Diabetic nephropathy is currently the leading cause of end-stage renal disease worldwide, and occurs in approximately one third of all diabetic patients. The molecular pathogenesis of diabetic nephropathy has not been fully characterized and novel mediators and drivers of the disease are still being described. Previous data from our laboratory has identified the developmentally regulated gene Gremlin as a novel target implicated in diabetic nephropathy in vitro and in vivo. We used bioinformatic analysis to examine whether Gremlin gene sequence and structure could be used to identify other genes implicated in diabetic nephropathy. The Notch ligand Jagged1 and its downstream effector, hairy enhancer of split-1 (Hes1), were identified as genes with significant similarity to Gremlin in terms of promoter structure and predicted microRNA binding elements. This led us to discover that transforming growth factor-beta (TGFbeta1), a primary driver of cellular changes in the kidney during nephropathy, increased Gremlin, Jagged1 and Hes1 expression in human kidney epithelial cells. Elevated levels of Gremlin, Jagged1 and Hes1 were also detected in extracts from renal biopsies from diabetic nephropathy patients, but not in control living donors. In situ hybridization identified specific upregulation and co-expression of Gremlin, Jagged1 and Hes1 in the same tubuli of kidneys from diabetic nephropathy patients, but not controls. Finally, Notch pathway gene clustering showed that samples from diabetic nephropathy patients grouped together, distinct from both control living donors and patients with minimal change disease. Together, these data suggest that Notch pathway gene expression is elevated in diabetic nephropathy, co-incident with Gremlin, and may contribute to the pathogenesis of this disease.

OBJECTIVEGremlin (grem1) is an antagonist of the bone morphogenetic protein family that plays a key role in limb bud development and kidney formation. There is a growing appreciation that altered grem1 expression may regulate the homeostatic constraints on damage responses in diseases such as diabetic nephropathy.RESEARCH DESIGN AND METHODSHere we explored whether knockout mice heterozygous for grem1 gene deletion (grem1+/−) exhibit protection from the progression of diabetic kidney disease in a streptozotocin-induced model of type 1 diabetes.RESULTSA marked elevation in grem1 expression was detected in the kidneys and particularly in kidney tubules of diabetic wild-type mice compared with those of littermate controls. In contrast, diabetic grem1+/− mice displayed a significant attenuation in grem1 expression at 6 months of diabetes compared with that in age- and sex-matched wild-type controls. Whereas the onset and induction of diabetes were similar between grem1+/− and wild-type mice, several indicators of diabetes-associated kidney damage such as increased glomerular basement membrane thickening and microalbuminuria were attenuated in grem1+/− mice compared with those in wild-type controls. Markers of renal damage such as fibronectin and connective tissue growth factor were elevated in diabetic wild-type but not in grem1+/− kidneys. Levels of pSmad1/5/8 decreased in wild-type but not in grem1+/− diabetic kidneys, suggesting that bone morphogenetic protein signaling may be maintained in the absence of grem1.CONCLUSIONSThese data identify grem1 as a potential modifier of renal injury in the context of diabetic kidney disease.

Transforming growth factor-beta (TGF-β1) is implicated in the onset and progression of renal fibrosis and diabetic nephropathy (DN), leading to a loss of epithelial characteristics of tubular cells. The transcriptional profile of renal tubular epithelial cells stimulated with TGF-β1 was assessed using RNA-Seq, with 2027 differentially expressed genes identified. Promoter analysis of transcription factor binding sites in the TGF-β1 responsive gene set predicted activation of multiple transcriptional networks, including NFκB. Comparison of RNA-Seq with microarray data from identical experimental conditions identified low abundance transcripts exclusive to RNA-Seq data. We compared these findings to human disease by analyzing transcriptomic data from renal biopsies of patients with DN versus control groups, identifying a shared subset of 179 regulated genes. ARK5, encoding an AMP-related kinase, and TGFBI - encoding transforming growth factor, beta-induced protein were induced by TGF-β1 and also upregulated in human DN. Suppression of ARK5 attenuated fibrotic responses of renal epithelia to TGF-β1 exposure; and silencing of TGFBI induced expression of the epithelial cell marker – E-cadherin. We identified low abundance transcripts in sequence data and validated expression levels of several transcripts (ANKRD56, ENTPD8) in tubular enriched kidney biopsies of DN patients versus living donors. In conclusion, we have defined a TGF-β1-driven pro-fibrotic signal in renal epithelial cells that is also evident in the DN renal transcriptome.

Transcriptome analysis using microarray technology represents a powerful unbiased approach for delineating pathogenic mechanisms in disease. Here molecular mechanisms of renal tubulointerstitial fibrosis (TIF) were probed by monitoring changes in the renal transcriptome in a glomerular disease-dependent model of TIF (adriamycin nephropathy) using Affymetrix (mu74av2) microarray coupled with sequential primary biological function-focused and secondary "baited"-global cluster analysis of gene expression profiles. Primary cluster analysis focused on mRNAs encoding matrix proteins and modulators of matrix turnover as classified by Onto-Compare and Gene Ontology and identified both molecules and pathways already implicated in the pathogenesis of TIF (e.g. transforming growth factor ␤1-CTGF-fibronectin-1 pathway) and novel TIF-associated genes (e.g. SPARC and Matrilin-2). Specific gene expression patterns identified by primary extracellular matrix-focused cluster analysis were then used as bioinformatic bait in secondary global clustering, with which to search the renal transcriptome for novel modulators of TIF. Among the genes clustering with ECM proteins in the latter analysis were endoglin, clusterin, and gelsolin. In several notable cases (e.g. claudin-1 and meprin-1␤) the pattern of gene expression identified in adriamycin nephropathy in vivo was replicated during transdifferentiation of renal tubule epithelial cells to a fibroblast-like phenotype in vitro on exposure to transforming growth factor-␤ and epidermal growth factor suggesting a role in fibrogenesis. The further exploration of these complex gene networks should shed light on the core molecular pathways that underpin TIF in renal disease.

Chronic fibrosis represents the final common pathway in progressive renal disease. Myofibroblasts deposit the constituents of renal scar, thus crippling renal function. It has recently emerged that an important source of these pivotal effector cells is the injured renal epithelium. This review concentrates on the process of epithelial-mesenchymal transition (EMT) and its regulation. The role of the developmental gene, gremlin, which is reactivated in adult renal disease, is the subject of particular focus. This member of the cysteine knot protein superfamily is critical to the process of nephrogenesis but quiescent in normal adult kidney. There is increasing evidence that gremlin expression reactivates in diabetic nephropathy, and in the diseased fibrotic kidney per se. Known to antagonize members of the bone morphogenic protein (BMP) family, gremlin may also act downstream of TGF-β in induction of EMT. An increased understanding of the extracellular modulation of EMT and, in particular, of the gremlin-BMP axis may result in strategies that can halt or reverse the devastating progression of chronic renal fibrosis.

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IntroductionShared treatment decision-making and planning of care are fundamental in advanced chronic kidney disease (CKD) management. There are limited data on several key outcomes for the elderly population including survival, quality of life, symptom burden, changes in physical functioning and experienced burden of healthcare. Patients, caregivers and clinicians consequently face significant uncertainty when making life-impacting treatment decisions. The Elderly Advanced CKD Programme includes quantitative and qualitative studies to better address challenges in treatment decision-making and planning of care among this increasingly prevalent elderly cohort.Methods and analysisThe primary component is OUTcomes of Older patients with Kidney failure (OUTLOOK), a multicentre prospective observational cohort study that will enrol 800 patients ≥75 years with kidney failure (estimated glomerular filtration rate ≤15 mL/min/1.73 m2) across a minimum of six sites in Australia. Patients entered are in the decision-making phase or have recently made a decision on preferred treatment (dialysis, conservative kidney management or undecided). Patients will be prospectively followed until death or a maximum of 4 years, with the primary outcome being survival. Secondary outcomes are receipt of short-term acute dialysis, receipt of long-term maintenance dialysis, changes in biochemistry and end-of-life care characteristics. Data will be used to formulate a risk prediction tool applicable for use in the decision-making phase. The nested substudies Treatment modalities for the InfirM ElderLY with end stage kidney disease (TIMELY) and Caregivers of The InfirM ElderLY with end stage kidney disease (Co-TIMELY) will longitudinally assess quality of life, symptom burden and caregiver burden among 150 patients and 100 caregivers, respectively. CONsumer views of Treatment options for Elderly patieNts with kiDney failure (CONTEND) is an additional qualitative study that will enrol a minimum of 20 patients and 20 caregivers to explore experiences of treatment decision-making and care.Ethics and disseminationEthics approval was obtained through Sydney Local Health District Human Research Ethics Committee (2019/ETH07718, 2020/ETH02226, 2021/ETH01020, 2019/ETH07783). OUTLOOK is approved to have waiver of individual patient consent. TIMELY, Co-TIMELY and CONTEND participants will provide written informed consent. Final results will be disseminated through peer-reviewed journals and presented at scientific meetings.