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

Identification of podocyte nuclei and cells in ASC biopsies.

<p>Upper panels show podocyte nuclei (red) identified by TLE4 immunofluoresce. Lower panels show Glepp1 immunoperoxidase (brown) in the same sections. Panels A and A* shows a normal glomerulus. Panels B to D and B* to D* show glomeruli with progressively reduced numbers of podocytes and Glepp1 peroxidase positive area (as a % of the glomerular area) representing progressive glomerular injury associated with AS. Parietal podocytes are excluded from analysis by delineating the area of interest using software so that the podocyte nuclear count and Glepp1 area quantitation is for the tuft area only. Magnification is the same for all panels where the bar shown represents 50um.</p

Accelerated Podocyte Detachment Early After Kidney Transplantation Is Related to Long-Term Allograft Loss of Function

BACKGROUND: Kidney allograft half-life has not improved despite excellent short-term survival. Recent long-term surveillance biopsy studies identify accumulating glomerulosclerosis (GS) to be associated with late allograft loss. While podocyte depletion is well known to drive proteinuria and GS in animal models and human glomerular diseases, its role in renal allograft loss of function is generally not recognized. METHODS: To address these questions, we collected urine from 125 kidney allograft recipients in the first posttransplant year for urine pellet messenger RNA (mRNA) and protein analysis, with a median follow up of 4.5 years. RESULTS: Using multivariable linear models adjusted for proteinuria, transplant, recipient and donor factors, we observed that the average urine pellet podocin mRNA normalized to urine creatinine (UPodCR) in the first posttransplant year was significantly associated with an estimated glomerular filtration rate (eGFR) decline (P = 0.001). The relationship between UPodCR and eGFR decline persisted even among recipients who were nonproteinuric and who had no recurrent or de novo glomerular disease identified on 1-year protocol biopsy. Finally, we identified recipient, donor and recipient:donor body surface area mismatch ratio to be independently associated with UPodCR early after transplantation. A larger donor was protective, while a larger recipient and increased recipient:donor size mismatch ratio were associated with increased UPodCR. CONCLUSIONS: These findings support the concept that in kidney allografts, accelerated podocyte loss precedes proteinuria and is associated with inferior long-term allograft outcomes as measured by eGFR decline and may be initiated by recipient:donor size mismatch. Modulating factors driving early podocyte detachment after kidney transplantation may help improve long-term outcomes