A Targeted Multiomics Approach to Identify Biomarkers Associated with Rapid eGFR Decline in Type 1 Diabetes

Background: Individuals with type 1 diabetes (T1D) demonstrate varied trajectories of estimated glomerular filtration rate (eGFR) decline. The molecular pathways underlying rapid eGFR decline in T1D are poorly understood, and individual-level risk of rapid eGFR decline is difficult to predict. Methods: We designed a case-control study with multiple exposure measurements nested within 4 well-characterized T1D cohorts (FinnDiane, Steno, EDC, and CACTI) to identify biomarkers associated with rapid eGFR decline. Here, we report the rationale for and design of these studies as well as results of models testing associations of clinical characteristics with rapid eGFR decline in the study population, upon which "omics" studies will be built. Cases (n = 535) and controls (n = 895) were defined as having an annual eGFR decline of >= 3 andPeer reviewe

An optimized classification system of acute kidney injury for predicting the short-term mortality after open heart surgery; comparison of current classification systems.

University of Minnesota M.S. thesis. May 2010. Major: Clinical Research. Advisor: Hassan N. Ibrahim MD, MS. 1 computer file (PDF); vii, 66 pages, appendices 1-14. Ill. (some col.)Epidemiologic studies need a unique operational definition of acute kidney injury (AKI) to compare outcomes. We aimed to compare prognostic value of change in serum creatinine with classification systems of AKI to predict 30day mortality after heart surgery. From VA database, 27410 eligible patients with stable baseline kidney function who had heart surgery from 1999 to 2005 were selected. There was a graded increase in mortality from stage A to stage C of all systems. Adjusted 30day mortality odds ratio starts to increase significantly after an acute rise ≥ 0.3 mg/dL of creatinine in CKD stages 1 and 2, and after 0.6 mg/dL increase in CKD stage 3. Area under ROC curve of change of creatinine from baseline was significantly higher than those of classification systems (P<0.001). In conclusion, compared to continuous increase of creatinine, classification systems of AKI misestimate mortality risk by collapsing predictive, clinically important data into categories

Acetyl Co-A Carboxylase Inhibition Halts Hyperglycemia Induced Upregulation of De Novo Lipogenesis in Podocytes and Proximal Tubular Cells

The effect of glycemic stress on de novo lipogenesis (DNL) in podocytes and tubular epithelial cells is understudied. This study is aimed (A) to show the effect of glycemic stress on DNL, and (B) to assess the effect of acetyl-Co A (ACC) inhibition on halting upregulation of DNL, on the expression of other lipid regulatory genes in the DNL pathway, and on markers of fibrosis and apoptosis in podocytes and tubular epithelial cells. We used cultured mouse primary tubular epithelial cells, mouse proximal tubular (BUMPT) cells, and immortal mouse podocytes and measured their percentage of labeled 13C2-palmitate as a marker of DNL after incubation with 13C2 acetate in response to high glucose concentration (25 mM). We then tested the effect of ACC inhibition by complimentary strategies utilizing CRISPR/cas9 deletion or incubation with Acaca and Acacb GapmeRs or using a small molecule inhibitor on DNL under hyperglycemic concentration. Exposure to high glucose concentration (25 mM) compared to osmotic controlled low glucose concentration (5.5 mM) significantly increased labeled palmitate after 24 h up to 72 h in podocytes and primary tubular cells. Knocking out of the ACC coding Acaca and Acacb genes by CRISPR/cas9, downregulation of Acaca and Acacb by specific antisense LNA GapmeRs and inhibition of ACC by firsocostat similarly halted/mitigated upregulation of DNL and decreased markers of fibrosis and programmed cell death in podocytes and various tubular cells. ACC inhibition is a potential therapeutic target to mitigate or halt hyperglycemia-induced upregulation of DNL in podocytes and tubular cells

Podocyte Depletion in Thin GBM and Alport Syndrome.

The proximate genetic cause of both Thin GBM and Alport Syndrome (AS) is abnormal α3, 4 and 5 collagen IV chains resulting in abnormal glomerular basement membrane (GBM) structure/function. We previously reported that podocyte detachment rate measured in urine is increased in AS, suggesting that podocyte depletion could play a role in causing progressive loss of kidney function. To test this hypothesis podometric parameters were measured in 26 kidney biopsies from 21 patients aged 2-17 years with a clinic-pathologic diagnosis including both classic Alport Syndrome with thin and thick GBM segments and lamellated lamina densa [n = 15] and Thin GBM cases [n = 6]. Protocol biopsies from deceased donor kidneys were used as age-matched controls. Podocyte depletion was present in AS biopsies prior to detectable histologic abnormalities. No abnormality was detected by light microscopy at 70% podocyte depletion. Low level proteinuria was an early event at about 25% podocyte depletion and increased in proportion to podocyte depletion. These quantitative data parallel those from model systems where podocyte depletion is the causative event. This result supports a hypothesis that in AS podocyte adherence to the GBM is defective resulting in accelerated podocyte detachment causing progressive podocyte depletion leading to FSGS-like pathologic changes and eventual End Stage Kidney Disease. Early intervention to reduce podocyte depletion is projected to prolong kidney survival in AS

ASC cohort characteristics at time of biopsy and treatment/outcome data.

<p>The deceased donor controls (n = 17) came from age 4–17 years deceased kidney donors with females/males = 12/5 similar to the ASC patient set (14/7).</p

Reduced eGFR is a late marker of podocyte depletion.

<p><b>Panel A</b>: Kidney function as measured by eGFR is detectably reduced only when podocyte depletion is reduced to the level of about 20% of normal (i.e. 80% depletion). <b>Panel B:</b> eGFR becomes measurably reduced when podocyte density falls below 100 per 10⁶ um³. <b>Panel C:</b> eGFR becomes measurably reduced when the Glepp1% area is reduced below about 15–20%. Two eGFR values of 380 and 400 ml/min from an infant aged 2 and 3 years with a serum creatinine of 0.1mg% and high level proteinuria at the time of biopsy were excluded from analysis because of the inherent inaccuracy of the eGFR estimate under those conditions. These data collectively show that reduction in eGFR below the normal range (60ml/min) is a late marker of podocyte depletion in ASC biopsies. Logarithmic equations using best fit curve estimation are used in all panels.</p

Clinical groupings showing podocyte depletion in ASC.

<p>Clinical parameters are shown to the left side and podometric parameters to the right side of the Table. Since age impacts podometric parameters the control group (n = 17 biopsies from deceased kidney donors aged 4–17 years) is appropriate for the ASC group (n = 26 biopsies from 21 patients aged 2–17 years). For cross-sectional analysis where more than one biopsy data set is available from one patient a single averaged value is used. Clinical parameters shown are from the time point at which the biopsy was performed. eGFR and urine protein:creatinine ratio (Urine PCR) data are not available (NA) for the control group. For each data set the mean (above in bold) and 1SD (below) are shown. Statistical comparisons of each data set compared to control using t-tests are shown by superscripted asterisks, while comparisons between sub-groups using analysis of variance are shown by asterisks in the area between the two sub-groups being compared. (* = P<0.05, ** = P <0.01). <b>Group Comparison:</b> All ASC subjects (n = 21) compared with control (n = 17) shows that glomerular volume was not different. However, podocyte number per glomerular tuft, podocyte nuclear density and podocyte cell (Glepp1 area density) were all decreased in ASC biopsies compared to control. Mean podocyte cell volume and mean podocyte nuclear diameter were not statistically significantly increased in ASC glomeruli. <b>GBM appearance:</b> Alport GBM is defined as typical alternating thin and thick GBM with lamellated lamina densa. Both the Thin GBM group (n = 5) and the Alport GBM group (n = 16) had significantly reduced podocyte number per tuft and Glepp1 area density compared to controls. The Thin GBM and Alport GBM groups were not significantly different from each other by any parameter (probably because one patient in the Thin GBM group progressed to ESKD in association with podocyte depletion). <b>Histologic appearance:</b> Biopsies containing only normal-appearing glomeruli without FSGS contained fewer podocytes and lower podocyte cell (Glepp1) area density than control. Biopsies with FSGS and/or global glomerulosclerosis (GGS) had significantly fewer podocytes (podocyte number per glomerulus, density or podocyte area density) than both control and ASC biopsies with normal appearing glomeruli. “<b>Non-progressors” vs Progressors</b>: Biopsies from people who did not progress (“Non-progressors”) as judged by retaining eGFR within the normal range by follow-up of 3 or more years (mean follow-up 7.4±3.9 years, n = 8) had significant podocyte depletion (reduced number of podocyte per glomerulus and reduced podocyte area [Glepp1 area density] compared to control. Biopsies from people who subsequently progressed to ESKD requiring renal replacement therapy (n = 7 patients) had significantly increased podocyte depletion at time of biopsy (reduced podocyte number per glomerulus, podocyte nuclear density and Glepp1 area density and larger podocytes) than those that did not progress.</p

Comparison of two methods for measuring podocyte density.

<p><b>Panel A</b>. Log podocyte density plotted versus Glepp1 area (%). The two methods correlate with R² = 0.70. <b>Panel B</b>. Podocyte density x glomerular volume gives the podocyte number per tuft. This value also correlates well with Glepp1 area (R² = 0.63). Bland-Altman comparison of podocyte density and podocyte number per glomerulus shows a mean difference of 106 (95% confidence interval of 115 to 327), and the Pitman’s test of difference in variance reveals r = 0.072, p = 0.728 indicating comparable results.</p