells is significantly reduced in <i>β-cat^s-/-</i> mutants and exhibited decreased cell proliferation and a specific loss of Cited 1, while maintaining the expression of other essential nephron progenitor proteins. <i>Wnt9b</i>, the key signal for the induction of nephron progenitors, was markedly reduced in adjacent ureteric epithelial cells in <i>β-cat^s-/-</i>. Analysis of Wnt9b-dependent genes in the neighboring nephron progenitors was significantly reduced while Wnt9b-independent genes remained unchanged. In contrast mice overexpressing β-catenin exclusively in the renal stroma demonstrated massive increases in the condensing mesenchyme population and <i>Wnt9b</i> was markedly elevated. We propose that β-catenin in the renal stroma modulates a genetic program in ureteric epithelium that is required for the induction of nephron progenitors.</p></div

β-catenin is expressed in distinctive patterns in the renal stroma.

<p>(A-I) Immunofluorescence demonstrating β-catenin spatial and temporal expression in stromal cells. (A) At E11.5 Pbx1 is expressed in the nucleus of stromal cells (arrow head-inset) surrounding the condensing mesenchyme. Some Pbx1 positive stromal cells locate within the condensing mesenchyme, directly adjacent to epithelial cells (arrows). (B,C) At E11.5 β-catenin is expressed in the condensing mesenchyme and ureteric epithelium, and co-localizes with Pbx1 demonstrating expression in the renal stroma. At E11.5, some Pbx1 cells co-localize with β-catenin in the nuclear compartment of stromal cells (arrowhead-inset). (D) At E13.5, Pbx1 is expressed in capsular and cortical stroma surrounding the condensing mesenchyme. (E, F) At E13.5, β-catenin co-localizes in the cytoplasm of capsular stromal cells. The stromal cells located between developing nephrons express β-catenin in the cytoplasmic (arrow-inset) and nuclear compartment (arrowhead-inset). (G) At E17.5, Pbx1 marks the capsular, cortical, and medullary stroma. (H-I) β-catenin is expressed in the medullary stroma and co-localizes strongly with Pbx1 in the nuclear compartment (arrowhead-inset). (scale bar = 100μm, s = stroma, cm = condensing mesenchyme, ub = ureteric epithelium, rc = renal capsule, ms = medullary stroma, rp = renal pelvis).</p

The condensing mesenchyme cell population is reduced in <i>β-cat</i>^<i>S-/</i>- mutant kidneys.

<p>(A,B) As compared to <i>WT</i>, which demonstrates 3–4 cell layers of aggregated condensing mesenchyme, <i>β-cat</i>^<i>S-/</i>- kidneys display a reduced, single cell layer of loosely aggregated condensing mesenchyme. (C-H) Analysis of cell proliferation in the condensing mesenchyme was performed using Brdu cell proliferation assay. (C-E) As compared to <i>WT</i>, <i>β-cat</i>^<i>S-/</i>- mutants demonstrated a 6.46% reduction in condensing mesenchyme cell proliferation at E14.5 (<i>WT</i>, 34.56%±1.45, n = 28 versus <i>β-cat</i>^<i>S-/</i>-, 28.02%±1.05, n = 27, p = 0.0006). (F-G) At E15.5 <i>β-cat</i>^<i>S-/</i>- mutants demonstrated a 7.35% reduction in condensing mesenchyme cell proliferation when compared to <i>WT</i> (<i>WT</i>, 34.09%±1.65, n = 17 versus <i>β-cat</i>^<i>S-/</i>-, 26.73%±2.21, n = 15, p = 0.01). (I,J) A TUNEL assay at E15.5 did not reveal any changes in apoptosis in the condensing mesenchyme between <i>WT</i> and <i>β-cat</i>^<i>S-/</i>-. Scale Bar = 50μm</p

<i>β-cat</i>^<i>S-/</i>- mutants demonstrate multiple kidney abnormalities.

<p>(A,B) Gross anatomy of PN0 <i>WT</i> and <i>β-cat</i>^<i>S-/</i>- kidneys show comparable size and shape. (C-J) In contrast to <i>WT</i>, histological analysis of <i>β-cat</i>^<i>S-/</i>- mutant kidneys demonstrate numerous kidney abnormalities. (C,D) As compared to <i>WT</i>, <i>β-cat</i>^<i>S-/</i>- mutant kidneys were lobular, lacked a distinct boarder, contained numerous cysts in the medulla and cortex (star), with an ill-defined cortical medullary axis and misplaced glomeruli (arrow). (E-J) In contrast to <i>WT</i>, high magnification of <i>β-cat</i>^<i>S-/</i>- kidneys at PN0 revealed a non-adherent sporadic renal capsule (F and J), misplaced tubules just under the renal capsule (F and J), glomeruli in the medulla (H) and a marked reduction in medullary stroma (H). (A, B scale bar = 1mm, C, D scale bar = 100μm, ad = adrenal gland, k = kidney, b = bladder, rc = renal capsule, cs = cortical stroma, ms = medullary stroma, g = glomerulus).</p

β-catenin in the renal stroma modulates Wnt9b expression in ureteric epithelial cells.

<p>(A-C) When compared to <i>WT</i>, In situ hybridization and real-time quantitative PCR for <i>Wnt9b</i> demonstrates <i>Wnt9b</i> mRNA expression is significantly reduced (1.00 versus 0.29, p=0.008) in E14.5 <i>β-cat^S-/-</i> kidneys. (D-I) In situ hybridization and Real-time quantitative PCR for <i>Ret</i> and <i>Wnt11</i> demonstrated no differences in mRNA expression between <i>WT</i> and <i>β-cat^S-/-</i> at E14.5. (J-K) Histological analysis of <i>β-cat^GOF-S</i> mutant kidneys demonstrate a marked increase in condensing mesenchyme population when compared to <i>WT</i> at E14.5. (L-N) In situ hybridization and quantitative PCR demonstrate <i>Wnt9b</i> expression in <i>β-cat^GOF-S</i> kidneys was significantly increased (1.02 versus 3.102, p=0.021) as compared to <i>WT</i> at E14.5. (scale bar = 50 μm, rc-renal capsule, cm= condensing mesenchyme, ub = ureteric epithelium).</p

Investigation of the renal stroma in <i>β-cat</i>^<i>S-/</i>- mutant kidneys.

<p>(A-L) Analysis of the renal stroma using stromal markers; Meis1/2, Pbx1, Foxd1, and TN-C at E15.5. As compared to <i>WT</i>, no overt changes were observed in the cortical stroma with respect to Meis1/2 (A, B), Pbx1 (E, F), Foxd1 (I, J), and TN-C (K, L). However, a reduction of Meis1/2 (C, D) and Pbx1 (G, H) was observed in the medullary region in <i>β-cat</i>^<i>S-/</i>- kidneys. (M, N) TUNEL assay at E15.5 reveals an increase in apoptosis in the medullary stroma of <i>β-cat</i>^<i>S-/</i>- kidneys compared to <i>WT</i>. (rc = renal capsule, cs = cortical stroma, ms = medullary stroma, A = apoptosis). Scale Bar = 50μm</p