Thesis (MAppSc (Medical Laboratory Sc)) --University of South Australia, 1993

Yi-Shen-Hua-Shi (YSHS) granule is a Chinese patent drug for treating chronic glomerulonephritis (CGN). It was marketed in 2009. However, up to now, there is no report about the quality and pharmacological activities of this product. In this work，we evaluated the quality and anti-CGN effects of the drug. To evaluate the quality of the granule, a qualitative and quantitative HPLC-DAD analytical method was developed. For qualitative analysis, HPLC fingerprint of ten batches of YSHS granule was established. The fingerprints were analyzed using similarity evaluation, hierarchical cluster analysis (HCA), principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) based on 15 characteristic fingerprint peaks. Similarity values of 10-batche samples were all above 0.960, indicating a stable quality. Minor differences were observed among batches by HCA and PCA. For quantification analysis, contents of six constituents in the granule were simultaneously measured. To establish the chemical profile of the granule, a HPLC-Q-TOF- MS/MS method was developed. A total of 105 peaks were detected using HPLC-Q-TOF-MS/MS in the granule, of which, 99 were tentatively identified as terpenoids, flavonoids, coumarins, alkaloids, phenols and other types of compounds, and 15 were further validated with reference substances. HPLC fingerprint chromatogram establishment, quantification analysis of 6 constituents and compound identification should improve the quality control of YSHS granule. To study the pharmacological activities of the granule, we investigated its anti-CGN effects and TGFβ signaling-related mechanism of action. A CGN rat model was established by injection of cationization-bovine serum albumin (C-BSA) for five weeks. After C-BSA injection, drugs were intragastrically administered to the rats once daily for four weeks. Clinical signs were recorded daily. Urine and serum biochemical parameters were analyzed using respective kits. Protein levels were examined by Western blotting. Pathological changes of renal tissues were evaluated using HE and Masson's trichrome staining. No significant differences in body weights and clinical signs were found among normal, model and drug treatment groups. Proteinuria; albuminuria; increased urine volume; elevated creatinine, urea nitrogen, triglyceride levels and total cholesterol in serum; decreased serum total protein and albumin; as well as renal pathological damages and fibrosis were observed in CGN model rats. YSHS granule ameliorated all the abnormal behavioral and biochemical changes in the model rats. Mechanistic investigations revealed that YSHS granule down-regulated proteins levels of TGFβ1, phospho-Smad2/3 (Thr 8) and Smad4 in rat renal tissues. These findings indicate that the drug has anti-CGN effects in rats, and inhibiting TGFβ signaling contributes to the underlying mechanisms. In summary, our chemical analytical studies will help in improving the quality control of YSHS granule. Our bioactivity and mechanistic studies provide a pharmacological basis for the clinical use of the granule in treating CGN.

Chronic kidney disease (CKD), defined as an irreversible reduction in glomerular filtration rate, is a large public health concern. Dissecting the genetic components of CKD is required to improve our understanding of disease pathogenesis. Researchers have identified that SHROOM3, has very high associations with kidney disease and function. Shroom3 encodes an actin-binding protein important in regulating cell and tissue morphogenesis. However, there is a lack of evidence supporting a role for Shroom3 in kidney function or disease. Here, I investigated the developmental and functional role of Shroom3 in the mammalian kidney. For the first time, I described the expression pattern of Shroom3 in the embryonic and adult mouse kidneys. By performing in situ hybridization and immunohistochemistry, I demonstrated that Shroom3 is expressed in the condensing mesenchyme, podocytes, and collecting ducts. I further showed that Shroom3 protein is localized in the foot processes of podocytes, utilizing immunogold labeling and transmission electron microscopy. In order to uncover a potential role of Shroom3 in the kidney, we utilized Shroom3 knockout mice. Shroom3 mutants demonstrated marked glomerular abnormalities including cystic and degenerating glomeruli, and reduced glomerular number. Scanning and transmission electron microscopic analyses of Shroom3 mutant glomeruli revealed disruptions in podocyte morphology characterized by disorganized foot processes with less interdigitation and segmental foot processes effacement. Furthermore, immunofluorescence analysis of mutant kidneys revealed aberrant distribution of podocyte actin-associated proteins. Elucidating the underlying molecular mechanism of this abnormal podocyte architecture; v we demonstrated that in the absence of Shroom3, Rho kinase is mislocalized in the apical membrane of podocytes. As a result, mislocalized Rho kinase failed to phosphorylate non-muscle myosin and induce actomyosin contraction resulting in a patchy granular distribution of actin in the podocytes of Shroom3 mutants. Taken together, our findings established that Shroom3 is essential for proper actin organization in the podocytes through interaction with Rock. Furthermore, we took advantage of a haploinsufficiency phenotype of Shroom3 heterozygote adult mice and demonstrated these mice develop glomerulosclerosis and proteinuria. In conclusion, our studies provided evidence to support a role for Shroom3 in kidney development and disease and support the GWAS studies that suggested a correlation between SHROOM3 variants and kidney function in humans. / Thesis / Master of Science (MSc)

No description available.

Thesis (Ph. D.)--University of Adelaide, 1986. / Includes bibliographical references (leaves 119-136).

Thesis (Ph. D.)--Case Western Reserve University, 2005. / Author's name on approval form and OhioLink ETD database: Maria Isabel Rico-Salas. [School of Medicine] Department of Physiology and Biophysics. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Abstract not available

Bibliography: leaves 119-136.

published_or_final_version / abstract / toc / Medicine / Master / Master of Philosophy

This KwaZulu-Natal (KZN) based study investigates hypertension, glomerulonephritides and the rarity of IgA Nephropathy (IgAN) in Africans in association with the Human Leukocyte Antigen (HLA). A retrospective hypertensive study found a positive association with HLA-B40 (P c<0.05) and HLA-B15 (Pc<0.02) in Indians and Africans respectively. No association was found in Whites. A prospective study showed glomerulonephritides to be positively associated with HLA-A33 in Indians (Pc 0.049). No associations were found with glomerulonephritides in Africans and Whites. Combined Race groups show no HLA associations. HLA-A30; HLA-A34; HLA-A29; HLA-B42; HLA-B58; HLA-B70 and HLA-DR11 were extremely significantly higher in Africans compared to Indians and Whites (all P<0.0001). In conclusion, HLA-B40 and I 1LA-B15 are possible disease susceptibility markers in Indian and African hypertensives; HLA-A33 is a possible disease susceptibility marker for glomerulonephritides in Indians and alleles in linkage might be responsible for the rarity of IgAN in Africans but further studies need to be employed. / Thesis (M.Med)-University of KwaZulu-Natal, 2007.